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Sample records for blotting northern

  1. Multiplexed miRNA northern blots via hybridization chain reaction.

    PubMed

    Schwarzkopf, Maayan; Pierce, Niles A

    2016-09-06

    Northern blots enable detection of a target RNA of interest in a biological sample using standard benchtop equipment. miRNAs are the most challenging targets as they must be detected with a single short nucleic acid probe. With existing approaches, it is cumbersome to perform multiplexed blots in which several RNAs are detected simultaneously, impeding the study of interacting regulatory elements. Here, we address this shortcoming by demonstrating multiplexed northern blotting based on the mechanism of hybridization chain reaction (HCR). With this approach, nucleic acid probes complementary to RNA targets trigger chain reactions in which fluorophore-labeled DNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability of HCR allows multiple amplifiers to operate simultaneously and independently within a blot, enabling straightforward multiplexing. We demonstrate simultaneous detection of three endogenous miRNAs in total RNA extracted from 293T and HeLa cells. For a given target, HCR signal scales linearly with target abundance, enabling relative and absolute quantitation. Using non-radioactive HCR, sensitive and selective miRNA detection is achieved using 2'OMe-RNA probes. The HCR northern blot protocol takes ∼1.5 days independent of the number of target RNAs.

  2. Detection of Long Noncoding RNA Expression by Nonradioactive Northern Blots.

    PubMed

    Hu, Xiaowen; Feng, Yi; Hu, Zhongyi; Zhang, Youyou; Yuan, Chao-Xing; Xu, Xiaowei; Zhang, Lin

    2016-01-01

    With the advances in sequencing technology and transcriptome analysis, it is estimated that up to 75 % of the human genome is transcribed into RNAs. This finding prompted intensive investigations on the biological functions of noncoding RNAs and led to very exciting discoveries of microRNAs as important players in disease pathogenesis and therapeutic applications. Research on long noncoding RNAs (lncRNAs) is in its infancy, yet a broad spectrum of biological regulations has been attributed to lncRNAs. As a novel class of RNA transcripts, the expression level and splicing variants of lncRNAs are various. Northern blot analysis can help us learn about the identity, size, and abundance of lncRNAs. Here we describe how to use northern blot to determine lncRNA abundance and identify different splicing variants of a given lncRNA.

  3. Detection of Long Non-coding RNA Expression by Non-radioactive Northern Blots

    PubMed Central

    Hu, Xiaowen; Feng, Yi; Hu, Zhongyi; Zhang, Youyou; Yuan, Chao-Xing; Xu, Xiaowei; Zhang, Lin

    2016-01-01

    With the advances in sequencing technology and transcriptome analysis, it is estimated that up to 75% of the human genome is transcribed into RNAs. This finding prompted intensive investigations on the biological functions of non-coding RNAs and led to very exciting discoveries of microRNAs as important players in disease pathogenesis and therapeutic applications. Research on long non-coding RNAs (lncRNAs) is in its infancy, yet a broad spectrum of biological regulations has been attributed to lncRNAs. As a novel class of RNA transcripts, the expression level and splicing variants of lncRNAs are various. Northern blot analysis can help us learn about the identity, size, and abundance of lncRNAs. Here we describe how to use northern blot to determine lncRNA abundance and identify different splicing variants of a given lncRNA. PMID:26721491

  4. An improved method for Southern DNA and Northern RNA blotting using a Mupid-2 Mini-Gel electrophoresis unit.

    PubMed

    Furuya, Hirokazu; Yamada, Takeshi; Ikezoe, Koji; Ohyagi, Yasumasa; Fukumaki, Yasuyuki; Fujii, Naoki

    2006-08-31

    An improved method for Southern DNA and Northern RNA blotting using the Mupid-2 Mini-Gel System is described. We get sharp and clear bands in Southern and Northern blotting after only 30 min short gel electrophoresis instead of the several hours large gel electrophoresis of conventional methods. The high electrical voltage with a pulse-like current of the Mupid-2 Mini-Gel System also allows reduction of the amount of formaldehyde, a harmful reagent, from the gel running buffer in RNA blotting. This minor modification of DNA and RNA blotting technique enables us to perform the complete experimental procedure more quickly economically in less space, than conventional Southern and Northern blotting, as well as using an extremely small amount of formaldehyde in RNA blotting.

  5. A sensitive non-radioactive northern blot method to detect small RNAs.

    PubMed

    Kim, Sang Woo; Li, Zhihua; Moore, Patrick S; Monaghan, A Paula; Chang, Yuan; Nichols, Mark; John, Bino

    2010-04-01

    The continuing discoveries of potentially active small RNAs at an unprecedented rate using high-throughput sequencing have raised the need for methods that can reliably detect and quantitate the expression levels of small RNAs. Currently, northern blot is the most widely used method for validating small RNAs that are identified by methods such as high-throughput sequencing. We describe a new northern blot-based protocol (LED) for small RNA (approximately 15-40 bases) detection using digoxigenin (DIG)-labeled oligonucleotide probes containing locked nucleic acids (LNA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide for cross-linking the RNA to the membrane. LED generates clearly visible signals for RNA amounts as low as 0.05 fmol. This method requires as little as a few seconds of membrane exposure to outperform the signal intensity using overnight exposure of isotope-based methods, corresponding to approximately 1000-fold improvement in exposure-time. In contrast to commonly used radioisotope-based methods, which require freshly prepared and hazardous probes, LED probes can be stored for at least 6 months, facilitate faster and more cost-effective experiments, and are more environmentally friendly. A detailed protocol of LED is provided in the Supplementary Data.

  6. The mouse prostaglandin E receptor EP2 subtype: cloning, expression, and northern blot analysis.

    PubMed

    Katsuyama, M; Nishigaki, N; Sugimoto, Y; Morimoto, K; Negishi, M; Narumiya, S; Ichikawa, A

    1995-09-25

    A functional cDNA clone for the mouse prostaglandin (PG) E receptor EP2 subtype was isolated from a mouse cDNA library. The mouse EP2 receptor consists of 362 amino acid residues with seven putative transmembrane domains. [3H]PGE2 bound specifically to the membrane of Chinese hamster ovary cells stably expressing the cloned receptor. This binding was displaced by unlabeled prostanoids in the order of PGE2 = PGE1 > iloprost, a stable PGI2 agonist > PGF2 alpha > PGD2. Binding was also inhibited by butaprost (an EP2 agonist) and to a lesser extent by M&B 28767 (an EP3 agonist), but not by sulprostone (an EP1 and EP3 agonist) or SC-19220 (an EP1 antagonist). PGE2 and butaprost increased the cAMP level in the Chinese hamster ovary cells in a concentration-dependent manner. Northern blot analysis revealed that EP2 mRNA is expressed most abundantly in the uterus, followed by the spleen, lung, thymus, ileum, liver, and stomach.

  7. Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides.

    PubMed

    Mishima, Eikan; Jinno, Daisuke; Akiyama, Yasutoshi; Itoh, Kunihiko; Nankumo, Shinnosuke; Shima, Hisato; Kikuchi, Koichi; Takeuchi, Yoichi; Elkordy, Alaa; Suzuki, Takehiro; Niizuma, Kuniyasu; Ito, Sadayoshi; Tomioka, Yoshihisa; Abe, Takaaki

    2015-01-01

    The biological roles of RNA modifications are still largely not understood. Thus, developing a method for detecting RNA modifications is important for further clarification. We developed a method for detecting RNA modifications called immuno-northern blotting (INB) analysis and herein introduce its various capabilities. This method involves the separation of RNAs using either polyacrylamide or agarose gel electrophoresis, followed by transfer onto a nylon membrane and subsequent immunoblotting using antibodies against modified nucleosides for the detection of specific modifications. We confirmed that INB with the antibodies for 1-methyladenosine (m1A), N6-methyladenosine (m6A), pseudouridine, and 5-methylcytidine (m5C) showed different modifications in a variety of RNAs from various species and organelles. INB with the anti-m5C antibody revealed that the antibody cross-reacted with another modification on DNA, suggesting the application of this method for characterization of the antibody for modified nucleosides. Additionally, using INB with the antibody for m1A, which is a highly specific modification in eukaryotic tRNA, we detected tRNA-derived fragments known as tiRNAs under the cellular stress response, suggesting the application for tracking target RNA containing specific modifications. INB with the anti-m6A antibody confirmed the demethylation of m6A by the specific demethylases fat mass and obesity-associated protein (FTO) and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m6A modification in small RNAs as well as in mRNA. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Therefore, this method would be useful for research on RNA modifications and metabolism.

  8. Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides

    PubMed Central

    Akiyama, Yasutoshi; Itoh, Kunihiko; Nankumo, Shinnosuke; Shima, Hisato; Kikuchi, Koichi; Takeuchi, Yoichi; Elkordy, Alaa; Suzuki, Takehiro; Niizuma, Kuniyasu; Ito, Sadayoshi; Tomioka, Yoshihisa; Abe, Takaaki

    2015-01-01

    The biological roles of RNA modifications are still largely not understood. Thus, developing a method for detecting RNA modifications is important for further clarification. We developed a method for detecting RNA modifications called immuno-northern blotting (INB) analysis and herein introduce its various capabilities. This method involves the separation of RNAs using either polyacrylamide or agarose gel electrophoresis, followed by transfer onto a nylon membrane and subsequent immunoblotting using antibodies against modified nucleosides for the detection of specific modifications. We confirmed that INB with the antibodies for 1-methyladenosine (m1A), N6-methyladenosine (m6A), pseudouridine, and 5-methylcytidine (m5C) showed different modifications in a variety of RNAs from various species and organelles. INB with the anti-m5C antibody revealed that the antibody cross-reacted with another modification on DNA, suggesting the application of this method for characterization of the antibody for modified nucleosides. Additionally, using INB with the antibody for m1A, which is a highly specific modification in eukaryotic tRNA, we detected tRNA-derived fragments known as tiRNAs under the cellular stress response, suggesting the application for tracking target RNA containing specific modifications. INB with the anti-m6A antibody confirmed the demethylation of m6A by the specific demethylases fat mass and obesity-associated protein (FTO) and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m6A modification in small RNAs as well as in mRNA. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Therefore, this method would be useful for research on RNA modifications and metabolism. PMID:26606401

  9. Quantitative Expression Analysis in Brassica napus by Northern Blot Analysis and Reverse Transcription-Quantitative PCR in a Complex Experimental Setting.

    PubMed

    Rumlow, Annekathrin; Keunen, Els; Klein, Jan; Pallmann, Philip; Riemenschneider, Anja; Cuypers, Ann; Papenbrock, Jutta

    Analysis of gene expression is one of the major ways to better understand plant reactions to changes in environmental conditions. The comparison of many different factors influencing plant growth challenges the gene expression analysis for specific gene-targeted experiments, especially with regard to the choice of suitable reference genes. The aim of this study is to compare expression results obtained by Northern blot, semi-quantitative PCR and RT-qPCR, and to identify a reliable set of reference genes for oilseed rape (Brassica napus L.) suitable for comparing gene expression under complex experimental conditions. We investigated the influence of several factors such as sulfur deficiency, different time points during the day, varying light conditions, and their interaction on gene expression in oilseed rape plants. The expression of selected reference genes was indeed influenced under these conditions in different ways. Therefore, a recently developed algorithm, called GrayNorm, was applied to validate a set of reference genes for normalizing results obtained by Northern blot analysis. After careful comparison of the three methods mentioned above, Northern blot analysis seems to be a reliable and cost-effective alternative for gene expression analysis under a complex growth regime. For using this method in a quantitative way a number of references was validated revealing that for our experiment a set of three references provides an appropriate normalization. Semi-quantitative PCR was prone to many handling errors and difficult to control while RT-qPCR was very sensitive to expression fluctuations of the reference genes.

  10. Quantitative Expression Analysis in Brassica napus by Northern Blot Analysis and Reverse Transcription-Quantitative PCR in a Complex Experimental Setting

    PubMed Central

    Rumlow, Annekathrin; Keunen, Els; Klein, Jan; Pallmann, Philip; Riemenschneider, Anja; Cuypers, Ann

    2016-01-01

    Analysis of gene expression is one of the major ways to better understand plant reactions to changes in environmental conditions. The comparison of many different factors influencing plant growth challenges the gene expression analysis for specific gene-targeted experiments, especially with regard to the choice of suitable reference genes. The aim of this study is to compare expression results obtained by Northern blot, semi-quantitative PCR and RT-qPCR, and to identify a reliable set of reference genes for oilseed rape (Brassica napus L.) suitable for comparing gene expression under complex experimental conditions. We investigated the influence of several factors such as sulfur deficiency, different time points during the day, varying light conditions, and their interaction on gene expression in oilseed rape plants. The expression of selected reference genes was indeed influenced under these conditions in different ways. Therefore, a recently developed algorithm, called GrayNorm, was applied to validate a set of reference genes for normalizing results obtained by Northern blot analysis. After careful comparison of the three methods mentioned above, Northern blot analysis seems to be a reliable and cost-effective alternative for gene expression analysis under a complex growth regime. For using this method in a quantitative way a number of references was validated revealing that for our experiment a set of three references provides an appropriate normalization. Semi-quantitative PCR was prone to many handling errors and difficult to control while RT-qPCR was very sensitive to expression fluctuations of the reference genes. PMID:27685087

  11. Analysis of solvent control and 1-nitrosopyrene-induced chinese hamster ovary cell mutants by southern and northern blots and the polymerase chain reaction

    SciTech Connect

    Newton, R.K.; Mittelstaedt, R.A.; Heflich, R.H. )

    1992-01-01

    1-Nitrosopyrene, a metabolite of the tumorigenic environmental pollutant 1-nitropyrene, is a potent mutagen at the hprt locus in Chinese hamster ovary (CHO) cells. A single DNA adduct, N-(deoxyguanosin-8-yl)-l-aminopyrene, is produced in CHO cells treated with 1-nitrosopyrene, and this adduct is found in rats and mice exposed to 1-nitropyrene. In this study, the structure of the hprt gene and the structure and amount of hprt mRNA were analyzed in 43 CHO cell mutants (16 isolated from solvent control cultures and 27 isolated from 1-nitrosopyrene-treated cultures). PstI- and BamHI-digested DNA from the mutants were subjected to Southern blot analysis using a hamster hprt cDNA probe. None of the 1-nitrosopyrene-induced mutants and only one of the control mutants displayed hybridization patterns that were different from the parent CHO cells. Northern blot analysis revealed that two control mutants had truncated hprt mRNAs, while 56% of the control mutants and 78% of the induced mutants had reduced levels of hprt mRNA. Using polymerase chain reaction amplification of cDNA synthesized from RNA, the hprt protein-coding region could be amplified from 23 of the 1-nitrosopyrene-induced mutants and 11 of the control mutants. The amplification products from 3 of the control mutants and 5 of the induced mutants were smaller than that found with RNA from parental CHO cells. These results indicate that the mutagenic DNA damage produced by 1-nitrosopyrene in CHO cells does not cause major structural alterations in the hprt gene and suggest that 1-nitrosopyrene acts as a point mutagen. A large number of both control and 1-nitrosopyrene-induced mutants exhibited a marked reduction in hprt mRNA concentration or possessed truncated mRNA hprt protein coding sequences. These alterations may contribute to the 6-thioguanine-resistant phenotype.

  12. MicroRNA fate upon targeting with anti-miRNA oligonucleotides as revealed by an improved Northern-blot-based method for miRNA detection

    PubMed Central

    Torres, Adrian G.; Fabani, Martin M.; Vigorito, Elena; Gait, Michael J.

    2011-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs involved in fine-tuning of gene regulation. Antisense oligonucleotides (ONs) are promising tools as anti-miRNA (anti-miR) agents toward therapeutic applications and to uncover miRNA function. Such anti-miR ONs include 2′-O-methyl (OMe), cationic peptide nucleic acids like K-PNA-K3, and locked nucleic acid (LNA)-based anti-miRs such as LNA/DNA or LNA/OMe. Northern blotting is a widely used and robust technique to detect miRNAs. However, miRNA quantification in the presence of anti-miR ONs has proved to be challenging, due to detection artifacts, which has led to poor understanding of miRNA fate upon anti-miR binding. Here we show that anti-miR ON bound to miR-122 can prevent the miRNA from being properly precipitated into the purified RNA fraction using the standard RNA extraction protocol (TRI-Reagent), yielding an RNA extract that does not reflect the real cellular levels of the miRNA. An increase in the numbers of equivalents of isopropanol during the precipitation step leads to full recovery of the targeted miRNA back into the purified RNA extract. Following our improved protocol, we demonstrate by Northern blotting, in conjunction with a PNA decoy strategy and use of high denaturing PAGE, that high-affinity anti-miRs (K-PNA-K3, LNA/DNA, and LNA/OMe) sequester miR-122 without causing miRNA degradation, while miR-122 targeting with a lower-affinity anti-miR (OMe) seems to promote degradation of the miRNA. The technical issues explored in this work will have relevance for other hybridization-based techniques for miRNA quantification in the presence of anti-miR ONs. PMID:21441346

  13. Whole genomic sequencing of RT98 mitochondria derived from Oryza rufipogon and northern blot analysis to uncover a cytoplasmic male sterility-associated gene.

    PubMed

    Igarashi, Keisuke; Kazama, Tomohiko; Motomura, Keiji; Toriyama, Kinya

    2013-02-01

    Cytoplasmic male sterility (CMS) is a maternally inherited trait resulting in the failure to produce functional pollen and is often observed when an alien cytoplasm is transferred into a cultivated species. An RT98A CMS line and an RT98C fertility restorer line were obtained by successive backcrossing between Oryza rufipogon W1109 and Oryza sativa cultivar Taichung 65. To uncover the CMS-associated mitochondrial genes, we determined the complete sequence of the RT98-CMS mitochondrial genome using next-generation pyrosequencing, and searched new open reading frames (orfs) absent in a reported mitochondrial genome of O. sativa Nipponbare. Then, six candidates were selected for the CMS-associated genes based on the criteria in which they were chimeric in structure or encoded a peptide with transmembrane domains. One of the candidates, orf113, showed different transcript sizes between RT98A and RT98C on Northern blot analysis. The orf113 gene was shown to be co-transcribed with atp4 and cox3 encoding ATP synthase F0 subunit 4 and Cyt c oxidase subunit 3, respectively, and their transcripts were distinctly processed in the presence of a fertility restorer gene. Our results indicate that orf113 is a CMS-associated gene of RT98-CMS.

  14. The western blot

    USDA-ARS?s Scientific Manuscript database

    Western blotting is a technique that involves the separation of proteins by gel electrophoresis, their blotting or transfer to a membrane, and selective immunodetection of an immobilized antigen. This is an important and routine method for protein analysis that depends on the specificity of antibod...

  15. The Dot Blot ELISA.

    ERIC Educational Resources Information Center

    Gerbig, Donald G., Jr.; Fenk, Christopher J.; Goodhart, Amy S.

    2000-01-01

    Uses two laboratory techniques, Enzyme Linked Immunosorbent Assay (ELISA) and Western Blot, to demonstrate antibody-antigen binding concepts. Includes a list of required materials and directions for the procedure, and makes suggestions for classroom applications. (Contains 13 references.) (YDS)

  16. The Dot Blot ELISA.

    ERIC Educational Resources Information Center

    Gerbig, Donald G., Jr.; Fenk, Christopher J.; Goodhart, Amy S.

    2000-01-01

    Uses two laboratory techniques, Enzyme Linked Immunosorbent Assay (ELISA) and Western Blot, to demonstrate antibody-antigen binding concepts. Includes a list of required materials and directions for the procedure, and makes suggestions for classroom applications. (Contains 13 references.) (YDS)

  17. Western blot analysis.

    PubMed

    Hirano, Seishiro

    2012-01-01

    Electrophoresis and the following western blot analysis are indispensable to investigate biochemical changes in cells and tissues exposed to nanoparticles or nanomaterials. Proteins should be extracted from the cells and tissues using a proper method, especially when phosphorylated proteins are to be detected. It is important to select a good blocking agent and an appropriate pair of primary and peroxidase-tagged secondary antibodies to obtain good results in western blot analysis. One thing that may be specific to nanomaterials, and that you should keep in mind, is that some proteins may be adsorbed on the surface of particulate nanomaterials. In this chapter the whole process of western blot analysis, from sample preparation to quantitative measurement of target proteins, is described.

  18. The Western Blot.

    PubMed

    Hnasko, Thomas S; Hnasko, Robert M

    2015-01-01

    Western blotting is a technique that involves the separation of proteins by gel electrophoresis, their blotting or transfer to a membrane, and selective immunodetection of an immobilized antigen. This is an important and routine method for protein analysis that depends on the specificity of antibody-antigen interaction and is useful for the qualitative or semiquantitative identification of specific proteins and their molecular weight from a complex mixture. This chapter will outline the requisite steps including gel electrophoresis of a protein sample, transfer of protein from a gel to a membrane support, and immunodetection of a target antigen.

  19. Western Blot Techniques.

    PubMed

    Kim, Brianna

    2017-01-01

    The Western blot is an important laboratory technique that allows for specific identification and characterization of proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins are electophoretically transferred to a polyvinylidene fluoride (PVDF) membrane which is then incubated with specific antibodies, then developed to show the protein of interest. Here, we describe the transfer and detection of Outer surface protein A (OspA), a protein only found on the surface of Borrelia burgdorferi, the bacteria responsible for Lyme disease.

  20. BLOT Ver. 1.65

    SciTech Connect

    MEYERS, RAY; GLICK, III, JOHN; FORSYTHE, CHRISTI; GILKEY, AMY; SJAARDEMA, GREGORY

    2009-03-24

    BLOT is a graphic program for post-processing finite element analyses output in the EXODUS II database format. It is command driven with free-format input and can drive graphics devices supported by the Sandia Virtual Device Interface. BLOT produces mesh plots of the analysis output variables including deformed mesh plots, line contours, filled (painted) contours, vector plots of two/three variables (velocity vectors), and symbol plots of scalar variables (discrete cracks). Features include pathlines of analysis variables drawn on the mesh, element selection by material, element birth and death, multiple views combining several displays on each plot, symmetry mirroring, and node and element numbering. X-Y plots of the analysis variables include time vs. variable plots or variable vs. variable plots, and distance vs. variable plots at selected time steps where distance is the accumulated distance between pairs of nodes or element centers. BLOT is written in as portable a form as possible. Fortran code is written in ANSI Standard FORTRAN-77. Machine-specific routines are limited in number and are grouped together to minimize the time required to adapt them to a new system. SEACAS codes have been ported to several Unix systems

  1. Single-cell western blotting

    PubMed Central

    Hughes, Alex J.; Spelke, Dawn P.; Xu, Zhuchen; Kang, Chi-Chih; Schaffer, David V.; Herr, Amy E.

    2014-01-01

    To measure cell-to-cell variation in protein-mediated functions — a hallmark of biological processes — we developed an approach to conduct ~103 concurrent single-cell western blots (scWesterns) in ~4 hours. A microscope slide supporting a 30 µm-thick photoactive polyacrylamide gel enables western blotting comprised of: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated blotting to immobilize proteins, and antibody probing. We apply this scWestern to monitor single rat neural stem cell differentiation and responses to mitogen stimulation. The scWestern quantifies target proteins even with off-target antibody binding, multiplexes to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supports analyses of low starting cell numbers (~200) when integrated with fluorescence activated cell sorting. The scWestern thus overcomes limitations in single-cell protein analysis (i.e., antibody fidelity, sensitivity, and starting cell number) and constitutes a versatile tool for the study of complex cell populations at single-cell resolution. PMID:24880876

  2. Lectin-probed western blot analysis.

    PubMed

    Sato, Takeshi

    2014-01-01

    Lectin-probed western blot analysis, the so-called lectin blot analysis, is a useful method to yield basic information on the glycan structures of glycoproteins, based on the carbohydrate-binding specificities of lectins. By lectin blot analysis, researchers can directly analyze the glycan structures without releasing the glycans from glycoproteins. Here, the author describes protocols for standard analysis, and applies analysis in combination with glycosidase digestion of blot.

  3. Detection of LINE-1 RNAs by Northern Blot.

    PubMed

    Deininger, Prescott; Belancio, Victoria P

    2016-01-01

    Repetitive genetic elements have had an unprecedented success populating phylogenetically diverse species making them a common feature of most genomes. Hundreds of thousands of copies of active and non-functional transposable elements representing different classes and families can reside within and outside of host genes. In addition to creating structural variations in genomic DNA, some of these loci are expressed to contribute to the continuing amplification cycle. Transposable elements, specifically Long Interspersed Element-1 (LINE-1) produce a spectrum of RNAs, some of which are important for their mobilization, while others are processed forms of LINE-1 transcription that may or may not play relevant functions. Additionally, many LINE-1 sequences integrated into cellular genes are included into cellular transcripts creating substantial background when L1-related RNA expression is detected by some conventional methods. This chapter provides an in-depth description of the complexity of L1-generated mRNAs as well as sources of cellular transcripts containing L1 sequences. It also highlights the strengths and weaknesses of conventional methods used to detect LINE-1 expression.

  4. Use of bidirectional blots in differential display analysis.

    PubMed

    Kestler, D P; Hill, M; Agarwal, S; Hall, R E

    2000-05-01

    We have used bidirectional transfer methods in concert with SMART total cDNA complex probes to sequentially screen differential display arrays. In this report we show the utility of this methodology in examining a manganese superoxide dismutase cDNA fragment which we detected while evaluating the effects of the proinflammatory cytokines IL1-beta, TNF-alpha, and IL6 on human umbilical vein endothelial cell (HUVEC) gene expression. By using parallel hybridization of the bidirectional blots with SMART total cDNA (32)P probes derived from untreated or cytokine-treated HUVECs, differential expression between cell treatments can be clearly evaluated. Subsequent screening using this bidirectional blot method results in detection of modulated cDNA clones. Northern and total cDNA blot hybridization with the cDNA clonal fragment confirmed both modulated expression and the efficacy of this screening method. These procedures allow one to use bidirectional blots to evaluate band modulation on agarose gels which are initially run to evaluate the reamplification of display fragments or to confirm cloned cDNA fragments. Thus, bidirectional blot analysis using SMART total cDNA probes allows direct evaluation of differential display bands from the initial reamplification through plasmid insert cloning, increasing the investigator's ability to eliminate false-positive bands during each step of analysis. Copyright 2000 Academic Press.

  5. Problem-Solving Test: Southwestern Blotting

    ERIC Educational Resources Information Center

    Szeberényi, József

    2014-01-01

    Terms to be familiar with before you start to solve the test: Southern blotting, Western blotting, restriction endonucleases, agarose gel electrophoresis, nitrocellulose filter, molecular hybridization, polyacrylamide gel electrophoresis, proto-oncogene, c-abl, Src-homology domains, tyrosine protein kinase, nuclear localization signal, cDNA,…

  6. Problem-Solving Test: Southwestern Blotting

    ERIC Educational Resources Information Center

    Szeberényi, József

    2014-01-01

    Terms to be familiar with before you start to solve the test: Southern blotting, Western blotting, restriction endonucleases, agarose gel electrophoresis, nitrocellulose filter, molecular hybridization, polyacrylamide gel electrophoresis, proto-oncogene, c-abl, Src-homology domains, tyrosine protein kinase, nuclear localization signal, cDNA,…

  7. Detection of Proteins on Blot Membranes.

    PubMed

    Goldman, Aaron; Harper, Sandra; Speicher, David W

    2016-11-01

    Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. © 2016 by John Wiley & Sons, Inc.

  8. Western blot: technique, theory, and trouble shooting.

    PubMed

    Mahmood, Tahrin; Yang, Ping-Chang

    2012-09-01

    Western blotting is an important technique used in cell and molecular biology. By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses three elements to accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize. This paper will attempt to explain the technique and theory behind western blot, and offer some ways to troubleshoot.

  9. Studying protein-protein interactions via blot overlay/far western blot.

    PubMed

    Hall, Randy A

    2015-01-01

    Blot overlay is a useful method for studying protein-protein interactions. This technique involves fractionating proteins on SDS-PAGE, blotting to nitrocellulose or PVDF membrane, and then incubating with a probe of interest. The probe is typically a protein that is radiolabeled, biotinylated, or simply visualized with a specific antibody. When the probe is visualized via antibody detection, this technique is often referred to as "Far Western blot." Many different kinds of protein-protein interactions can be studied via blot overlay, and the method is applicable to screens for unknown protein-protein interactions as well as to the detailed characterization of known interactions.

  10. Streamlined Strategies to Better Visualize Southern Blotting

    ERIC Educational Resources Information Center

    Dean, Derek M.

    2012-01-01

    In this article, I describe an animated slideshow of Southern blotting that I have made freely available to other instructors. My hope is to provide a clear visualization of the logistics behind the technique so that instructors have a solid basis--as well as time freed up--to discuss its applications with students.

  11. Blot hybridisation analysis of genomic DNA.

    PubMed Central

    Vandenplas, S; Wiid, I; Grobler-Rabie, A; Brebner, K; Ricketts, M; Wållis, G; Bester, A; Boyd, C; Måthew, C

    1984-01-01

    Restriction endonuclease analysis of specific gene sequences is proving to be a valuable technique for characterisation and diagnosis of inherited disorders. This paper describes detailed protocols for isolation, restriction, and blot hybridisation of genomic DNA. Problems and alternatives in the procedure are discussed and a troubleshooting guide has been provided to help rectify faults. Images PMID:6086927

  12. Streamlined Strategies to Better Visualize Southern Blotting

    ERIC Educational Resources Information Center

    Dean, Derek M.

    2012-01-01

    In this article, I describe an animated slideshow of Southern blotting that I have made freely available to other instructors. My hope is to provide a clear visualization of the logistics behind the technique so that instructors have a solid basis--as well as time freed up--to discuss its applications with students.

  13. Single cell–resolution western blotting

    PubMed Central

    Kang, Chi-Chih; Yamauchi, Kevin A; Vlassakis, Julea; Sinkala, Elly; Duncombe, Todd A; Herr, Amy E

    2017-01-01

    This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). the gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. to extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. once the microdevice has been fabricated, the assay can be completed in 4–6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. the technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine. PMID:27466711

  14. TLC-Blot (Far-Eastern Blot) and Its Application to Functional Lipidomics.

    PubMed

    Taki, Takao

    2015-01-01

    A simple method for transfer of lipids-including phospholipids, glycolipids, and neutral lipids-from a high performance thin layer chromatography (HPTLC) plate to a polyvinylidene difluoride (PVDF) membrane, TLC-Blot (Far-Eastern Blot), and its biochemical applications are presented. This chapter presents the conventional procedures for separating lipid from tissue samples, cultured cells, and serum and the subsequent development of TLC. Individual lipids separated on an HPTLC plate can be transferred to the PVDF membrane quantitatively and also isolated from the lipid-blotted membrane by a one-step purification procedure. Immunodetection with monoclonal antibodies and treatment with lipid-metabolizing enzymes on the lipid-blotted membrane are possible. The method for identification of individual lipids transferred on the PVDF membrane using matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (TLC-Blot/MALDI-TOF MS) is shown as a functional lipidomics application.

  15. BLOT II Ver.1.39

    SciTech Connect

    2003-06-03

    BLOT II is a graphic program for post-processing finite element analyses output in the EXODUS II database format. It is command driven with free-format input and can drive graphics devices supported by the Sandia Virtual Device Interface. BLOT produces mesh plots of the analysis output variables including deformed mesh plots, line contours, filled (painted) contours, vector plots of two/three variables (velocity vectors), and symbol plots of scalar variables (discrete cracks). Features include pathlines of analysis variables drawn on the mesh, element selection by material, element birth and death, multiple views combining several displays on each plot, symmetry mirroring, and node and element numbering. X-Y plots of the analysis variables include time vs. variable plots or variable vs. variable plots, and distance vs. variable plots at selected time stips where distance is the accumulated distance between pairs of nodes or element centers.

  16. Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS)

    PubMed Central

    Gilda, Jennifer E.; Ghosh, Rajeshwary; Cheah, Jenice X.; West, Toni M.; Bodine, Sue C.; Gomes, Aldrin V.

    2015-01-01

    Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis. PMID:26287535

  17. Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS).

    PubMed

    Gilda, Jennifer E; Ghosh, Rajeshwary; Cheah, Jenice X; West, Toni M; Bodine, Sue C; Gomes, Aldrin V

    2015-01-01

    Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis.

  18. Line blot and western blot immunoassays for diagnosis of Mediterranean spotted fever.

    PubMed Central

    Raoult, D; Dasch, G A

    1989-01-01

    The line blot, a new immunoassay in which antigens are placed on nitrocellulose as narrow lines, was evaluated for its sensitivity and specificity relative to the microimmunofluorescence assay for the diagnosis of Mediterranean spotted fever (MSF). The line blot assay was only slightly less sensitive and less specific than the microimmunofluorescence assay for detection of immunoglobulin M (IgM) or IgG in 100 serum specimens from 42 patients with MSF. No line blot reactions were observed among 50 control serum specimens from febrile patients with other illnesses. The line blot assay was largely group reactive for spotted fever rickettsiae, but 26% of the positive serum specimens also cross-reacted by IgM with Rickettsia typhi. Western immunoblotting was used to characterize the antigenic components recognized by 19 MSF serum specimens. For both IgM and IgG, lipopolysaccharide was the cross-reactive group antigen, whereas the high-molecular-weight species-specific protein antigens (SPAs) were the only reactive proteins. Relative to the other nine rickettsiae, Rickettsia bellii was unique both in exhibiting no SPA reactions and in having a lipopolysaccharide with a predominantly high-molecular-weight distribution. Although most of the 19 MSF serum specimens examined by Western blotting exhibited preferential reactivity to SPAs of two strains of R. conorii and weaker reactions to the other rickettsiae, 2 serum specimens exhibited SPA reactions consistent with typhus infections. In comparison with other assays, the line blot and Western blot immunoassays have advantages which may permit an improvement in the general availability and commercialization of assays for the serodiagnosis of rickettsial infections. Images PMID:2506223

  19. Western blot analysis of adhesive interactions under fluid shear conditions: the blot rolling assay.

    PubMed

    Sackstein, Robert; Fuhlbrigge, Robert

    2015-01-01

    Western blotting has proven to be an important technique in the analysis of receptor-ligand interactions (i.e., by ligand blotting) and for identifying molecules mediating cell attachment (i.e., by cell blotting). Conventional ligand blotting and cell blotting methods employ non-dynamic (static) incubation conditions, whereby molecules or cells of interest are placed in suspension and overlaid on membranes. However, many cell-cell and cell-matrix adhesive interactions occur under fluid shear conditions, and shear stress itself mediates and/or facilitates the engagement of these physiologically appropriate receptors and ligands. Notably, shear forces critically influence the adhesion of circulating cells and platelets to vessel walls in physiologic cell migration and hemostasis, as well as in inflammatory and thrombotic disorders, cancer metastasis, and atherosclerosis. Use of non-dynamic blotting conditions to analyze such interactions can introduce bias, overtly missing relevant effectors and/or exaggerating the relative role(s) of non-physiologic adhesion molecules. To address this shortfall, we have developed a new technique for identifying binding interactions under fluid shear conditions, the "blot rolling assay." Using this method, molecules in a complex mixture are resolved by gel electrophoresis, transferred to a membrane that is rendered semitransparent, and the membrane is then incorporated into a parallel-plate flow chamber apparatus. Under controlled flow conditions, cells or particles bearing adhesion proteins of interest are then introduced into the chamber and interactions with individual immobilized molecules (bands) can be visualized in real time. The substrate molecule(s) supporting adhesion under fluid shear can then be identified by staining with specific antibodies or by excising the relevant band(s) and performing mass spectrometry or microsequencing of the isolated material. This method thus allows for the identification, within a complex

  20. Western blot analysis of adhesive interactions under fluid shear conditions: the blot rolling assay.

    PubMed

    Sackstein, Robert; Fuhlbrigge, Robert

    2009-01-01

    Western blotting has proven to be an important technique in analysis of receptor-ligand interactions (i.e., by ligand blotting) and for identifying molecules mediating cell attachment (i.e., by cell blotting). Conventional ligand blotting and cell blotting methods employ nondynamic (static) incubation conditions, whereby molecules or cells of interest are placed in suspension and overlaid on membranes. However, many cell-cell and cell-matrix adhesive interactions occur under fluid shear conditions, and shear stress itself mediates and/or facilitates the engagement of these physiologically appropriate receptors and ligands. Notably, shear forces critically influence the adhesion of circulating cells and platelets to vessel walls in physiologic cell migration and hemostasis, as well as in inflammatory and thrombotic disorders, cancer metastasis, and atherosclerosis. Use of nondynamic blotting conditions to analyze such interactions can introduce bias, overtly missing relevant effectors and/or exaggerating the relative role(s) of nonphysiologic adhesion molecules. To address this shortfall, we have developed a new technique for identifying binding interactions under fluid shear conditions, the "blot rolling assay." Using this method, molecules in a complex mixture are resolved by gel electrophoresis, transferred to a membrane that is rendered semi-transparent, and the membrane is then incorporated into a parallel-plate flow chamber apparatus. Under controlled flow conditions, cells or particles bearing adhesion proteins of interest are then introduced into the chamber and interactions with individual immobilized molecules (bands) can be visualized in real-time. The substrate molecule(s) supporting adhesion under fluid shear can then be identified by staining with specific antibodies or by excising the relevant band(s) and performing mass spectrometry or microsequencing of the isolated material. This method thus allows for the identification, within a complex mixture

  1. SDS -PAGE and Western Blotting Techniques.

    PubMed

    Blancher, C; Jones, A

    2001-01-01

    The goal of Western blotting, or more correctly, immunoblotting, is to identify with a specific antibody a particular antigen within a complex mixture of proteins that has been fractionated in a polyacrylamide gel and immobilized onto a membrane. Immunoblotting can be used to determine a number of important characteristics of protein antigens-the presence and quantity of an antigen, the relative molecular weight of the polypeptide chain, and the efficiency of extraction of the antigen.Immunoblotting occurs in six stages: (1) extraction and quantification of protein samples; (2) resolution of the protein sample in sodium dodecyl sulfatepolyacrylamide denaturing gel electrophoresis (SDS-PAGE); (3) transfer of the separated polypeptides to a membrane support; (4) blocking nonspecific binding sites on the membrane; (5) addition of antibodies; and (6) detection.Sample preparation is important for obtaining accurate separation of the proteins on the basis of molecular weight. Depending on whether an antigen is primarily extracellular, cytoplasmic, or membrane-associated different procedures might be required to prepare the sample initially. Although there are exceptions, many soluble nuclear and cytoplasmic proteins can be solubilized by lysis buffers that contain the nonionic detergent Nonidet P-40 (NP-40) and either no salt at all or relatively high concentrations of salt (e.g., 0.5 M NaCl). However, the efficiency of extraction is often greatly affected by pH of the buffer and the presence or absence of chelating agents such EDTA.

  2. Repeated probing of Southwestern blots using alkaline phosphatase stripping.

    PubMed

    Jia, Yinshan; Jiang, Daifeng; Jarrett, Harry W

    2010-11-05

    Southwestern blotting is when a DNA sequence is used to probe DNA-binding proteins on an electrophoretic gel blot. It would be highly desirable to be able to probe a blot repeatedly with different DNA sequences. Alkaline phosphatase can remove 5'-phosphoryl groups from DNA and radiolabeled 5'-(32)P-DNA probes are commonly used in Southwestern blotting. Here is shown that once probed, the radioisotope signal on the blot can be effectively removed by brief digestion with alkaline phosphatase, and the blot can then be repeatedly probed at least six times with different DNA probes. This exceeds the repetitions possible with another commonly used method using SDS. The technique can be used with either one-dimensional or multi-dimensional Southwestern blots and does not have a large effect on the phosphorylation state of the blotted proteins. An alternative method using T4 polynucleotide kinase stripping is also introduced but was less well characterized.

  3. Repeated probing of Southwestern blots using alkaline phosphatase stripping

    PubMed Central

    Jia, Yinshan; Jiang, Daifeng; Jarrett, Harry W.

    2010-01-01

    Southwestern blotting is when a DNA sequence is used to probe DNA-binding proteins on an electrophoretic gel blot. It would be highly desirable to be able to probe a blot repeatedly with different DNA sequences. Alkaline phosphatase can remove 5′-phosphoryl groups from DNA and radiolabeled 5′-32P-DNA probes are commonly used in Southwestern blotting. Here is shown that once probed, the radioisotope signal on the blot can be effectively removed by brief digestion with alkaline phosphatase, and the blot can then be repeatedly probed at least six times with different DNA probes. This exceeds the repetitions possible with another commonly used method using SDS. The technique can be used with either one-dimensional or multi-dimensional Southwestern blots and does not have a large effect on the phosphorylation state of the blotted proteins. An alternative method using T4 polynucleotide kinase stripping is also introduced but was less well characterized. PMID:20926088

  4. Dealing with large sample sizes: comparison of a new one spot dot blot method to western blot.

    PubMed

    Putra, Sulistyo Emantoko Dwi; Tsuprykov, Oleg; Von Websky, Karoline; Ritter, Teresa; Reichetzeder, Christoph; Hocher, Berthold

    2014-01-01

    Western blot is the gold standard method to determine individual protein expression levels. However, western blot is technically difficult to perform in large sample sizes because it is a time consuming and labor intensive process. Dot blot is often used instead when dealing with large sample sizes, but the main disadvantage of the existing dot blot techniques, is the absence of signal normalization to a housekeeping protein. In this study we established a one dot two development signals (ODTDS) dot blot method employing two different signal development systems. The first signal from the protein of interest was detected by horseradish peroxidase (HRP). The second signal, detecting the housekeeping protein, was obtained by using alkaline phosphatase (AP). Inter-assay results variations within ODTDS dot blot and western blot and intra-assay variations between both methods were low (1.04-5.71%) as assessed by coefficient of variation. ODTDS dot blot technique can be used instead of western blot when dealing with large sample sizes without a reduction in results accuracy.

  5. BLOTS AND ALL: A HISTORY OF THE RORSCHACH INK BLOT TEST IN BRITAIN.

    PubMed

    Hubbard, Katherine; Hegarty, Peter

    2016-01-01

    Despite the easily recognizable nature of the Rorschach ink blot test very little is known about the history of the test in Britain. We attend to the oft-ignored history of the Rorschach test in Britain and compare it to its history in the US. Prior to the Second World War, Rorschach testing in Britain had attracted advocates and critiques. Afterward, the British Rorschach Forum, a network with a high proportion of women, developed around the Tavistock Institute in London and The Rorschach Newsletter. In 1968, the International Rorschach Congress was held in London but soon after the group became less exclusive, and fell into decline. A comparative account of the Rorschach in Britain demonstrates how different national institutions invested in the 'projective hypothesis' according to the influence of psychoanalysis, the adoption of a nationalized health system, and the social positioning of 'others' throughout the twentieth century. In comparing and contrasting the history of the Rorschach in Britain and the US, we decentralize and particularize the history of North American Psychology.

  6. The design of a quantitative western blot experiment.

    PubMed

    Taylor, Sean C; Posch, Anton

    2014-01-01

    Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013) and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting.

  7. The Design of a Quantitative Western Blot Experiment

    PubMed Central

    Taylor, Sean C.; Posch, Anton

    2014-01-01

    Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013) and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting. PMID:24738055

  8. A defined methodology for reliable quantification of Western blot data.

    PubMed

    Taylor, Sean C; Berkelman, Thomas; Yadav, Geetha; Hammond, Matt

    2013-11-01

    Chemiluminescent western blotting has been in common practice for over three decades, but its use as a quantitative method for measuring the relative expression of the target proteins is still debatable. This is mainly due to the various steps, techniques, reagents, and detection methods that are used to obtain the associated data. In order to have confidence in densitometric data from western blots, researchers should be able to demonstrate statistically significant fold differences in protein expression. This entails a necessary evolution of the procedures, controls, and the analysis methods. We describe a methodology to obtain reliable quantitative data from chemiluminescent western blots using standardization procedures coupled with the updated reagents and detection methods.

  9. Interpretation criteria in Western blot diagnosis of Lyme borreliosis.

    PubMed

    Mavin, S; McDonagh, S; Evans, R; Milner, R M; Chatterton, J M W; Ho-Yen, D O

    2011-01-01

    This study reviews the Lyme borreliosis Western blot interpretation process, including what bands are classed as specific, the number of bands needed for a positive result, the role of band intensity and the use of clinical information. In 2008, 3688 patients (4223 serum samples) were tested by enzyme immunoassay (EIA), with 832 patients tested by confirmatory in-house IgG Western blot: 272 patients were Western blot-positive, 170 were weak positive, 156 were equivocal and 234 were negative. These results were assessed, and a review of interpretation criteria from both the USA and Europe was carried out. New interpretation criteria and a testing algorithm were developed. The revised criteria changed the results in 109/3688 (3%) patients and produced significantly more Western blot-positive and weak-positive patients than with the current criteria (485 vs. 442, P < 0.0001). In total, 76 patients who were negative/equivocal became positive, which may have led to a change in their management. Conversely, 33 patients who were weak-positive became equivocal but their management may not have been affected. The authors believe that the revised criteria have simplified blot interpretation and improved the sensitivity and robustness of their Western blot method. Using a protocol tailored to patients that incorporates clinical characteristics means that the entire process will be easier and will aid the management of patients.

  10. Skin blotting: a noninvasive technique for evaluating physiological skin status.

    PubMed

    Minematsu, Takeo; Horii, Motoko; Oe, Makoto; Sugama, Junko; Mugita, Yuko; Huang, Lijuan; Nakagami, Gojiro; Sanada, Hiromi

    2014-06-01

    The skin performs important structural and physiological functions, and skin assessment represents an important step in identifying skin problems. Although noninvasive techniques for assessing skin status exist, no such techniques for monitoring its physiological status are available. This study aimed to develop a novel skin-assessment technique known as skin blotting, based on the leakage of secreted proteins from inside the skin following overhydration in mice. The applicability of this technique was further investigated in a clinical setting. Skin blotting involves 2 steps: collecting proteins by attaching a damp nitrocellulose membrane to the surface of the skin, and immunostaining the collected proteins. The authors implanted fluorescein-conjugated dextran (F-DEX)-containing agarose gels into mice and detected the tissue distribution of F-DEX under different blotting conditions. They also analyzed the correlations between inflammatory cytokine secretion and leakage following ultraviolet irradiation in mice and in relation to body mass index in humans. The F-DEX in mice was distributed in the deeper and shallower layers of skin and leaked through the transfollicular and transepidermal routes, respectively. Ultraviolet irradiation induced tumor necrosis factor secretion in the epidermis in mice, which was detected by skin blotting, whereas follicular tumor necrosis factor was associated with body mass index in obese human subjects. These results support the applicability of skin blotting for skin assessment. Skin blotting represents a noninvasive technique for assessing skin physiology and has potential as a predictive and diagnostic tool for skin disorders.

  11. Mixed lubrication after rewetting of blotted pleural mesothelium.

    PubMed

    Bodega, Francesca; Sironi, Chiara; Porta, Cristina; Pecchiari, Matteo; Zocchi, Luciano; Agostoni, Emilio

    2013-01-15

    Coefficient of kinetic friction (μ) of pleural mesothelium blotted with filter paper, and rewetted with Ringer solution markedly increases; this increase is removed if a sufficient amount of sialomucin or hyaluronan is added to Ringer (Bodega et al., 2012. Respiratory Physiology and Neurobiology 180, 34-39). In this research we found that μ of pleural mesothelium blotted, rewetted, and sliding at physiological velocities and loads, decreased with increase of velocity, mainly at low velocities. Despite this decrease, μ at highest velocity was still double that before blotting. With small concentration of sialomucin or hyaluronan μ was markedly smaller at each velocity, decreased less with increase of velocity, and at highest velocity approached preblotting value. These findings indicate a regime of mixed lubrication in post-blotting Ringer, at variance with boundary lubrication occurring before blotting or postblotting with sufficient macromolecule addition. Greater roughness of mesothelial surface, caused by blotting, likely induces zones of elastohydrodynamic lubrication, which increase with velocity, while contact area decreases.

  12. The line blot assay: problems with titrating first and second antibodies for Western blot and immunohistochemistry assays?

    PubMed

    Rojas-Espinosa, O; Silva-Miranda, M; Wek-Rodriguez, K; Arce-Paredes, P

    2006-01-01

    We describe a technique designed to assess the optimal dilution of primary and secondary antibodies, to be used in Western blot, dot blot, the multi-antigen print immunoassay (MAPIA) and immunohistochemistry assays. The method that we call "line blot" is not an alternative but a practical, complementary tool for the above techniques that assures definitive results are obtained from single assays, so there is no need to repeat the assay. As with most immunoenzymatic assays, the line blot assay is very sensitive, allowing the detection of absolute amounts of antigen as low as 2.5 ng in the 0.5 cm-long segment line (see Results), depending on the strength of the secondary, enzyme-labelled antibody.

  13. Western Blot of Stained Proteins from Dried Polyacrylamide Gels

    NASA Technical Reports Server (NTRS)

    Gruber, Claudia; Stan-Lotter, Helga

    1996-01-01

    Western blotting of proteins is customarily performed following their separation on polyacrylamide gels, either prior to staining (1) or, as recently reported, following staining (2). We describe here Western blotting with stained gels, which had been dried and some of which had been stored for years. This procedure permits immunological analysis of proteins, to which antisera may have become available only later, or where the application of newly developed sensitive detection methods is desired. Once rehydration of the gels is achieved, proteins can be-transferred to blotting membranes by any appropriate protocol. Proteins stained with Coomassie Blue have to be detected with a non-chromogenic method, such as the film-based enhanced chemiluminescence (ECL)2) procedure (3). Silver stained proteins, which transfer in the colorless form, may be visualized by any detection method, although, because of the usually very low amounts of proteins, detection by ECL is preferable. Blotting of stained proteins from rehydrated gels is as rapid and as quantitative as from freshly prepared gels, in contrast to blotting from wet stained gels, which requires extensive washing and results in low transfer efficiency (2). Together with a photographic record of the gel pattern, unambiguous identification of immunoreactive proteins from complex mixtures is possible. Some further applications of this work are discussed.

  14. Direct detection of idiotypic determinants on blotted monoclonal antibodies.

    PubMed

    Petit, C; Sauron, M E; Gilbert, M; Thèze, J

    1982-01-01

    The protein-blotting technique has been tested as a mean to study the expression of idiotypic determinants. A monoclonal BALB/c antipoly (Glu60-Ala30-Tyr10) GAT antibody (G5) was caused to migrate on SDS gel and transferred to a nitrocellulose filter. To facilitate the renaturation of the idiotypic determinants, the blotted proteins were incubated in NP40 buffer, immediately after the transfer. The ability of two anti-idiotypic sera to detect two defined idiotypic specificities of the blotted G5 molecules was investigated. When G5 was electrophoresed on SDS gel under non-reducing conditions, a specific detection of two idiotypic specificities of the G5-blotted molecules was obtained. On the other hand, when G5 was migrated under reducing conditions, none of the two antiidiotypic sera gave a staining of the heavy and the light chains. This result indicates that molecules expressing conformational idiotypic determinants can be detected by protein-blotting technique after migration on SDS gel. Moreover, this suggests the possible interest of this technique to analyse non-antibody molecules bearing idiotypic determinants.

  15. Blame it on Southern, but it's a western blot.

    PubMed

    Klionsky, Daniel J

    2017-01-02

    Edwin M. Southern is a professor emeritus at the University of Oxford. He is perhaps best known for development of the "Southern blot" (Dr. Southern was at the University of Edinburgh when he wrote his landmark paper). The Southern blot provided a scientific breakthrough by allowing scientists to detect a particular DNA sequence without first purifying it from the rest of the genome; the basic method involves the transfer of the DNA to a membrane, followed by detection with a specific probe. Although few people perform Southern blots as originally carried out by Southern, due in part to the more recent technique of the polymerase chain reaction, the basic concept continues to play an important role in molecular biology.

  16. Bioconjugation of quantum dot luminescent probes for Western blot analysis.

    PubMed

    Makrides, Savvas C; Gasbarro, Christina; Bello, Job M

    2005-10-01

    Western blot analysis is a widely used technique for protein immunodetection. Its current format, however is unsuitable for multiplex detection of proteins, primarily due to intrinsic limitations of standard organic dyes employed as probes. Quantum dot (QD) semiconductor nanoparticles exhibit significant advantages over organic dyes, including their broad absorption bands, narrow, tunable, and symmetric emission spectra, large Stokes shifts, and excellent photostability. Here we describe a novel method for the functionalization of streptavidin-coated QDs with an in vivo biotinylated peptide (head-to-tail dimerized Z domain derived from protein A) that permits the facile conjugation of antibodies to QDs. In this study, we demonstrate the simultaneous detection of two different types of protein in a Western blot. The bioconjugation of QDs described here makes it possible to achieve multiplex detection of proteins in Western blot analysis in a more straightforward manner.

  17. Clinical, immunohistochemical, Western blot, and genetic analysis in dystrophinopathy.

    PubMed

    Na, Sang-Jun; Kim, Won-Joo; Kim, Seung Min; Lee, Kee Ook; Yoon, Bora; Choi, Young-Chul

    2013-08-01

    Dystrophin-deficient muscular dystrophies (dystrophinopathies) are the most common form of muscular dystrophy, with variable clinical phenotypes ranging from the severe Duchenne (DMD) to the milder Becker (BMD) forms. In this study, we investigated the relationship between clinical characteristics, findings at immunohistochemistry (IHC) and Western blot, and the pattern of exon deletions in 24 male patients with dystrophinopathies. We retrospectively reviewed findings from clinical and laboratory examinations, IHC for dystrophin of muscle biopsy tissue, Western blot analysis, and multiplex polymerase chain reaction (PCR) examination of genomic DNA. All tests were performed in every patient. PCR examination revealed exon deletions in 13 patients (54.2%). At Western blot analysis, 15 patients (62.5%) were negative at all three dystrophin domains. Most of these patients had a clinical presentation consistent with the DMD phenotype. Nine (37.5%) others were weakly positive at one or more domains. Most of these patients presented clinically as BMD phenotype. One patient whose clinical presentation was consistent with BMD phenotype had normal findings at IHC and was weakly positive at all three domains on Western blot analysis; however, with the exception of this patient, the findings at IHC and Western blot were consistent for individual patients. Based on these findings, we conclude that Western blot analysis appears useful for confirmation of dystrophinopathy in BMD patients with normal staining on IHC. Exon deletion analysis by multiplex PCR using peripheral blood is also a simple and useful test for the diagnosis of dystrophinopathy, although it has limited sensitivity. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Application of Intermittent Microwave Irradiation to Western Blot Analysis.

    PubMed

    Liu, Yu-Ting; Toyokuni, Shinya

    2015-01-01

    We established a shortened protocol for Western blot analysis using intermittent microwave irradiation. With this method, the procedure is completed within 1 h after applying the primary antibody, and thus greatly saves time. This procedure appears to be applicable to any antibody based on our experience of several years.

  19. Coomassie staining as loading control in Western blot analysis.

    PubMed

    Welinder, Charlotte; Ekblad, Lars

    2011-03-04

    In Western blotting, immunodetection of housekeeping proteins is routinely performed to detect differences in electrophoresis loading. The present work describes a much faster and simpler protein staining method, which is compatible with ordinary blocking conditions. In addition, the method can be used after immunodetection with superior linearity compared to ordinary staining methods. After immunoblotting and staining, protein bands can be further identified using peptide mass fingerprinting.

  20. Development of new staining technology "eastern blotting" using monoclonal antibody.

    PubMed

    Morinaga, Osamu; Shoyama, Yukihiro

    2011-03-01

    Ginsenosides contained in Panax species were separated by silica gel TLC blotted to a polyvinylidene difluoride (PVDF) membrane which was dipped in a sodium periodide (NaIO(4)) solution and reacted with protein, preparing a ginsenoside-protein conjugate for binding a ginsenoside on a PVDF membrane. The blotted spots were stained by anti-ginsenoside-Rb1 monoclonal antibody (MAb) and anti-ginsenoside-Rg1MAb, respectively. The newly established immunostaining method, eastern blotting was applied for the determination of ginsenosides possessing protopanaxadiol and/or protopanaxatriol. Double staining of eastern blotting for ginsenosides using anti-ginsenoside-Rb1 MAb and anti-ginsenoside-Rg1 MAb promoted complete identification of ginsenosides in Panax species. This technique has been devised for the chromatographic separation and identification of ginsenosides using polyethersulfone (PES) membrane. It caused an acceptable separation of ginsenoside-Rb1, -Rc and -Rd in various ginseng extracts. Newly developed technique is quite simple and applies for immunoassay system. Ginsenosides separated using a PES membrane were directly treated with a NaIO(4) solution and then reacted with bovine serum albumin (BSA) for making a ginsenoside-protein conjugate. After the blocking, anti-ginsenoside-Rb1 MAb recognized a ginsenoside on a PES membrane and then a sec-ond antibody labeled with enzyme reacted to the first antibody. Finally a substrate was oxidized with the enzyme and de-veloped the staining of ginsenosides. The staining spots of ginsenosides on membrane were quantitatively evaluated by NIH Image indicating at least 62.5 ng of each ginsenoside-Rb1, -Rc and -Rd were detected with clarity. The determination range of three ginsenosides was from 0.125 to 2.0 µg of direct amount on PES membrane.

  1. Validation of western blot for Histoplasma capsulatum antibody detection assay.

    PubMed

    Almeida, Marcos de Abreu; Pizzini, Cláudia Vera; Damasceno, Lisandra Serra; Muniz, Mauro de Medeiros; Almeida-Paes, Rodrigo; Peralta, Regina Helena Saramago; Peralta, José Mauro; Oliveira, Raquel de Vasconcelos Carvalhaes; Vizzoni, Alexandre Gomes; de Andrade, Carla Lourenço Tavares; Zancopé-Oliveira, Rosely Maria

    2016-02-24

    Histoplasmosis is worldwide systemic mycoses caused by the dimorphic fungus Histoplasma capsulatum. The isolation and identification of H. capsulatum in culture is the reference test for histoplasmosis diagnosis confirmation. However, in the absence of it, serology has been used as a presumptive diagnosis through antibody and antigen detection. The purpose of the present study was to validate an immunoassay method (western blot) for antibodies detection in the diagnosis of histoplasmosis. To validate the western blot (WB) a study was conducted using 118 serum samples from patients with histoplasmosis and 118 serum controls collected from January 2000 to December 2013 in residents of the Rio de Janeiro State, Brazil. Diagnostic validation parameters were calculated based on the categorization of results obtained in a 2 × 2 table and subjected to statistical analysis. In addition, the viability of deglycosylated histoplasmin antigen (ptHMIN) onto nitrocellulose membranes previously sensitized was evaluated during the same period. The WB test showed sensitivity of 94.9 %, specificity of 94.1 %, positive predictive value of 94.1 %, negative predictive value of 94.9 %, accuracy of 94.5 %, and almost perfect precision. Besides, the strips have proved to be viable for using at least 5 years after ptHMIN antigen sensitization. Western blot test using ptHMIN provides sensitive, specific, and faster results. Therefore, could be considered a useful tool in the diagnosis of histoplasmosis being used by public health system, even in situations where laboratory facilities are relatively limited.

  2. Lipid A binding proteins in macrophages detected by ligand blotting

    SciTech Connect

    Hampton, R.Y.; Golenbock, D.T.; Raetz, C.R.H.

    1987-05-01

    Endotoxin (LPS) stimulates a variety of eukaryotic cells. These actions are involved in the pathogenesis of Gram-negative septicemia. The site of action of the LPS toxic moiety, lipid A (LA), is unclear. Their laboratory has previously identified a bioactive LA precursor lipid IV/sub A/, which can be enzymatically labeled with /sup 32/P/sub i/ (10/sup 9/ dpm/nmole) and purified (99%). They now show that this ligand binds to specific proteins immobilized on nitrocellulose (NC) from LPS-sensitive RAW 264.7 cultured macrophages. NC blots were incubated with (/sup 32/P)-IV/sub A/ in a buffer containing BSA, NaCl, polyethylene glycol, and azide. Binding was assessed using autoradiography or scintillation counting. Dot blot binding of the radioligand was inhibited by excess cold IV/sub A/, LA, or ReLPS but not by phosphatidylcholine, cardiolipin, phosphatidylinositol, or phosphatidic acid. Binding was trypsin-sensitive and dependent on protein concentration. Particulate macrophage proteins were subjected to SDS-PAGE and then electroblotted onto NC. Several discrete binding proteins were observed. Identical treatment of fetal bovine serum or molecular weight standards revealed no detectable binding. By avoiding high nonspecific binding of intact membranes, this ligand blotting assay may be useful in elucidating the molecular actions of LPS.

  3. Failure analysis of blots for diesel engine intercooler

    NASA Astrophysics Data System (ADS)

    Ren, Ping; Li, Zongquan; Wu, Jiangfei; Guo, Yibin; Li, Wanyou

    2017-05-01

    In diesel generating sets, it will lead to the abominable working condition if the fault couldn’t be recovered when the bolt of intercooler cracks. This paper aims at the fault of the blots of diesel generator intercooler and completes the analysis of the static strength and fatigue strength. Static intensity is checked considering blot preload and thermal stress. In order to obtain the thermal stress of the blot, thermodynamic of intercooler is calculated according to the measured temperature. Based on the measured vibration response and the finite element model, using dynamic load identification technique, equivalent excitation force of unit was solved. In order to obtain the force of bolt, the excitation force is loaded into the finite element model. By considering the thermal stress and preload as the average stress while the mechanical stress as the wave stress, fatigue strength analysis has been accomplished. Procedure of diagnosis is proposed in this paper. Finally, according to the result of intensity verification the fatigue failure is validation. Thereby, further studies are necessary to verification the result of the intensity analysis and put forward some improvement suggestion.

  4. Mechanism of DNA (Southern) and protein (Western) blotting on cellulose nitrate and other membranes.

    PubMed

    Van Oss, C J; Good, R J; Chaudhury, M K

    1987-03-27

    The transfer of DNA fractions from hydrophilic gels to nitrocellulose membranes (Southern blotting) which was soon followed by the description of an analogous procedure for RNA (Northern blotting), and somewhat later for proteins (Western blotting), has rapidly become an important separation and characterization method in molecular biology, genetic engineering, and immunological detection. Surface tension measurements have shown that the interfacial attraction between DNA and cellulose esters (-delta G132) in aqueous media can be considerable. The weaker binding energy of proteins to cellulose nitrate and to cellulose acetate may be compared to hydrophobic interaction chromatography, as on account of the somewhat lower [-delta G132] values, it often is necessary to "fix" them more tightly onto nitrocellulose by using high salt concentrations. The binding energy of RNA to both cellulose esters also is rather low. In addition to the effect of high ionic strength, the effect of adding methanol, and the effects of denaturation, heating and drying on the energy of attachment of the biopolymers to cellulose esters, have been studied. Cationized nylon membranes have been advocated recently, especially for electrophoretic transfer of nucleic acids (in which process high salt concentrations cannot easily be used). With positively charged nylon membranes, the attachment mainly occurs through the electrostatic attraction between the strongly negatively charged nucleic acids (or proteins) and the positively charged membrane. Also, more apolar membranes (of polyvinyl difluoride) have been proposed, which manifest a strong interfacial (hydrophobic) attraction to all the above biopolymers (regardless of their electrostatic charge). However, with these two novel membrane types it is no longer possible to exploit the large difference in binding energy between DNA and RNA, which makes cellulose nitrate membranes so uniquely suited for RNA-DNA hybridization assays.

  5. Evaluating strategies to normalise biological replicates of Western blot data.

    PubMed

    Degasperi, Andrea; Birtwistle, Marc R; Volinsky, Natalia; Rauch, Jens; Kolch, Walter; Kholodenko, Boris N

    2014-01-01

    Western blot data are widely used in quantitative applications such as statistical testing and mathematical modelling. To ensure accurate quantitation and comparability between experiments, Western blot replicates must be normalised, but it is unclear how the available methods affect statistical properties of the data. Here we evaluate three commonly used normalisation strategies: (i) by fixed normalisation point or control; (ii) by sum of all data points in a replicate; and (iii) by optimal alignment of the replicates. We consider how these different strategies affect the coefficient of variation (CV) and the results of hypothesis testing with the normalised data. Normalisation by fixed point tends to increase the mean CV of normalised data in a manner that naturally depends on the choice of the normalisation point. Thus, in the context of hypothesis testing, normalisation by fixed point reduces false positives and increases false negatives. Analysis of published experimental data shows that choosing normalisation points with low quantified intensities results in a high normalised data CV and should thus be avoided. Normalisation by sum or by optimal alignment redistributes the raw data uncertainty in a mean-dependent manner, reducing the CV of high intensity points and increasing the CV of low intensity points. This causes the effect of normalisations by sum or optimal alignment on hypothesis testing to depend on the mean of the data tested; for high intensity points, false positives are increased and false negatives are decreased, while for low intensity points, false positives are decreased and false negatives are increased. These results will aid users of Western blotting to choose a suitable normalisation strategy and also understand the implications of this normalisation for subsequent hypothesis testing.

  6. Evaluating Strategies to Normalise Biological Replicates of Western Blot Data

    PubMed Central

    Degasperi, Andrea; Birtwistle, Marc R.; Volinsky, Natalia; Rauch, Jens; Kolch, Walter; Kholodenko, Boris N.

    2014-01-01

    Western blot data are widely used in quantitative applications such as statistical testing and mathematical modelling. To ensure accurate quantitation and comparability between experiments, Western blot replicates must be normalised, but it is unclear how the available methods affect statistical properties of the data. Here we evaluate three commonly used normalisation strategies: (i) by fixed normalisation point or control; (ii) by sum of all data points in a replicate; and (iii) by optimal alignment of the replicates. We consider how these different strategies affect the coefficient of variation (CV) and the results of hypothesis testing with the normalised data. Normalisation by fixed point tends to increase the mean CV of normalised data in a manner that naturally depends on the choice of the normalisation point. Thus, in the context of hypothesis testing, normalisation by fixed point reduces false positives and increases false negatives. Analysis of published experimental data shows that choosing normalisation points with low quantified intensities results in a high normalised data CV and should thus be avoided. Normalisation by sum or by optimal alignment redistributes the raw data uncertainty in a mean-dependent manner, reducing the CV of high intensity points and increasing the CV of low intensity points. This causes the effect of normalisations by sum or optimal alignment on hypothesis testing to depend on the mean of the data tested; for high intensity points, false positives are increased and false negatives are decreased, while for low intensity points, false positives are decreased and false negatives are increased. These results will aid users of Western blotting to choose a suitable normalisation strategy and also understand the implications of this normalisation for subsequent hypothesis testing. PMID:24475266

  7. Protein detection by Western blot via coiled-coil interactions.

    PubMed

    Boucher, Cyril; St-Laurent, Gilles; Jolicoeur, Mario; Crescenzo, Gregory De; Durocher, Yves

    2010-04-01

    We propose an approach for the detection of proteins by Western blot that takes advantage of the high-affinity interaction occurring between two de novo designed peptides, the E and K coils. As a model system, K coil-tagged epidermal growth factor (EGF) was revealed with secreted alkaline phosphatase (SeAP) tagged with E coil (SeAP-Ecoil) as well as with biotinylated E coil. In that respect, we first produced purified SeAP-Ecoil and verified its ability to interact with K coil peptides by surface plasmon resonance biosensing. We demonstrated that protein detection with Ecoil-biotin was more specific than with SeAP-Ecoil. We then showed that our approach is as sensitive as conventional detection strategies relying on nickel-nitrilotriacetic acid-horseradish peroxidase (Ni-NTA-HRP), anti-His-HRP, or anti-EGF. Altogether, our results indicate that the E/K coiled-coil system is a good alternative for protein detection by Western blot. Crown Copyright 2009. Published by Elsevier Inc. All rights reserved.

  8. Microfluidic Western Blotting of Low-Molecular-Mass Proteins

    PubMed Central

    2015-01-01

    We describe a microfluidic Western blot assay (μWestern) using a Tris tricine discontinuous buffer system suitable for analyses of a wide molecular mass range (6.5–116 kDa). The Tris tricine μWestern is completed in an enclosed, straight glass microfluidic channel housing a photopatterned polyacrylamide gel that incorporates a photoactive benzophenone methacrylamide monomer. Upon brief ultraviolet (UV) light exposure, the hydrogel toggles from molecular sieving for size-based separation to a covalent immobilization scaffold for in situ antibody probing. Electrophoresis controls all assay stages, affording purely electronic operation with no pumps or valves needed for fluid control. Electrophoretic introduction of antibody into and along the molecular sieving gel requires that the probe must traverse through (i) a discontinuous gel interface central to the transient isotachophoresis used to achieve high-performance separations and (ii) the full axial length of the separation gel. In-channel antibody probing of small molecular mass species is especially challenging, since the gel must effectively sieve small proteins while permitting effective probing with large-molecular-mass antibodies. To create a well-controlled gel interface, we introduce a fabrication method that relies on a hydrostatic pressure mismatch between the buffer and polymer precursor solution to eliminate the interfacial pore-size control issues that arise when a polymerizing polymer abuts a nonpolymerizing polymer solution. Combined with a new swept antibody probe plug delivery scheme, the Tris tricine μWestern blot enables 40% higher separation resolution as compared to a Tris glycine system, destacking of proteins down to 6.5 kDa, and a 100-fold better signal-to-noise ratio (SNR) for small pore gels, expanding the range of applicable biological targets. PMID:25268977

  9. Antibody performance in western blot applications is context-dependent.

    PubMed

    Algenäs, Cajsa; Agaton, Charlotta; Fagerberg, Linn; Asplund, Anna; Björling, Lisa; Björling, Erik; Kampf, Caroline; Lundberg, Emma; Nilsson, Peter; Persson, Anja; Wester, Kenneth; Pontén, Fredrik; Wernérus, Henrik; Uhlén, Mathias; Ottosson Takanen, Jenny; Hober, Sophia

    2014-03-01

    An important concern for the use of antibodies in various applications, such as western blot (WB) or immunohistochemistry (IHC), is specificity. This calls for systematic validations using well-designed conditions. Here, we have analyzed 13 000 antibodies using western blot with lysates from human cell lines, tissues, and plasma. Standardized stratification showed that 45% of the antibodies yielded supportive staining, and the rest either no staining (12%) or protein bands of wrong size (43%). A comparative study of WB and IHC showed that the performance of antibodies is application-specific, although a correlation between no WB staining and weak IHC staining could be seen. To investigate the influence of protein abundance on the apparent specificity of the antibody, new WB analyses were performed for 1369 genes that gave unsupportive WBs in the initial screening using cell lysates with overexpressed full-length proteins. Then, more than 82% of the antibodies yielded a specific band corresponding to the full-length protein. Hence, the vast majority of the antibodies (90%) used in this study specifically recognize the target protein when present at sufficiently high levels. This demonstrates the context- and application-dependence of antibody validation and emphasizes that caution is needed when annotating binding reagents as specific or cross-reactive. WB is one of the most commonly used methods for validation of antibodies. Our data implicate that solely using one platform for antibody validation might give misleading information and therefore at least one additional method should be used to verify the achieved data. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Serological Diagnosis of Paracoccidioidomycosis through a Western Blot Technique

    PubMed Central

    Perenha-Viana, M. C. Z.; Gonzales, I. A. A.; Brockelt, S. R.; Machado, L. N. C.

    2012-01-01

    Paracoccidioidomycosis (PCM) is a serious infectious disease that progresses toward death if untreated. Its confirmatory diagnosis is made by the detection of the fungus Paracoccidioides brasiliensis in a direct mycological examination or by histopathology. However, these techniques are of low sensitivity. Serological tests seem to be more promising. The objective of this study was to test Western blot (WB) analysis using sera from patients suspected of PCM to determine whether it represents a safe and sensitive serological technique for a rapid and effective diagnosis for this disease. Sera from 517 patients were analyzed through WB analysis and double-immunodiffusion (DID) techniques using a crude exoantigen of P. brasiliensis 339. DID gave positive reactions for 140 sera (27%) and WB for 250 sera (48.4%). All sera that had a positive reaction by DID also had a positive result with a 43-kDa glycoprotein by WB analysis. Among the 377 samples that were negative by DID, 29.1% were reactive in WB analysis. For the cutoff dilution used (1:400), a positive reaction was not observed with any of the 102 sera from patients with other diseases in regions where such diseases are endemic and 30 healthy individuals tested as negative controls. These results prove WB analysis to be a sensitive technique and suggest its inclusion among routine laboratory assays as a safe method for PCM diagnosis. PMID:22301695

  11. Cy5 total protein normalization in Western blot analysis.

    PubMed

    Hagner-McWhirter, Åsa; Laurin, Ylva; Larsson, Anita; Bjerneld, Erik J; Rönn, Ola

    2015-10-01

    Western blotting is a widely used method for analyzing specific target proteins in complex protein samples. Housekeeping proteins are often used for normalization to correct for uneven sample loads, but these require careful validation since expression levels may vary with cell type and treatment. We present a new, more reliable method for normalization using Cy5-prelabeled total protein as a loading control. We used a prelabeling protocol based on Cy5 N-hydroxysuccinimide ester labeling that produces a linear signal response. We obtained a low coefficient of variation (CV) of 7% between the ratio of extracellular signal-regulated kinase (ERK1/2) target to Cy5 total protein control signals over the whole loading range from 2.5 to 20.0μg of Chinese hamster ovary cell lysate protein. Corresponding experiments using actin or tubulin as controls for normalization resulted in CVs of 13 and 18%, respectively. Glyceraldehyde-3-phosphate dehydrogenase did not produce a proportional signal and was not suitable for normalization in these cells. A comparison of ERK1/2 signals from labeled and unlabeled samples showed that Cy5 prelabeling did not affect antibody binding. By using total protein normalization we analyzed PP2A and Smad2/3 levels with high confidence. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Comparisons of double immunodiffusion, ELISA, western blot and CAPE blot for the detection of anti-SSA antibody: study of anti-SSA prevalence in systemic lupus erythematosus.

    PubMed

    Chretien, P; Soulie, E; Johanet, C; Abuaf, N

    1994-06-01

    The specificity and sensitivity of anti-SSA (Ro) antibody detection by double immunodiffusion, ELISA, western blot and a new method named CAPE blot were studied. Using 79 normal sera, 61 sera positive for anti-SSA (Ro) antibodies (double immunodiffusion), and 39 sera without anti-SSA (Ro) antibodies but containing anti-Sm, anti-RNP, anti-SSB (La), anti-Scl 70 and anti-PCNA antibodies, we compared ELISA, western blot and CAPE blot with the immunodiffusion assay. The sensitivities of the three methods were 100, 62 and 100%, respectively. Sera from 196 patients with systemic lupus erythematosus (SLE) were tested. The prevalence of anti-SSA (Ro) antibodies in this group was scored as 26% by double immunodiffusion, 47% by ELISA, 25% by western blot and 38% by CAPE blot.

  13. Cell adhesion to proteins separated by lithium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto a polyvinylidene difluoride membrane: a new cell-blotting technique.

    PubMed

    Seshi, B

    1994-12-02

    Cell blotting, although conceptually simple, has failed to achieve wide practical application. Described here is a new cell-blotting technique which involves cell adhesion to protein bands after separation by lithium dodecyl sulfate-polyacrylamide gel electrophoresis (LDS-PAGE) and blotting onto polyvinylidene difluoride (PVDF) membrane at 4 degrees C. Cell bands adherent on PVDF are detected using hematoxylin, or propidium iodide (PI) staining followed by viewing under ultraviolet (UV) light. The technique allows quick microscopic visualization of adherent cells composing the bands, without requiring clearing of the membrane. Representative cell adhesion proteins from different sources, i.e., plant lectins (e.g., phytohemagglutinin, PHA; concanavalin A, ConA; and wheat germ agglutinin, WGA); extracellular matrix (ECM) proteins; and integral membrane proteins (e.g., recombinant soluble vascular cell adhesion molecule-1, rs VCAM-1) were tested for cell binding by the new cell-blotting technique using human lymphoid progenitor (NALM-6) and myeloid progenitor (KG1a) cell lines. Cell adhesion proteins retained their adhesion function in all cases tested. Specificity of cell binding on PVDF blot was demonstrated by inhibition of cell adhesion to WGA protein bands using an appropriate sugar, i.e., N-acetyl D-glucosamine. The cell blotting assay was comparable in sensitivity to Coomassie blue staining of protein bands. The ability to conduct protein extraction, separation and blotting at low temperature avoids thermal denaturation, thereby preserving the adhesion properties of the proteins. The electrophoretic/blotting system has unique detergent removal/protein renaturation properties and the ability to preserve functionally active adhesion protein complexes. The cell-blotting technique described is sufficiently robust for routine application in the investigation of novel cell adhesion proteins.

  14. Reversible Ponceau staining as a loading control alternative to actin in Western blots.

    PubMed

    Romero-Calvo, Isabel; Ocón, Borja; Martínez-Moya, Patricia; Suárez, María Dolores; Zarzuelo, Antonio; Martínez-Augustin, Olga; de Medina, Fermín Sánchez

    2010-06-15

    It is becoming standard practice to measure a housekeeping gene, typically actin, in Western blots, as it is the rule in RNA blots. We have applied reversible Ponceau staining to check equal loading of gels and measured actin in parallel under different conditions. Our results show that densitometric analysis is comparable with both techniques. Therefore, routine quantitation of Ponceau staining before antibody probing is validated as an alternative to actin blotting.

  15. A comparison of the Genie and western blot assays in confirmatory testing for HIV-1 antibody.

    PubMed

    Chan, E L; Sidaway, F; Horsman, G B

    1996-03-01

    The Genie HIV-1/2 kit (Sanofi Diagnostics Pasteur, Montreal, Quebec), a synthetic-peptide solid-phase enzyme immunoassay, was evaluated as a confirmatory assay for HIV-1 antibodies in comparison with Western blot (BioRad, Hercules, CA, USA) on 50 stored HIV-1 antibody-positive sera and the 137 sera yielding repeated positive results in the conventional EIA screen out of 13405 fresh patient sera from Saskatchewan in 1993. The stored HIV-1-positive sera were uniformly positive in the Genie test. Of the 137 EIA screen-positive sera, 33 were uniformly positive and 64 were uniformly negative in Genie and Western blot; 36 were Genie-negative and indeterminate by Western blot; and four were Genie indeterminate, of which one was negative and three were indeterminate by Western blot. All HIV-1 Western blot-indeterminate and Genie-interdeterminate sera were negative in radio-immunoprecipitation assay (RIPA) and Western blot for HIV-1 and HIV-2 antibodies performed by a reference laboratory. Genie gave an accurate definitive result for 97% of EIA positive sera compared with 71% for Western blot. There was excellent correlation between Genie, Western blot and RIPA results. However, the Genie assay was faster, less costly and yielded fewer indeterminate results than Western blot in confirmatory testing for HIV-1 antibodies.

  16. Parafilm-M®, An Available Cost-Effective Alternative for Immuno-blot Pouches.

    PubMed

    Quadri, Syed M S

    2015-01-01

    Commercially available standard immuno-blot pouches do play an efficient role in antibody incubation in performing an immuno-blot, but are not readily available in the laboratory and have to be specifically ordered. We have developed an equally efficient technique to make an immune-blot more cost-effective with more conservation of antibodies by using a common and readily available laboratory product Parafilm-M(®). Parafilm-M(®) which serves as a sealant for various items of laboratory equipment can be used for antibody incubation. Manually made Parafilm-M(®) pouch has a clear advantage over standard immuno-blot pouches in terms of availability, cost-effectiveness, and consumption of antibodies that ultimately reduces the cost of an immuno-blot. We have performed a series of experiments to check the efficacy of both the techniques. Samples with equal amount of protein were analyzed on separate SDS PAGE gels. The proteins were transferred electrophoretically to the nitrocellulose membrane using Trans-Blot(®) Turbo™ Mini Nitrocellulose Transfer Pack. Antibody incubation was done using standard immuno-blot pouch, standard container and Parafilm-M(®) sealed pouch. The expression of protein was determined and the results of immuno-blots were compared. We found that antibodies are binding the membrane in Parafilm-M(®) pouches as efficiently as in container method or in standard immuno-blot pouches. By restricting the membrane, the surface area of the manually made Parafilm-M(®) pouch can be reduced, less diluent is required to cover the membrane as a result less antibodies are consumed. We also calculated that each immuno-blot pouch cost around $0.1906, whereas the cost for Parafilm-M(®) pouch is 0.0695 which is almost one-third the price of an immuno-blot pouch. Thus, Parafilm-M(®) method distinctly provides a cost-effective solution for antibody incubation.

  17. A Laboratory Exercise Illustrating the Sensitivity and Specificity of Western Blot Analysis

    ERIC Educational Resources Information Center

    Chang, Ming-Mei; Lovett, Janice

    2011-01-01

    Western blot analysis, commonly known as "Western blotting," is a standard tool in every laboratory where proteins are analyzed. It involves the separation of polypeptides in polyacrylamide gels followed by the electrophoretic transfer of the separated polypeptides onto a nitrocellulose or polyvinylidene fluoride membrane. A replica of the…

  18. Microfluidic integration of Western blotting is enabled by electrotransfer-assisted sodium dodecyl sulfate dilution.

    PubMed

    Hou, Chenlu; Herr, Amy E

    2013-01-07

    We integrate sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with subsequent antibody probing in a single, monolithic microdevice to realize microfluidic Western blotting. A hurdle to successful on-chip Western blotting lies in restoring antibody recognition of previously sized (denatured, reduced) proteins. To surmount this hurdle, we locally dilute free SDS from SDS-protein complexes using differential electromigration of the species during electrotransfer between SDS-PAGE and blotting regions of a microchamber. Local dilution of SDS minimizes re-association of SDS with proteins offering means to restore antibody binding affinity to proteins after SDS-PAGE. To achieve automated, programmable operation in a single instrument, we utilize a 1 × 2 mm(2) glass microchamber photopatterned with spatially distinct, contiguous polyacrylamide regions for SDS-PAGE, electrotransfer, and antibody blotting. Optimization of both the SDS-PAGE and electrotransfer conditions yields transfer distances of <1 mm (40 s). The Western blot is completed in 180 s, with fully automated assay operation using programmable voltage control. After SDS-PAGE and electrotransfer, we observe ~80% capture of protein band mass on the blotting region for a model protein, C-reactive protein. This novel microfluidic Western blot approach introduces fine transport control for in-transit protein handling to form the basis for an automated, rapid alternative to conventional slab-gel Western blotting.

  19. Luminex xMAP combined with Western blot improves HIV diagnostic sensitivity.

    PubMed

    Kong, Weiwei; Li, Yan; Cheng, Shaohui; Yan, Chen; An, Shiping; Dong, Zheng; Yan, Lina; Yuan, Yuhua

    2016-01-01

    Currently, Western blot is used to confirm the initial serodiagnosis of HIV infection by antibody detection. However, a major deficiency of the Western blot relates to a lack of sufficient sensitivity in detecting HIV antibodies. This report describes a simple, sensitive and inexpensive bead-based assay for detection of early HIV infection. A panel of 138 positive specimens including 105 blood donors and 33 MSM with known Western blot results were evaluated using Luminex xMAP at Tianjin Centers for Disease Control and Prevention (CDC). We demonstrate a superior sensitivity of Luminex xMAP compared with Western blot. Of the 87 confirmed HIV positive blood donors, Western blot only confirmed 65 cases with 74.7% (65/87) sensitivity while Luminex xMAP identified 72 cases with 82.8% (72/87) sensitivity (p<0.05). Western blot and Luminex xMAP verified 13 and 19 of 33 MSM specimens, respectively. The sensitivity was 39.4% (13/33) for Western blot and 57.6% (19/33) for Luminex xMAP (p<0.1). Luminex xMAP combined with Western blot improves the diagnostic sensitivity of HIV infection at an early stage, and reduces the chances of missed diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Identification of immunodiagnostic antigens for cerebrospinal filariasis in horses by western blot analysis.

    PubMed

    Takesue, Masataka; Osaka, Yuki; Muranaka, Masanori; Katayama, Yoshinari; Ikadai, Hiromi

    2016-01-01

    In the present study, the serum and cerebrospinal fluid of horses diagnosed with Setaria digitata cerebrospinal filariasis were analyzed by western blot. The results revealed S. digitata protein bands measuring 65, 34, 22, and 18 kDa in molecular weight. In particular, the 18 kDa band is a possible candidate for clinical immunodiagnosis on the basis of western blot findings.

  1. A Laboratory Exercise Illustrating the Sensitivity and Specificity of Western Blot Analysis

    ERIC Educational Resources Information Center

    Chang, Ming-Mei; Lovett, Janice

    2011-01-01

    Western blot analysis, commonly known as "Western blotting," is a standard tool in every laboratory where proteins are analyzed. It involves the separation of polypeptides in polyacrylamide gels followed by the electrophoretic transfer of the separated polypeptides onto a nitrocellulose or polyvinylidene fluoride membrane. A replica of the…

  2. A protocol for stripping and reprobing of Western blots originally developed with colorimetric substrate TMB.

    PubMed

    Kar, Parmita; Agnihotri, Saurabh Kumar; Sharma, Archana; Sachan, Rekha; Lal Bhatt, Madan; Sachdev, Monika

    2012-10-01

    Western blotting is a widely used analytical technique for detection of specific protein(s) in a given sample of tissue/cell homogenate or extract. Both chemiluminescence (CL) and colorimetric detections can be used for imaging Western blots. Colorimetric substrates offer background free, sensitive, and clean imaging results directly on the blotted membrane and provides more accurate profile with respect to prestained marker. However, blots stained with colorimetric substrates cannot be reused since no stripping protocols have been reported for such blots, thus limiting their reuse for detection of another protein. In the present study, for the first time, we report a novel method of stripping Western blots developed with the colorimetric substrate TMB for detection of a low-abundant protein and reprobing of these blots after stripping for detection of a more abundant protein through CL procedure. The stripping procedure utilizes a stripping buffer consisting of β-mercaptoethanol, SDS, and Tris-HCl and a washing buffer consisting of PBS added with 0.1% Tween-20 involves a series of steps and facilitates accurate detection of the second protein (i.e., more abundant protein) in the stripped blot through CL. The protocol is reproducible and facilitates saving of precious clinical samples, in addition to saving cost and time as compared to the existing procedures. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Detection of antibodies to Hypoderma lineatum in cattle by Western blotting with recombinant hypodermin C antigen.

    PubMed

    Boldbaatar, D; Xuan, X; Kimbita, E; Huang, X; Igarashi, I; Byambaa, B; Battsetseg, B; Battur, B; Battsetseg, G; Batsukh, Z; Nagasawa, H; Fujisaki, K; Mikami, T

    2001-08-01

    The cDNA encoding the entire mature hypodermin C (HC) of Hypoderma lineatum was cloned and expressed in Escherichia coli as a glutathione S-transferase fusion protein using pGEX vector. The recombinant HC protein (rHC) was tested by Western blotting to detect antibodies to H. lineatum in cattle. Western blotting with rHC as antigen clearly differentiated between H. lineatum-infested cattle sera and normal cattle sera. Forty-six out of forty-eight serum samples from cattle in Central Mongolia were positive, whereas all 30 serum samples from cows in Hokkaido, Japan, were negative by Western blotting. The result of Western blotting was identical to that of a previously developed enzyme-linked immunosorbent assay. These data demonstrated that Western blotting, with rHC expressed in E. coli, might be a useful method for the diagnosis of cattle hypodermosis.

  4. Charge standards as reference points in two-dimensional western blot patterns

    SciTech Connect

    Zhang, J.S.; Tollaksen, S.L.; Giometti, C.S.

    1986-01-01

    High-resolution two-dimensional electrophoresis can be used to separate hundreds or even thousands of proteins from complex mixtures. Specific proteins in 2DE patterns can be identified by using Western Blotting. Correlation between a single stained spot on a nitrocellulose blot and the same spot in a stained 2DE gel pattern can be difficult since no reference points are available on the blot. Inclusion of a protein or proteins to serve as reference or standard points in both the blotted pattern and the stained gel pattern would make pattern recognition much more rapid and reliable. In this paper the use of a commercial charge standard (carbamylated creatine phosphokinase mix) was used to produce reference points in both gel patterns and corresponding nitrocellulose blots to simplify recognition of individual protein spots in complicated gel patterns. 8 refs., 2 figs.

  5. The fastest Western in town: a contemporary twist on the classic Western blot analysis.

    PubMed

    Silva, Jillian M; McMahon, Martin

    2014-02-05

    The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

  6. GTP-blot analysis of small GTP-binding proteins. The C-terminus is involved in renaturation of blotted proteins.

    PubMed

    Klinz, F J

    1994-10-01

    Recombinant c-Ha-ras, ralA and rap2, but not rap1A or rap1B proteins retained their ability to bind [alpha-32P]GTP after SDS/PAGE and transfer to nitrocellulose. Recombinant c-Has-ras missing the C-terminal 23 amino acid residues failed to bind [alpha-32P]GTP after the blot, and the ability of recombinant ralA missing the C-terminal 28 amino acid residues to bind [alpha-32P]GTP was decreased many-fold. The presence of nonionic detergents of the polyoxyethylene type such as Tween 20, Triton X-100, Nonidet P40 or Lubrol PX in the incubation buffer was necessary to induce renaturation of blotted recombinant c-Ha-ras protein, whereas other types of detergents were ineffective. We propose that detergents of the polyoxyethylene type induce the refolding of some types of blotted small GTP-binding proteins and that the C-terminus is involved in the refolding process. Membranes from NIH3T3 fibroblasts overexpressing c-Ha-ras protein showed much weaker binding of [alpha-32P]GTP as expected from the level of ras immunoreactivity. Treatment of fibroblasts with lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, caused the accumulation of the unfarnesylated form of c-Ha-ras in the cytosol. Examination of [alpha-32P]GTP-binding and immunoreactivity for cytosolic and membrane-bound c-Ha-ras revealed that binding of [alpha-32P]GTP to unprocessed c-Ha-ras was increased about threefold compared to the same amount of processed c-Ha-ras. Our results demonstrate that detection and quantification of small GTP-binding proteins in eukaryotic cells by GTP-blot analysis is hampered by the fact that these proteins differ strongly in their ability to renature after blotting to nitrocellulose.

  7. Application of SYPRO Ruby- and Flamingo-stained polyacrylamide gels to Western blot analysis.

    PubMed

    Hagiwara, Makoto; Kobayashi, Ken-Ichi; Tadokoro, Tadahiro; Yamamoto, Yuji

    2010-02-15

    Western blots are widely used for analysis of the expression levels of specific proteins. Blotting is conducted after sodium dodecyl sulfate or native polyacrylamide gel electrophoresis without staining the gel. However, when it is necessary to analyze the gel, duplicate polyacrylamide gels (one of which is stained) usually must be prepared, leading to the consumption of precious sample. Thus, we have developed a convenient and efficient Western blot method using a stained gel. This simple modification should be beneficial for the analysis of samples that are limited in quantity and/or samples for which the stained gel serves as the loading control.

  8. Detection and quantitation of HPV DNA replication by Southern blotting and real-time PCR.

    PubMed

    Morgan, Iain M; Taylor, Ewan R

    2005-01-01

    This provides a brief introduction into the mechanism of DNA replication by the E1 and E2 proteins and describes the traditional Southern blotting technique that is used to monitor E1- and E2-mediated DNA replication. It also includes a novel real-time polymerase chain reaction (PCR) approach for monitoring E1- and E2-mediated DNA replication that has enhanced sensitivity and quantitation compared with Southern blotting, and a discussion of when to use the Southern blotting and real-time PCR techniques.

  9. Northern Nutrition.

    ERIC Educational Resources Information Center

    Northwest Territories Dept. of Education, Yellowknife.

    This guide contains nutrition information and nutrition education strategies aimed at residents of the Canadian Arctic. Section I: (1) defines nutrition terms; (2) describes the sources and functions of essential nutrients; (3) explains Canada's food guide and special considerations for the traditional northern Native diet and for lactose…

  10. Northern Nutrition.

    ERIC Educational Resources Information Center

    Northwest Territories Dept. of Education, Yellowknife.

    This guide contains nutrition information and nutrition education strategies aimed at residents of the Canadian Arctic. Section I: (1) defines nutrition terms; (2) describes the sources and functions of essential nutrients; (3) explains Canada's food guide and special considerations for the traditional northern Native diet and for lactose…

  11. Automated capillary Western dot blot method for the identity of a 15-valent pneumococcal conjugate vaccine.

    PubMed

    Hamm, Melissa; Ha, Sha; Rustandi, Richard R

    2015-06-01

    Simple Western is a new technology that allows for the separation, blotting, and detection of proteins similar to a traditional Western except in a capillary format. Traditionally, identity assays for biological products are performed using either an enzyme-linked immunosorbent assay (ELISA) or a manual dot blot Western. Both techniques are usually very tedious, labor-intensive, and complicated for multivalent vaccines, and they can be difficult to transfer to other laboratories. An advantage this capillary Western technique has over the traditional manual dot blot Western method is the speed and the automation of electrophoresis separation, blotting, and detection steps performed in 96 capillaries. This article describes details of the development of an automated identity assay for a 15-valent pneumococcal conjugate vaccine, PCV15-CRM197, using capillary Western technology. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Comparisons of ELISA and Western blot assays for detection of autophagy flux.

    PubMed

    Oh, Sung-Hee; Choi, Yong-Bok; Kim, June-Hyun; Weihl, Conrad C; Ju, Jeong-Sun

    2017-08-01

    We analyzed autophagy/mitophagy flux in vitro (C2C12 myotubes) and in vivo (mouse skeletal muscle) following the treatments of autophagy inducers (starvation, rapamycin) and a mitophagy inducer (carbonyl cyanide m-chlorophenylhydrazone, CCCP) using two immunodetection methods, ELISA and Western blotting, and compared their working range, accuracy, and reliability. The ELISAs showed a broader working range than that of the LC3 Western blots (Table 1). Table 2 showed that data value distribution was tighter and the average standard error from the ELISA was much smaller than those of the Western blot, directly relating to the accuracy of the assay. Test-retest reliability analysis showed good reliability for three individual ELISAs (interclass correlation, ≥ 0.7), but poor reliability for three individual Western blots (interclass correlation, ≤ 0.4) (Table 3).

  13. Multiplexed microfluidic blotting of proteins and nucleic acids by parallel, serpentine microchannels.

    PubMed

    He, Sha; Zhang, Yi; Wang, Pei; Xu, Xingzhi; Zhu, Kui; Pan, Wenying; Liu, Wenwen; Cai, Kaiyong; Sun, Jiashu; Zhang, Wei; Jiang, Xingyu

    2015-01-07

    This work develops a high-throughput, high-efficiency and straightforward microfluidic blotting method for analyzing proteins and nucleic acids. Sample solutions containing antibodies (for protein detection) or hybridization probes (for nucleic acid detection) are introduced into the parallel, serpentine microchannels to specifically recognize the immobilized targets on the substrate, achieving the identification of multiple targets in multiple samples simultaneously. The loading control, molecular weight markers, and antigen/antibody titration are designed and integrated into the microfluidic chip, thus allowing for the quantification of proteins and nucleic acids. Importantly, we could easily distinguish the adjacent blotting bands inside parallel microchannels, which may be difficult to achieve in conventional blotting. The small dimensions of microfluidic channels also help to reduce the amount of probing molecules and to accelerate the biochemical reaction. Our microfluidic blotting could bypass the steps of blocking and washing, further reducing the operation time and complexity.

  14. Detection of Blotted Proteins on Nitrocellulose/PVDF Membranes by Alta.

    PubMed

    Pal, Jayanta K; Rao, Shilpa J; Godbole, Dhanashri J

    2015-01-01

    We describe here a simple method of staining nitrocellulose/PVDF blots by Alta. This red-colored stain which is used as a cosmetic contains Crocein scarlet and Rhodamine B as the principal components. It is very cheap, is available as a ready-to-use liquid, and is as sensitive as the most commonly used stain Ponceau Red S. We further demonstrate that Crocein scarlet (one of the principal components) alone can be used for staining the blots with equal efficiency as well. The stained protein profile can be detected on a white light plate and documented in the usual manner. Detailed analysis indicates that this stain does not interfere with subsequent immunoreactions. Moreover, Alta is almost 100 times cheaper to the routinely used stain Ponceau Red S, and thus is an ideal alternative to the Ponceau Red S for staining blots during western blot analysis.

  15. Identification of immunodiagnostic antigens for cerebrospinal filariasis in horses by western blot analysis

    PubMed Central

    TAKESUE, Masataka; OSAKA, Yuki; MURANAKA, Masanori; KATAYAMA, Yoshinari; IKADAI, Hiromi

    2016-01-01

    ABSTRACT In the present study, the serum and cerebrospinal fluid of horses diagnosed with Setaria digitata cerebrospinal filariasis were analyzed by western blot. The results revealed S. digitata protein bands measuring 65, 34, 22, and 18 kDa in molecular weight. In particular, the 18 kDa band is a possible candidate for clinical immunodiagnosis on the basis of western blot findings. PMID:27073332

  16. Accuracy of Reverse Dot-Blot PCR in Detection of Different β-Globin Gene Mutations.

    PubMed

    El-Fadaly, N; Abd-Elhameed, A; Abd-Elbar, E; El-Shanshory, M

    2016-06-01

    Prevention programs for β-thalassemia based on molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation screening methods. The aim of this study was to compare between direct DNA sequencing, and reverse dot-blot PCR in detection of different β-globin gene mutations in Egyptian children with β-thalassemia. Forty children with β-thalassemia were subjected to mutation analysis, performed by both direct DNA sequencing and β-globin Strip Assay MED™ (based on reverse dot-blot PCR). The most frequent mutant alleles detected by reverse dot-blot PCR were; IVSI-110 G>A (31.25 %), IVS I-6 T > C (21.25 %), and IVS I-1 G>A (20 %). Relatively less frequent mutant alleles detected by reverse dot-blot PCR were "IVSII-1 G>A (5 %), IVSII-745 C>G (5 %), IVSII-848 C>A (2.5 %), IVSI-5 G>C (2.5 %), -87 C>G(2.5 %), and cd39 C>T (2.5 %)", While the genotypes of three patients (6 alleles 7.5 %) were not detected by reverse dot-blot PCR. Mutant alleles detected by direct DNA sequencing were the same as reverse dot-blot PCR method except it revealed the genotypes of 3 undetected patients (one patient was homozygous IVSI-110 G>A, and two patients were homozygous IVS I-1 G>A. Sensitivity of the reverse dot-blot PCR was 92.5 % when compared to direct DNA sequencing for detecting β-thalassemia mutations. Our results therefore suggest that, direct DNA sequencing may be preferred over reverse dot-blot PCR in critical diagnostic situations like genetic counseling for prenatal diagnosis.

  17. Using a large area CMOS APS for direct chemiluminescence detection in Western blotting electrophoresis

    NASA Astrophysics Data System (ADS)

    Esposito, Michela; Newcombe, Jane; Anaxagoras, Thalis; Allinson, Nigel M.; Wells, Kevin

    2012-03-01

    Western blotting electrophoretic sequencing is an analytical technique widely used in Functional Proteomics to detect, recognize and quantify specific labelled proteins in biological samples. A commonly used label for western blotting is Enhanced ChemiLuminescence (ECL) reagents based on fluorescent light emission of Luminol at 425nm. Film emulsion is the conventional detection medium, but is characterized by non-linear response and limited dynamic range. Several western blotting digital imaging systems have being developed, mainly based on the use of cooled Charge Coupled Devices (CCDs) and single avalanche diodes that address these issues. Even so these systems present key drawbacks, such as a low frame rate and require operation at low temperature. Direct optical detection using Complementary Metal Oxide Semiconductor (CMOS) Active Pixel Sensors (APS)could represent a suitable digital alternative for this application. In this paper the authors demonstrate the viability of direct chemiluminescent light detection in western blotting electrophoresis using a CMOS APS at room temperature. Furthermore, in recent years, improvements in fabrication techniques have made available reliable processes for very large imagers, which can be now scaled up to wafer size, allowing direct contact imaging of full size western blotting samples. We propose using a novel wafer scale APS (12.8 cm×13.2 cm), with an array architecture using two different pixel geometries that can deliver an inherently low noise and high dynamic range image at the same time representing a dramatic improvement with respect to the current western blotting imaging systems.

  18. Northern Pintail

    USGS Publications Warehouse

    Clark, Robert G.; Fleskes, Joseph P.; Guyn, Karla L.; Haukos, David A.; Austin, Jane E.; Miller, Michael R.

    2014-01-01

    This medium-sized dabbling duck of slender, elegant lines and conservative plumage coloration is circumpolar in distribution and abundant in North America, with core nesting habitat in Alaska and the Prairie Pothole Region of southern Canada and the northern Great Plains. Breeders favor shallow wetlands interspersed throughout prairie grasslands or arctic tundra. An early fall migrant, the species arrives on wintering areas beginning in August, after wing molt, often forming large roosting and feeding flocks on open, shallow wetlands and flooded agricultural fields. The birds consume grains, marsh plant seeds, and aquatic invertebrates throughout fall and winter.Northern Pintails are among the earliest nesting ducks in North America, beginning shortly after ice-out in many northern areas. Individuals form new pair bonds each winter but are highly promiscuous during the nesting season, with mated and unmated males often involved in vigorous, acrobatic Pursuit Flights. Annual nest success and productivity vary with water conditions, predation, and weather. Females build nests on the ground, often far from water. Only the female incubates; her mate leaves shortly after incubation begins. Ducklings hatch together in one day, follow the female to water after a day in the nest, and fledge by July or August. Adults and ducklings consume mainly aquatic invertebrates during the breeding season.Predators and farming operations destroy many thousands of Northern Pintail nests annually; farming has also greatly reduced the amount of quality nesting cover available. Winter habitats are threatened by water shortages, agricultural development, contamination, and urbanization. Periods of extended drought in prairie nesting regions have caused dramatic population declines, usually followed by periods of recovery. Over the long term, however, the continental population of Northern Pintails has declined significantly from 6 million birds in the early 1970s to less than 3 million in

  19. Immunoconfirmation of central nervous system tuberculosis by blotting: A study of 300 cases.

    PubMed

    Patil, Shripad; Giribhattanavar, Prashant; Patil, Madhusudan; Kumar, Kavitha

    2015-06-01

    Tuberculous meningitis (TBM) is a serious form of disease of the central nervous system. Early and accurate diagnosis of the disease and effective treatment are key important factors to contain the disease. The disease presents as chronic meningitis where other partners such as fungal meningitis, neurosyphilis, cysticercal meningitis, carcinomatous meningitis and partially treated pyogenic meningitis share a similar clinical picture making the diagnosis complicated. Culturing of the pathogen Mycobacterium tuberculosis (MTB) from the cerebrospinal fluid (CSF) sample has shown a poor response. The main immunological method for the immunodiagnosis of TBM is the detection of an antibody response in the CSF. In the present study, total MTB sonicated extract antigen was used for ELISA and Western blot. ELISA shows overall immune response of the test sample, whereas Western blotting reveals the specific reactivity to a particular molecular weight antigen. This would also reveal the immunodominant antigen. A total of 300 CSF samples were analyzed by both ELISA and Western blotting. Of the 240 clinically suspected TBM cases, 111 samples were positive by ELISA and 81 samples by Western blot. A total of 76 CSF samples were positive by both ELISA and Western blot. None of the control samples showed positivity either by ELISA or by Western blot. TBM patients revealed major antibody reactivity to 30-40 kD region, followed by 14 kD region. ELISA is sensitive with mild non-specific binding, but Western blot is specific in detecting the immune response. The findings will be useful in definitive immunodiagnosis of TBM.

  20. HTLV-I/II seroindeterminate Western blot reactivity in a cohort of patients with neurological disease.

    PubMed

    Soldan, S S; Graf, M D; Waziri, A; Flerlage, A N; Robinson, S M; Kawanishi, T; Leist, T P; Lehky, T J; Levin, M C; Jacobson, S

    1999-09-01

    The human T-cell lymphotropic virus type I (HTLV-I) is associated with a chronic, progressive neurological disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis. Screening for HTLV-I involves the detection of virus-specific serum antibodies by EIA and confirmation by Western blot. HTLV-I/II seroindeterminate Western blot patterns have been described worldwide. However, the significance of this blot pattern is unclear. We identified 8 patients with neurological disease and an HTLV-I/II seroindeterminate Western blot pattern, none of whom demonstrated increased spontaneous proliferation and HTLV-I-specific cytotoxic T lymphocyte activity. However, HTLV-I tax sequence was amplified from the peripheral blood lymphocytes of 4 of them. These data suggest that patients with chronic progressive neurological disease and HTLV-I/II Western blot seroindeterminate reactivity may harbor either defective HTLV-I, novel retrovirus with partial homology to HTLV-I, or HTLV-I in low copy number.

  1. A guide to modern quantitative fluorescent western blotting with troubleshooting strategies.

    PubMed

    Eaton, Samantha L; Hurtado, Maica Llavero; Oldknow, Karla J; Graham, Laura C; Marchant, Thomas W; Gillingwater, Thomas H; Farquharson, Colin; Wishart, Thomas M

    2014-11-20

    The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term "western blot" suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many "optimized" western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

  2. Protein analysis through Western blot of cells excised individually from human brain and muscle tissue.

    PubMed

    Koob, A O; Bruns, L; Prassler, C; Masliah, E; Klopstock, T; Bender, A

    2012-06-15

    Comparing protein levels from single cells in tissue has not been achieved through Western blot. Laser capture microdissection allows for the ability to excise single cells from sectioned tissue and compile an aggregate of cells in lysis buffer. In this study we analyzed proteins from cells excised individually from brain and muscle tissue through Western blot. After we excised individual neurons from the substantia nigra of the brain, the accumulated surface area of the individual cells was 120,000, 24,000, 360,000, 480,000, 600,000 μm2. We used an optimized Western blot protocol to probe for tyrosine hydroxylase in this cell pool. We also took 360,000 μm2 of astrocytes (1700 cells) and analyzed the specificity of the method. In muscle we were able to analyze the proteins of the five complexes of the electron transport chain through Western blot from 200 human cells. With this method, we demonstrate the ability to compare cell-specific protein levels in the brain and muscle and describe for the first time how to visualize proteins through Western blot from cells captured individually. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Telomere length measurement by a novel Luminex-based assay: a blinded comparison to Southern blot.

    PubMed

    Pierce, Brandon L; Jasmine, Farzana; Roy, Shantanu; Zhang, Chenan; Aviv, Abraham; Hunt, Steven C; Ahsan, Habibul; Kibriya, Muhammad G

    2016-01-01

    Telomere length (TL) is a potential biomarker of aging and age-related disease risk. We recently published a novel Luminex-based method for high-throughput, low-cost TL measurement. Here we describe a blinded comparison of the Luminex method to Southern blot, the most precise TL measurement method. Luminex and Southern blot measurements for the same 50 DNA samples were taken in two independent laboratories; each sample was measured twice, several months apart. The inter-assay CV for Luminex ranged from 5.5 to 9.1 (depending on CV estimation method), and Southern blot CV from 1.0 to 1.7. Both measures were inversely associated with age. The correlation between the repeated measurements was 0.66 for Luminex and 0.97 for Southern blot. The correlation between Southern blot and Luminex was 0.65 in round 1 and 0.75 in round 2, and the relationship showed no evidence of non-linearity. Our results demonstrate that the Luminex assay is a valid and reproducible method for high-throughput TL measurement. The Luminex assay involves no DNA amplification, which may make Luminex an attractive alternative to PCR-based TL measurement.

  4. Value of Western blotting in the clinical follow-up of canine leishmaniasis.

    PubMed

    Fernández-Pérez, F J; Méndez, S; de la Fuente, C; Cuquerella, M; Gómez, M T; Alunda, J M

    1999-03-01

    Specific serum antibody levels in Leishmania infantum-infected dogs treated with a combination of glucantime and allopurinol were estimated by indirect immunofluorescence and Western blotting. The sensitivity of Western blot was greater than that obtained with immunofluorescence titration. In general, both diagnostic methods concurred with the post-treatment clinical status of the animals. Clinical improvement of successfully treated dogs was related to lower immunofluorescence titers and simpler and/or less reactive immunodetection patterns in Western blotting. The recognition, by infected dogs, of certain low molecular weight antigens, particularly one of approximately 26 kDa, was restricted to pretreatment samples and a single animal in relapse thus apparently constituting an active infection marker.

  5. Quantum dot-based western blot for sensitive detection of pig serum antibody to actinobacillus pleuropneumoniae

    NASA Astrophysics Data System (ADS)

    Cişmileanu, Ana; Sima, Cornelia; Grigoriu, Constantin

    2007-08-01

    A quantum dot - immunoglobulin conjugate specific for pig IgG, was obtained by carbodiimide chemistry. We used a Western blot technique for detecting specific antibodies against Actinobacillus pleuropneumoniae (A. pp), which cause porcine pleuropneumonia. The antigen used in this technique was Apx haemolysin which is an important virulence factor of A. pp and it induces protective immunity in vaccined pigs. The detection on Western blot membrane was possible at 1/50 dilution of quantum dot conjugate at a dilution of pig serum till 1/6400. The results for pig serum demonstrated a higher sensitivity of QD-based Western blot technique for the presence of antibodies specific for Apx haemolysin in comparison with similar classical techniques (with coloured substrate for enzyme present in secondary antibody conjugate).

  6. Western blotting using in-gel protein labeling as a normalization control: stain-free technology.

    PubMed

    Gilda, Jennifer E; Gomes, Aldrin V

    2015-01-01

    Western blotting is a commonly used laboratory technique for semi-quantifying protein amounts. It is important when quantifying protein expression to account for differences in the amount of total protein loaded onto the gel using a loading control. Common loading controls include housekeeping proteins, such as β-actin or GAPDH, quantified by Western blot, or total protein, quantified using a stain such as Coomassie Brilliant Blue or Ponceau S. A more recently developed method for total protein quantification utilizes stain-free technology, which has a linear dynamic detection range and allows for protein detection on both gels and membranes. Here, we describe the theory and use of stain-free gels for total protein quantification and normalization of Western blots.

  7. Application of SYPRO Ruby- and Flamingo-stained polyacrylamide gels to Western blot analysis.

    PubMed

    Hagiwara, Makoto; Kobayashi, Ken-Ichi; Tadokoro, Tadahiro; Yamamoto, Yuji

    2009-06-15

    Western blot analysis has been a useful method for analysis of expression levels of specific proteins and is conducted after sodium dodecyl sulfate (SDS) or native polyacrylamide gel electrophoresis without staining the gel. However, when it is necessary to analyze the gel, duplicate polyacrylamide gels usually must be prepared, one of which is stained, leading to the consumption of precious sample. Thus, we developed a convenient and efficient Western blotting method using a stained gel. This simple modification should be beneficial for analyzing samples that are limited in quantity and/or samples for which the stained gel serves as the loading control.

  8. Identification of claudins by western blot and immunofluorescence in different cell lines and tissues.

    PubMed

    González-Mariscal, Lorenza; Garay, Erika; Quirós, Miguel

    2011-01-01

    Claudins are integral proteins of the TJ. Each epithelia in the organism expresses a unique set of claudins that determines the degree of sealing of the paracellular pathway and the ionic selectivity of the tissue. TJs are dynamic structures whose organization and composition change in response to alterations in the environment as well as under physiological and pathological conditions. Changes in claudin expression and subcellular distribution can be analyzed in western blot and immunofluorescence experiments, employing a wide array of available specific antibodies against claudins. In this chapter, we describe in detail protocols used for western blot and immunofluorescence detection of claudins in epithelial cell lines and in various tissue samples.

  9. Quantitation of protein on gels and blots by infrared fluorescence of Coomassie blue and Fast Green.

    PubMed

    Luo, Shen; Wehr, Nancy B; Levine, Rodney L

    2006-03-15

    Coomassie blue staining of gels and blots is commonly employed for detection and quantitation of proteins by densitometry. We found that Coomassie blue or Fast Green FCF bound to protein fluoresces in the near infrared. We took advantage of this property to develop a rapid and sensitive method for detection and quantitation of proteins in gels and on blots. The fluorescence response is quantitative for protein content between 10 ng and 20 microg per band or spot. Staining and destaining require only 30 min, and the method is compatible with subsequent immunodetection.

  10. Investigation of immunofluorescence cross-reactions against Trichinella spiralis by western blot (immunoblot) analysis.

    PubMed Central

    Robert, F; Weil, B; Kassis, N; Dupouy-Camet, J

    1996-01-01

    Immunofluorescence cross-reactions in Trichinella spiralis serodiagnosis are sometimes difficult to identify. We compared the results of an indirect immunofluorescence assay and the profiles obtained by Western blot (immunoblot) analysis for three groups of patients: 10 T. spiralis-infected patients, 10 patients with autoimmune diseases, and 7 patients with parasitic diseases other than trichinellosis. The degree of immunofluorescence cross-reaction was variable. Western blotting allowed us to differentiate Trichinella infection from other parasitic diseases. In 3 of 10 serum samples from patients with autoimmune diseases, bands which had the same sizes as Trichinella bands were observed, and they could correspond to shared epitopes such as heat shock proteins. PMID:8877138

  11. Quantitative Immunofluorescent Blotting of the Multidrug Resistance-associated Protein 2 (MRP2)

    PubMed Central

    Gerk, Phillip M.

    2011-01-01

    Introduction Quantitation of the expression levels of proteins involved in drug transport and disposition is needed to overcome limitations of film-based detection of chemiluminescent immunoblots. Purpose The purpose was to describe and validate a quantitative immunofluorescent blotting method for detection of ATP-Binding Cassette Transporter Isoform C2/Multidrug Resistance-associated Protein 2 (ABCC2/MRP2). Methods Western blotting was performed by electrophoresis of membrane vesicle protein isolated from Sf9 cells overexpressing MRP2 subsequently blotting with infrared labeled secondary antibody. The bound complex was detected using the Odyssey Infrared Imaging System (Li-Cor; Lincoln, NE). The images were analyzed using the Odyssey Application Software to obtain the integrated intensities, followed by linear regression of the intensity data. Results The limits of quantitation for the time-insensitive technique described here were from 0.001μg to 0.5μg of total membrane protein, the coefficient of variation of the slope was 8.9%; r2 values were 0.986 ± 0.012. The utility and sensitivity of this technique was demonstrated in quantitating expression of MRP2 in human placental tissue samples, in which MRP2 was present in low abundance. Discussion The immunofluorescent blotting technique described provides sensitive, reproducible, and quantitative determinations of large, integral membrane proteins such as MRP2, all with potential long-term cost savings. PMID:21277982

  12. A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

    PubMed Central

    Eaton, Samantha L.; Hurtado, Maica Llavero; Oldknow, Karla J.; Graham, Laura C.; Marchant, Thomas W.; Gillingwater, Thomas H.; Farquharson, Colin; Wishart, Thomas M.

    2014-01-01

    The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term “western blot” suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many “optimized” western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies. PMID:25490604

  13. COMPARISONS OF ELISA AND WESTERN BLOT ASSAYS FOR DETECTION OF CRYPTOSPORIDIUM ANTIBODY

    EPA Science Inventory

    A seroprevalence survey was conducted using ELISA and Western blot (WB) assays for antibody to three Cryptosporidium antigens on 380 blood donors in Jackson County, Oregon. The purpose was to determine if either assay could detect serological evidence of an outbreak which occurre...

  14. Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting.

    PubMed

    Heda, Ghanshyam D; Omotola, Oluwabukola B; Heda, Rajiv P; Avery, Jamie

    2016-09-01

    SDS-PAGE and Western blotting are 2 of the most commonly used biochemical methods for protein analysis. Proteins are electrophoretically separated based on their MWs by SDS-PAGE and then electrotransferred to a solid membrane surface for subsequent protein-specific analysis by immunoblotting, a procedure commonly known as Western blotting. Both of these procedures use a salt-based buffer, with the latter procedure consisting of methanol as an additive known for its toxicity. Previous reports present a contradictory view in favor or against reusing electrotransfer buffer, also known as Towbin's transfer buffer (TTB), with an aim to reduce the toxic waste. In this report, we present a detailed analysis of not only reusing TTB but also gel electrophoresis buffer (EB) on proteins of low to high MW range. Our results suggest that EB can be reused for at least 5 times without compromising the electrophoretic separation of mixture of proteins in an MW standard, BSA, and crude cell lysates. Additionally, reuse of EB did not affect the quality of subsequent Western blots. Successive reuse of TTB, on the other hand, diminished the signal of proteins of different MWs in a protein standard and a high MW membrane protein cystic fibrosis transmembrane-conductance regulator (CFTR) in Western blotting.

  15. A Study of Rubisco through Western Blotting and Tissue Printing Techniques

    ERIC Educational Resources Information Center

    Ma, Zhong; Cooper, Cynthia; Kim, Hyun-Joo; Janick-Buckner, Diane

    2009-01-01

    We describe a laboratory exercise developed for a cell biology course for second-year undergraduate biology majors. It was designed to introduce undergraduates to the basic molecular biology techniques of Western blotting and immunodetection coupled with the technique of tissue printing in detecting the presence, relative abundance, and…

  16. COMPARISONS OF ELISA AND WESTERN BLOT ASSAYS FOR DETECTION OF CRYPTOSPORIDIUM ANTIBODY

    EPA Science Inventory

    A seroprevalence survey was conducted using ELISA and Western blot (WB) assays for antibody to three Cryptosporidium antigens on 380 blood donors in Jackson County, Oregon. The purpose was to determine if either assay could detect serological evidence of an outbreak which occurre...

  17. Two-dimensional gel-based protein standardization verified by western blot analysis.

    PubMed

    Haniu, Hisao; Watanabe, Daisuke; Kawashima, Yusuke; Matsumoto, Hiroyuki

    2015-01-01

    In data presentation of biochemical investigation the amount of a target protein is shown in the y-axis against the x-axis representing time, concentrations of various agents, or other parameters. Western blot is a versatile and convenient tool in such an analysis to quantify and display the amount of proteins. In western blot, so-called housekeeping gene product(s), or "housekeeping proteins," are widely used as internal standards. The rationale of using housekeeping proteins for standardization of western blot is based on the assumption that the expression of chosen housekeeping gene is always constant, which could be false under certain physiological or pathological conditions. We have devised a two-dimensional gel-based standardization method in which the protein content of each sample is determined by scanning the total protein density of two-dimensional gels and the expression of each protein is quantified as the density ratio of each protein divided by the density of the total proteins on the two-dimensional gel. The advantage of this standardization method is that it is not based on any presumed "housekeeping proteins" that are supposed to be being expressed constantly under all physiological conditions. We will show that the total density of a two-dimensional gel can render a reliable protein standardization parameter by running western blot analysis on one of the proteins analyzed by two-dimensional gels.

  18. Use of Nonradioactive Detection Method for North- and South-Western Blot.

    PubMed

    Franke, Claudia; Gräfe, Daniel; Bartsch, Holger; Bachmann, Michael P

    2015-01-01

    Many proteins bind to nucleic acids. For the first characterization of novel proteins, a fast and simple technique for testing their nucleic acid binding capabilities is desirable. Here we describe the use of a North-western and South-western blot protocol for the evaluation of the DNA and RNA binding abilities of a novel putative methyl transferase HSPC133 (METTL5).

  19. A Study of Rubisco through Western Blotting and Tissue Printing Techniques

    ERIC Educational Resources Information Center

    Ma, Zhong; Cooper, Cynthia; Kim, Hyun-Joo; Janick-Buckner, Diane

    2009-01-01

    We describe a laboratory exercise developed for a cell biology course for second-year undergraduate biology majors. It was designed to introduce undergraduates to the basic molecular biology techniques of Western blotting and immunodetection coupled with the technique of tissue printing in detecting the presence, relative abundance, and…

  20. A Streamlined Western Blot Exercise: An Efficient and Greener Approach in the Laboratory Classroom

    ERIC Educational Resources Information Center

    Ness, Traci L.; Robinson, Rebekah L.; Mojadedi, Wais; Peavy, Lydia; Weiland, Mitch H.

    2015-01-01

    SDS-PAGE and western blotting are two commonly taught protein detection techniques in biochemistry and molecular biology laboratory classrooms. A pitfall associated with incorporating these techniques into the laboratory is the significant wait times that do not allow students to obtain timely results. The waiting associated with SDS-PAGE comes…

  1. A Streamlined Western Blot Exercise: An Efficient and Greener Approach in the Laboratory Classroom

    ERIC Educational Resources Information Center

    Ness, Traci L.; Robinson, Rebekah L.; Mojadedi, Wais; Peavy, Lydia; Weiland, Mitch H.

    2015-01-01

    SDS-PAGE and western blotting are two commonly taught protein detection techniques in biochemistry and molecular biology laboratory classrooms. A pitfall associated with incorporating these techniques into the laboratory is the significant wait times that do not allow students to obtain timely results. The waiting associated with SDS-PAGE comes…

  2. Ferulic acid enhances IgE binding to peanut allergens in western blots.

    USDA-ARS?s Scientific Manuscript database

    Phenolic compounds at high concentrations are known to form insoluble complexes with proteins. We hypothesized that this complex formation could interfere with Western blot and ELISA assays for peanut allergens. To verify this, three simple phenolic compounds (ferulic, caffeic, and chlorogenic acids...

  3. Ferulic acid enhances IgE binding to peanut allergens in western blots.

    USDA-ARS?s Scientific Manuscript database

    Because phenolic compounds can precipitate or complex with proteins, we postulated that interactions of phenolics with IgE antibodies help enhance IgE binding to peanut allergens in Western blots. Three different phenolics, such as, ferulic, caffeic and chlorogenic acids were examined. Each was mixe...

  4. Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting

    PubMed Central

    Omotola, Oluwabukola B.; Heda, Rajiv P.; Avery, Jamie

    2016-01-01

    SDS-PAGE and Western blotting are 2 of the most commonly used biochemical methods for protein analysis. Proteins are electrophoretically separated based on their MWs by SDS-PAGE and then electrotransferred to a solid membrane surface for subsequent protein-specific analysis by immunoblotting, a procedure commonly known as Western blotting. Both of these procedures use a salt-based buffer, with the latter procedure consisting of methanol as an additive known for its toxicity. Previous reports present a contradictory view in favor or against reusing electrotransfer buffer, also known as Towbin’s transfer buffer (TTB), with an aim to reduce the toxic waste. In this report, we present a detailed analysis of not only reusing TTB but also gel electrophoresis buffer (EB) on proteins of low to high MW range. Our results suggest that EB can be reused for at least 5 times without compromising the electrophoretic separation of mixture of proteins in an MW standard, BSA, and crude cell lysates. Additionally, reuse of EB did not affect the quality of subsequent Western blots. Successive reuse of TTB, on the other hand, diminished the signal of proteins of different MWs in a protein standard and a high MW membrane protein cystic fibrosis transmembrane-conductance regulator (CFTR) in Western blotting. PMID:27582639

  5. Biofilm detection by wound blotting can predict slough development in pressure ulcers: A prospective observational study.

    PubMed

    Nakagami, Gojiro; Schultz, Gregory; Gibson, Daniel J; Phillips, Priscilla; Kitamura, Aya; Minematsu, Takeo; Miyagaki, Tomomitsu; Hayashi, Akitatsu; Sasaki, Sanae; Sugama, Junko; Sanada, Hiromi

    2016-12-26

    Bacteria have been found to form multicellular aggregates which have collectively been termed "biofilms." It is hypothesized that biofilm formation is a means to protect bacterial cells including protection form the immune response of humans. This protective mechanism is believed to explain persistent chronic wound infections. At times, the biofilms are abundant enough to see, and remove by simple wiping. However, recent evidence has shown that the removal of these visible portions are not sufficient, and that biofilms can continue to form even with daily wiping. In this work, we tested an approach to detect the biofilms which are present after clinically wiping or sharp wound debridement. Our method is based on a variation of impression cytology in which a nitrocellulose membrane was used to collect surface biofilm components, which were then differentially stained. In this prospective study, members of an interdisciplinary pressure ulcer team at a university hospital tested our method's ability to predict the generation of wound slough in the week that followed each blotting. A total of 70 blots collected from 23 pressure ulcers produced 27 wounds negative for staining and 43 positive. In the negative blots 55.6% were found to have decreased wound slough, while 81.4% with positive staining had either increase or unchanged wound slough generation. These results lead to an odds ratio of positive blotting cases of 9.37 (95% confidence intervals: 2.47-35.5, p = 0.001) for slough formation; suggesting that the changes in wound slough formation can be predicted clinically using a non-invasive wound blotting method.

  6. Design, preparation and use of ligated phosphoproteins: a novel approach to study protein phosphatases by dot blot array, ELISA and Western blot assays.

    PubMed

    Sun, Luo; Ghosh, Inca; Barshevsky, Tanya; Kochinyan, Samvel; Xu, Ming-Qun

    2007-07-01

    The study of substrate specificity of protein phosphatases (PPs) is very challenging since it is difficult to prepare a suitable phosphorylated substrate. Phosphoproteins, phosphorylated by a protein kinase, or chemically synthesized phosphopeptides are commonly used substrates for PPs. Both types of these substrates have their advantages and limitations. Phosphoproteins mimic more closely the physiologically relevant PP substrates, but their preparation is technically demanding. Synthetic phosphopeptides present advantages over proteins because they can be easily produced in large quantity and their amino acid sequence can be designed to contain potential determinants of substrate specificity. However, short peptides are less optimal compared to in vivo PP substrates and often display poor and variable binding to different matrices, resulting in low sensitivity in analysis of PP activity on solid support. In this work we utilize the intein-mediated protein ligation (IPL) technique to generate substrates for PPs, combining the advantages of proteins and synthetic peptides in one molecule. The ligation of a synthetic phosphopeptide to an intein-generated carrier protein (CP) with a one-to-one stoichiometry results in the formation of a ligated phosphoprotein (LPP). Three widely used assays, dot blot array, Western blot and ELISA were employed to study the PP activity on LPP substrates. Dephosphorylation was measured by detection of the remaining phosphorylation, or lack of it, with a phospho-specific antibody. The data show the advantage of LPPs over free peptides in assays on solid supports. LPPs exhibited enhanced binding to the matrices used in the study, which significantly improved sensitivity and consistency of the assays. In addition, saturation of the signal was circumvented by serial dilution of the assay samples. This report describes detailed experimental procedures for preparation of LPP substrates and their use in PP assays based on immobilization on

  7. Nanogold Immunodetection Detection Systems for the Identification of Autoantigens by Western Blotting.

    PubMed

    Moore, Jacen S; Scofield, R Hal

    2015-01-01

    Nanogold-conjugated immunodetection systems are now widely and commercially available for use in a number of research applications including electron microscopy, light microscopy, and western blotting. Nanogold clusters are small, uniform in size, and stable, unlike gold colloids historically used in protein detection. Covalent linkage of nanogold particles to secondary antibodies prevents dissociation of the gold particles during the staining process, making protein detection reliable, antigen specific, and highly sensitive. Nanogold labeling is extremely versatile and can be used in conjunction with other staining methodologies including Alexa Fluor immunofluorescence detection to perform coupled staining procedures. Silver enhancement increases the limits of sensitivity for nanogold staining, thus improving detection signals for antigens with reduced expression levels. Herein, we describe the use of nanogold-silver detection as an immunodetection system for standard western blotting of autoantigens.

  8. Native Electrophoresis and Western Blot Analysis (NEWeB): Methods and Applications.

    PubMed

    Manoussopoulos, Ioannis N; Tsagris, Mina

    2015-01-01

    Native Electrophoresis and Western Blot Analysis (NEWeB) has been developed for the study of plant virus characteristics, among others, virus particle-protein interactions, electrophorotype formation, and strain separation. The method is based on the property of electrophoretic mobility of virus particles (VP) and proteins and combines the analytical capacity of electrophoresis with the specificity of western blot. One of its advantages is that it deals with entire VP that can be studied in cause and effect or in time-interval experiments. Some of the most interesting approaches include VP structural studies, VP interaction with host or viral proteins, and also the characterization of VP-protein complexes. In this protocol, NEWeB is used to demonstrate the interaction of Plum pox virus particles with the helper component, a virus encoded protein. It is expected that the method could be used in analogous studies of other viruses or large protein complexes, where similar principles apply.

  9. Direct detection of molluscum contagiosum virus in clinical specimens by dot blot hybridization.

    PubMed Central

    Hurst, J W; Forghani, B; Chan, C S; Oshiro, L; Darai, G

    1991-01-01

    A dot blot hybridization protocol was developed for the direct detection of molluscum contagiosum virus (MCV) DNA in clinical specimens submitted for virus isolation. Samples were concentrated by high-speed centrifugation and treated with proteinase K; this was followed by a single phenol-chloroform extraction step. The DNA was denatured, and the entire volume was spotted onto a nitrocellulose membrane. A biotinylated DNA probe specific for the BamHI-C region of MCV type 1 was used for hybridization. Evidence of MCV DNA was visualized by using streptavidin alkaline phosphatase conjugate and 5-bromo-4-chloro-3-indolyl phosphate-nitroblue tetrazolium as the substrate. Results showed that nonspecific hybridization does not occur with herpes simplex virus- or orf virus-infected clinical specimens and that dot blotting is more sensitive and reproducible than electron microscopy. Images PMID:1774321

  10. Mapping of radiolabeled peptides derived from proteolysis of polypeptides bound to nitrocellulose after Western blotting

    SciTech Connect

    Carrey, E.A.; Hardie, D.G.

    1986-11-01

    Sections of nitrocellulose containing bound /sup 32/P-labeled polypeptides were excised from Western blots and exhaustively digested by trypsin in order to analyze the distribution of phosphorylation sites between the products of limited proteolysis of the multifunctional protein CAD. Using the criterion of analytical isoelectric focusing, the /sup 32/P-peptides obtained by this method were found to be similar, although not identical, to peptides obtained by a more conventional digestion of trichloroacetic acid precipitates. Digestion on Western blots is more straightforward than electrophoretic elution of individual gel slices, gives better recoveries than direct digestion of gel slices, and is particularly suitable for peptide mapping of small peptides which bind to nitrocellulose but would diffuse out of polyacrylamide gels during the commonly used fixing and staining procedures.

  11. Mapping Point Mutations in the Drosophila Rosy Locus Using Denaturing Gradient Gel Blots

    PubMed Central

    Gray, M.; Charpentier, A.; Walsh, K.; Wu, P.; Bender, W.

    1991-01-01

    Mutations within the rosy locus of Drosophila were mapped using blots of genomic DNA fragments separated on denaturing gradient gels. DNA sequence differences between otherwise identical small rosy DNA fragments were detected among the mutants as mobility shifts on the blots. Mutations were mapped to within a few hundred base pairs of rosy sequence in 100 of 130 mutants tested--a 77% detection rate. The sequence changes in 43 rosy mutations are presented; all but six of these were single base changes. Thirty-four of 36 sequenced mutations induced by the alkylating agents N-ethyl-N-nitrosourea and ethyl methanesulfonate were transitions. All of the mutations mapped in the rosy transcription unit. Twenty-three of the 43 sequenced mutations change the predicted rosy gene polypeptide sequence; the remainder would interrupt protein translation (17), or disrupt mRNA processing (3). PMID:1901817

  12. Western blot membrane composed of electrospun polyvinylidene fluoride nanofiber membrane and polyethylene terephthalate sheet.

    PubMed

    Cho, Eugene; Kim, Chan; Hwang, Cheol Ho; Chang, Duck Rye; Kook, Joong-Ki

    2013-06-01

    In a previous study, an electrospun polyvinylidene fluoride (PVDF) nanofiber membrane was developed for Western blotting. The membrane exhibited high sensitivity and high binding capacity for the detection of protein bands that was unlike that observed for conventional, microphase separation-based porous PVDF membranes. Nevertheless, the PVDF nanofiber membrane is quite expensive. The objective of this study was to develop an economical Western blot membrane using a hybrid electrospun PVDF nanofiber and polyethylene terephthalate (PET) sheet. The results showed that the detection sensitivity of the 4 gram per square meter (gsm) membrane was similar to those of the electrospun PVDF nanofiber membrane only, and the 7 gsm PVDF nanofiber membranes on a PET sheet and the electrospun PVDF nanofiber membrane. This means the protein detection sensitivity is not proportional to the thickness of the PVDF nanofiber membrane. The 4 gsm PVDF nanofiber membrane on a PET sheet can be used to detect proteins with high sensitivity and economic efficiency.

  13. Development and Evaluation of a Western Blot Kit for Diagnosis of Human Trichinellosis

    PubMed Central

    Yera, Hélène; Andiva, Shakir; Perret, Catherine; Limonne, Denis; Boireau, Pascal; Dupouy-Camet, Jean

    2003-01-01

    We evaluated industrially prepared Western blot strips designed to avoid the cross-reactions observed with indirect immunofluorescence and enzyme-linked immunosorbent assays used for the serodiagnosis of trichinellosis. The antigen preparations were crude extracts of Trichinella spiralis. The Western blot profile characteristic of trichinellosis was characterized by comparing 60 sera from patients infected by Trichinella to 11 sera from healthy subjects, 51 sera from patients with other proven parasitic diseases (cysticercosis, schistosomiasis, strongyloidosis, fascioliasis, toxocariasis, liver amebiasis, anisakiasis, filariasis, toxoplasmosis, hydatidosis, or malaria), and 23 sera from patients with autoantibodies. Specific 43- to 44-kDa and 64-kDa bands were obtained with all of the sera from 51 patients with acute trichinellosis, in 4 out of 9 patients at the early stages of the disease, and in only 1 control patient, who had suspected anisakiasis and in whom trichinellosis could not be ruled out by muscle biopsy. PMID:12965906

  14. Northern cardiometeopathies.

    PubMed

    Hasnulin, V I; Sevost'yanova, E V; Hasnulina, A V

    2001-04-01

    Our research in high latitudes has allowed the identification of a special class of deadaptive disorders, northern cardiometeopathies, which integrates cardiovascular functional violations connected to biologically significant modifications of meteorological, geomagnetic, electrical, gravitational, rhythmological and other geo-ecological factors of the North. Cardiac and cerebral disorder complexes, and also some psychoemotional modifications manifest cardiometeopathies. Cardiometeopathies can occur with developing of pathology, and in such a case they can be selected in the independent form of deadaptive disease. At the same time, cardiometeopathies in case where cardiovascular pathology already exists could cause complications and become a particular risk factor for the development of injuries and myocardial heart attack. The most important mechanism of cardiometeopathies' formation is the organism's reaction to modifications of the Earth's electromagnetic field, based on internal electromagnetic fields' (first of all pulsing field of heart) dependence on the magnitude and directness of modification of the external electromagnetic fields. The analysis of the high degree of dependence of the blood circulation effiency during geomagnetic perturbations in the North of the modification of electromagnetic heart activity allows us to speak of the discovery of an earlier unknown electromagnetic blood pump.

  15. Serological Differentiation of Murine Typhus and Epidemic Typhus Using Cross-Adsorption and Western Blotting

    PubMed Central

    La Scola, Bernard; Rydkina, Lena; Ndihokubwayo, Jean-Bosco; Vene, Sirkka; Raoult, Didier

    2000-01-01

    Differentiation of murine typhus due to Rickettsia typhi and epidemic typhus due to Rickettsia prowazekii is critical epidemiologically but difficult serologically. Using serological, epidemiological, and clinical criteria, we selected sera from 264 patients with epidemic typhus and from 44 patients with murine typhus among the 29,188 tested sera in our bank. These sera cross-reacted extensively in indirect fluorescent antibody assays (IFAs) against R. typhi and R. prowazekii, as 42% of the sera from patients with epidemic typhus and 34% of the sera from patients with murine typhus exhibited immunoglobulin M (IgM) and/or IgG titers against the homologous antigen (R. prowazekii and R. typhi, respectively) that were more than one dilution higher than those against the heterologous antigen. Serum cross-adsorption studies and Western blotting were performed on sera from 12 selected patients, 5 with murine typhus, 5 with epidemic typhus, and 2 suffering from typhus of undetermined etiology. Differences in IFA titers against R. typhi and R. prowazekii allowed the identification of the etiological agent in 8 of 12 patients. Western blot studies enabled the identification of the etiological agent in six patients. When the results of IFA and Western blot studies were considered in combination, identification of the etiological agent was possible for 10 of 12 patients. Serum cross-adsorption studies enabled the differentiation of the etiological agent in all patients. Our study indicates that when used together, Western blotting and IFA are useful serological tools to differentiate between R. prowazekii and R. typhi exposures. While a cross-adsorption study is the definitive technique to differentiate between infections with these agents, it was necessary in only 2 of 12 cases (16.7%), and the high costs of such a study limit its use. PMID:10882661

  16. PCR versus Southern blot detection of somatic mosaicism in fragile X syndrome

    SciTech Connect

    Mueller, O.T.; Amar, M.J.A.; Gallardo, L.A.; Kousseff, B.G.

    1994-09-01

    The incidence of somatic mosaicism in males with fragile X syndrome has been reported to be as high as 17% of all clinically affected males. Mosaic cases usually do not show the cytogenetic fragile site at Xq27.3 and generally are fully affected according to the clinical criteria, although some subjects show somewhat milder symptoms. Detection of mosaicism relies on the identification of multiple distinct sizes of the CGG size anomaly in the 5{prime} untranslated exon of the FMR-1 gene. Some alleles identified are of a premutation size with trinucleotides numbering 50 to 80 X CGG. In addition, there is a greatly expanded allele with well over 200 copies of the CGG repeat detected. We screened 314 subjects for fragile X syndrome over a three year period. Cases were routinely screened by Southern blotting using the StB12.3 probe as well as by polymerase chain reaction. The PCR amplification products were electrophoresed in agarose, blotted and hybridized with a (CGG){sub 5} oligonucleotide followed by chemiluminescent detection. Seventeen males and 16 females were identified with a CGG expansion, including two males with premutations with repeat sizes of between 50 and 100 CGG trinucleotides and three cases exhibiting somatic mosaicism. The mosaic cases had both a premutation-sized allele and one or more expanded alleles of over 250 CGG copies. The mosaic cases were usually undetected with Southern blotting but easily identified with this PCR protocol. The relative proportion of the expanded allele as determined by scanning densitometry were 70%, 35%, and 5% in the three cases. All three cases were cytogenetically negative. The clinical severity of the mosaic cases was variable, with symptoms ranging from severe MR with most of the physical stigmata to mild learning disability. In our experience, Southern blotting allows more accurate sizing of the expanded allele; however, PCR is essential to identify cases that exhibit mosaicism.

  17. Indeterminate HIV-1 Western Blots: Etiology, Natural History and Psychological Reactions

    DTIC Science & Technology

    1991-09-24

    yes ;9=unk;.=ND ME.1A Allergies O=no;l=yes, not deseris 2=yes, desens ME.1B Surgery 0=no; l=yes ME.1C Hospitalizations 0-no, l=yes Specify...ioYDETERMINATE WESTERN BLOT PSYCHOLOGICAL QUESTIONNAIRE 0-prior to 1st visit; 1-initial; 3.3 mos; 6= Gmos ; 9=9mos 0-case; .. control 1 - AIDS

  18. Use of a Western blot technique for the serodiagnosis of glanders

    PubMed Central

    2011-01-01

    Background The in vivo diagnosis of glanders relies on the highly sensitive complement fixation test (CFT). Frequently observed false positive results are troublesome for veterinary authorities and cause financial losses to animal owners. Consequently, there is an urgent need to develop a test with high specificity. Hence, a Western blot assay making use of a partly purified lipopolysaccaride (LPS) containing antigen of three Burkholderia mallei strains was developed. The test was validated investigating a comprehensive set of positive and negative sera obtained from horses and mules from endemic and non endemic areas. Results The developed Western blot assay showed a markedly higher diagnostic specificity when compared to the prescribed CFT and therefore can be used as a confirmatory test. However, the CFT remains the test of choice for routine testing of glanders due to its high sensitivity, its feasibility using standard laboratory equipment and its worldwide distribution in diagnostic laboratories. Conclusions The CFT should be amended by the newly validated Western blot to increase the positive likelihood ratio of glanders serodiagnosis in non endemic areas or areas with low glanders prevalence. Its use for international trade of horses and mules should be implemented by the OIE. PMID:21247488

  19. Differentiation of larva migrans caused by Baylisascaris procyonis and Toxocara species by Western blotting.

    PubMed

    Dangoudoubiyam, Sriveny; Kazacos, Kevin R

    2009-11-01

    Baylisascaris procyonis and Toxocara species are two important causes of larva migrans in humans. Larva migrans caused by Toxocara spp. is well known and is diagnosed serologically by enzyme immunoassay. Over a dozen cases of larva migrans and associated eosinophilic encephalitis caused by B. procyonis have also been reported, and at least a dozen additional cases are known. An enzyme-linked immunosorbent assay (ELISA) using the excretory-secretory (ES) antigen of B. procyonis larvae is currently being used in our laboratory as an aid in the diagnosis of this infection in humans. Clinically affected individuals show very high reactivity (measured as the optical density) on this ELISA; however, a one-way cross-reactivity with Toxocara spp. has been observed. As an approach to differentiate these two infections based on serology, we performed Western blots, wherein the B. procyonis ES antigen was reacted with serum samples from individuals known to be positive for either Toxocara spp. or B. procyonis larva migrans. Western blot results showed that B. procyonis antigens of between 30 and 45 kDa were specifically identified only by the sera from individuals with Baylisascaris larva migrans, thus allowing for differentiation between the two infections. This included human patient serum samples submitted for serologic testing, as well as sera from rabbits experimentally infected with B. procyonis. When used in conjunction with the ELISA, Western blotting could be an efficient tool for diagnosis of this infection in humans.

  20. Use of western blot to study Microsporum canis antigenic proteins in canine dermatophytosis.

    PubMed

    Peano, Andrea; Min, Annarita Molinar; Beccati, Massimo; Menzano, Arianna; Pasquetti, Mario; Gallo, Maria Grazia

    2011-05-01

    Western blotting was used to describe the Microsporum canis proteins with antigenic activity in dogs with dermatophytosis. Electrophoretic separation of whole fungal strain extract cultured from a cat was performed under denaturing conditions. The proteins were blotted onto nitrocellulose and probed with sera collected from 22 dogs with dermatophytosis (18 M. canis, 3 M. gypseum, 1 Trichophyton mentagrophytes; group A), 20 dogs with skin diseases other than dermatophytosis, and 22 dogs with no clinical cutaneous signs (group B, n = 42). Nine principal IgG-binding proteins with apparent molecular weights of 180, 144, 130, 120, 102, 96, 80, 68, and 48 kD were visualised on group A blots. For these proteins, serological cross-reactivity with different strains of M. canis may be indirectly confirmed, whereas additional proteins were found to react with sera from individual dogs. The proteins visualised in this study may represent diagnostic markers of dermatophyte infection. The proteins should be further evaluated for their role in the cellular immune response of dogs with dermatophytosis. © 2009 Blackwell Verlag GmbH.

  1. A streamlined Western blot exercise: An efficient and greener approach in the laboratory classroom.

    PubMed

    Ness, Traci L; Robinson, Rebekah L; Mojadedi, Wais; Peavy, Lydia; Weiland, Mitch H

    2015-01-01

    SDS-PAGE and western blotting are two commonly taught protein detection techniques in biochemistry and molecular biology laboratory classrooms. A pitfall associated with incorporating these techniques into the laboratory is the significant wait times that do not allow students to obtain timely results. The waiting associated with SDS-PAGE comes from staining and destaining, whereas with western blotting it is the times required for antibody incubations and the numerous wash steps. This laboratory exercise incorporates 2,2,2-trichloroethanol (TCE) into the SDS-PAGE gel allowing for visualization of migrated proteins in a matter of minutes, saving both the time and chemical waste associated with traditional Coomassie staining. Additionally, TCE staining does not affect protein transfer eliminating the requirement for duplicated gels for total protein and western analyses. Protein transfer can be confirmed immediately without the use of Ponceau S staining. Lastly, this western blot procedure has been further shortened by using an HRP-conjugated primary antibody, which eliminates the secondary antibody incubation and washes, and uses a colorimetric detection to allow for visualization by students without the need for specialized equipment. © 2015 The International Union of Biochemistry and Molecular Biology.

  2. Imported intraocular gnathostomiasis with subretinal tracks confirmed by western blot assay.

    PubMed

    Yang, Ji Ho; Kim, Moosang; Kim, Eung Suk; Na, Byoung-Kuk; Yu, Seung-Young; Kwak, Hyung-Woo

    2012-03-01

    We report a case of intraocular gnathostomiasis diagnosed by western blot assay in a patient with subretinal tracks. A 15-year-old male patient complained of blurred vision in the right eye, lasting for 2 weeks. Eight months earlier, he had traveled to Vietnam for 1 week and ate raw wild boar meat and lobster. His best-corrected visual acuity was 20/20 in both eyes and anterior chamber examination revealed no abnormalities. Fundus examination showed subretinal tracks in the right eye. Fluorescein angiography and indocyanine green angiography showed linear hyperfluorescence of the subretinal lesion observed on fundus in the right eye. Ultrasound examination revealed no abnormalities. Blood tests indicated mild eosinophilia (7.5%), and there was no abnormality found by systemic examinations. Two years later, the patient visited our department again for ophthalmologic evaluation. Visual acuity remained 20/20 in both eyes and the subretinal tracks in the right eye had not changed since the previous examination. Serologic examination was performed to provide a more accurate diagnosis, and the patient's serum reacted strongly to the Gnathostoma nipponicum antigen by western blot assay, which led to a diagnosis of intraocular gnathostomiasis. This is the first reported case of intraocular gnathostomiasis with subretinal tracks confirmed serologically using western blot in Korea.

  3. Characterization of a biopharmaceutical protein and evaluation of its purification process using automated capillary Western blot.

    PubMed

    Xu, Dong; Mane, Sarthak; Sosic, Zoran

    2015-01-01

    This paper describes the application of an automated size-based capillary Western blot system (Sally instrument) from ProteinSimple, Inc., for biopharmaceutical fusion-Fc protein characterization and evaluation of its purification process. The fusion-Fc protein column purification from an excess of single chain Fc polypeptide and removal of an enzyme coexpressed for protein maturation have been demonstrated using an automated capillary Western system. The clearance of a selected host cell protein (HCP) present in cell culture of fusion-Fc protein was also quantitatively monitored throughout the protein purification process. Additionally, the low levels of fusion-Fc product-related impurities detected by traditional slab gel Western blot were confirmed by the automated capillary Western system. Compared to the manual approach, the automated capillary Western blot provides the advantages of ease of operation, higher sample throughput, greater linearity range, and higher precision for protein quantitation. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Direct Blue 71 staining as a destaining-free alternative loading control method for Western blotting.

    PubMed

    Zeng, Li; Guo, Jing; Xu, Hong-Bo; Huang, Rongzhong; Shao, Weihua; Yang, Liu; Wang, Mingju; Chen, Jianjun; Xie, Peng

    2013-08-01

    In Western blotting, a suitable loading control is indispensable for correcting errors in the total amount of loaded protein. Immunodetection of housekeeping proteins and total protein staining have traditionally been used as loading control methods. Direct Blue 71 (DB71) staining-a novel, sensitive, dye-binding staining method compatible with immunodetection-may offer advantages over these traditional loading control methods. Three common neuroscientific samples (human plasma, human oligodendrocytes, and rat brain) were employed to assess DB71 staining as a loading control method for Western blotting. DB71, CBB, one traditional housekeeping protein, and one protein of interest were comparatively assessed for reliability and repeatability and linear dynamic range over 2.5-40 μg of protein loaded. DB71's effect on the reliability and repeatability and linear dynamic range of immunoreaction were also assessed. Across all three sample types, DB71 was either equivalent or superior to CBB and housekeeping protein-based methods in terms of reliability and repeatability and linear dynamic range. Across all three sample types, DB71 staining did not impair the reliability and repeatability or linear dynamic range of immunoreaction. Our results demonstrate that the DB71 staining can be used as a destaining-free alternative loading control method for Western blotting. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Banding pattern indicative of echinococcosis in a commercial cysticercosis western blot

    PubMed Central

    2009-01-01

    Objective A commercial cysticercosis Western blot was evaluated for serological cross-reactivity of sera from patients with alveolar (AE) and cystic echinococcosis (CE). Methods A total of 161 sera were examined, including 31 sera from AE-patients, 11 sera from CE-patients, 9 sera from patients with other parasitic diseases and 109 sera from patients with unrelated medical conditions. All AE-and CE-sera were also examined by the echinococcosis Western blot. Results More sera from patients with AE than with CE showed cross-reactivity in the form of ladder-like patterns ("Mikado aspect") and untypical bands at 6-8 kDa (71% and 77.4% versus 27.3% and 45.5%, respectively). In contrast, triplets of bands in the area above 50 kDa and between 24 and 39-42 kDa were more frequent in CE than in AE sera. The fuzzy band at 50-55 kDa typical for cysticercosis was absent in all AE and CE sera. Conclusions Atypical banding patterns in the cysticercosis Western blot should raise the suspicion of a metacestode infection different from Taenia solium, i.e. Echinococcus multilocularis or E. granulosus, especially when the Mikado aspect and an altered 6-8 kDa band is visible in the absence of a fuzzy 50-55 kDa band. PMID:19748853

  6. Using genomic slot blot hybridization to assess intergeneric Saccharum x Erianthus hybrids (Andropogoneae - Saccharinae).

    PubMed

    Besse, P; McIntyre, C L; Burner, D M; Almeida, C G

    1997-08-01

    The use of genomic slot blot hybridization enabled the differentiation of hybrids from selfs in Saccharum x Erianthus intergeneric crosses in which Saccharum was used as the female parent. Based on the genomic in situ hybridization technique, slot blots of DNA from the parents and the progeny were blocked with the Saccharum parent DNA and hybridized with the labelled male Erianthus genomic DNA. This technique allowed a rapid screening for hybrids and was sensitive enough to detect a 1/20 dilution of Erianthus in Saccharum DNA, which should enable the detection of most partial hybrids. The genomic slot blot hybridization technique was shown to be potentially useful for assessing crosses involving Saccharum species with either Old World Erianthus section Ripidium or North American Erianthus (= Saccharum) species. The effectiveness of the technique was assessed on 144 progeny of a Saccharum officinarum x Erianthus arundinaceus cross, revealing that 43% of the progeny were selfs. The importance of this test as a tool to support intergeneric breeding programs is discussed.

  7. Fluorescent Labeling of Proteins and Its Application to SDS-PAGE and Western Blotting.

    PubMed

    Alba, F Javier; Bartolomé, Salvador; Bermúdez, Antonio; Daban, Joan-Ramon

    2015-01-01

    This chapter describes very simple fluorescent methods developed in our laboratory allowing the rapid monitoring of total protein patterns on both sodium dodecyl sulfate (SDS) polyacrylamide gels and western blots. The noncovalent dye Nile red (9-diethylamino-5H-benzo[α]phenoxazine-5-one) is used for the sensitive staining of proteins in SDS gels. This method is compatible with the electroblotting of protein bands and with the staining of the resulting blot with the covalent dye MDPF (2-methoxy-2,4-diphenyl-3(2H)-furanone). These staining procedures are applied sequentially; there is no need to run a duplicate unstained gel for protein blotting. Furthermore, since only the adduct formed by the reaction of MDPF with proteins is fluorescent, there is no need to destain the membrane after protein labeling. In addition, MDPF staining is compatible with further immunodetection of specific bands with polyclonal antibodies. Finally, using the adequate conditions described below, MDPF staining does not preclude the N-terminal sequence analysis of proteins in selected bands.

  8. Specific Western Blot Bands Are Associated with Initial CD4+ Lymphocyte Counts in Human Immunodeficiency Virus Seroconverters.

    DTIC Science & Technology

    1992-09-30

    SPECIFIC WESTERN BLOT BANDS ARE ASSOCIATED WITH INITIAL CD4+ LYMPHOCYTE COUNTS IN HUMAN IMMUNODEFICIENCY VIRUS SEROCONVERTERS DTI C ELEkCTE AUG...NAVAL MEDICAL RESEARCH AND DEVELOPMENT COMMAND BETHESDA, MARYLAND Specific Western Blot Bands Are Associated With Initial CD4+ Lymphocyte Counts in...and Ms. Susan Yu also provided - quality assurance review of Western blot results. Mr. Jerry Talicurian, Mr. Juan Jurado, Mr. David Morgan, and

  9. Autoantibody profiling of patients with primary biliary cirrhosis using a multiplexed line-blot assay.

    PubMed

    Villalta, Danilo; Sorrentino, Maria Concetta; Girolami, Elia; Tampoia, Marilina; Alessio, Maria Grazia; Brusca, Ignazio; Daves, Massimo; Porcelli, Brunetta; Barberio, Giuseppina; Bizzaro, Nicola

    2015-01-01

    To evaluate the autoantibody profile in patients with primary biliary cirrhosis (PBC) using a new multiplexed line-blot assay specifically designed for the diagnosis of autoimmune liver diseases. Sera of 58 consecutive PBC patients and 191 disease controls (144 with autoimmune liver diseases other than PBC, and 67 with non-autoimmune chronic liver diseases) were tested by both the multiplexed line-blot Autoimmune Liver Disease Profile 2 (ALD2) and by IIF on HEp-2 cells and on rat kidney/liver/stomach tissues. ALD2 contains the following PBC-associated antigens: AMA-M2, natively purified from bovine heart; M2-E3, a recombinant fusion protein including the E2 subunits of PDC, BCOADC and OGDC; sp100, PML and gp210 recombinant proteins. With the ALD2 assay, a positive reaction to AMA-M2, M2-E3, sp100, PML and gp210 in PBC patients was observed in 77.6%, 84.5%, 34.5%, 15.1% and 18.9%, respectively, of the PBC sera. The overall sensitivity and specificity for PBC were 98.3% and 93.7%. Using IIF, positivity rates to AMA, and to antinuclear autoantibodies with membranous/rim-like and multiple nuclear dot patterns were 86.2%, 8.6% and 22.4%, respectively. The overall sensitivity and specificity for PBC of the IIF method were 86.2% and 97.9%, respectively. The ALD2 line-blot showed a good diagnostic accuracy for PBC and a higher sensitivity than the IIF method to detect sp100 and gp210 autoantibodies. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Fluorescent detection of Southern blots and PCR-based genetic typing tests

    SciTech Connect

    Mansfield, E.S.; Worley, J.M.; Zimmerman, P.A.

    1994-09-01

    The Southern blot is used to study gene organization, to identify disease-causing genomic rearrangements, or for typing RFLP markers in forensic, paternity, or prenatal diagnostic testing. Fluorescence offers a much greater dynamic range and a more linear response than film used in radioactive or chemiluminescent detection of RFLPs. We therefore investigated using the Fluorimager{trademark} 575 (Molecular Dynamics, Inc.) for analyzing Southern blots. Using a single-locus probe to D2S44 (YNH24) (Promega Corp.), we detect as little as 100 ng (0.05 attomole) genomic DNA. The alkaline phosphatase-labeled probe is detected using AttoPhos (JBL Scientific), and the developed membrane is scanned with the Fluorimager. Biotinylated hybridization probes can also be developed using a streptavidin-alkaline phosphatase conjugate and AttoPhos. The instrument scan parameters can be adjusted to prevent overexposure and accompanying loss of resolution in images of blots, gels, or 96-well microplates. We have used these other sample formats in PCR-based genetic typing assays. We use FluorKit DQS (Molecular Dynamics) to accurately quantify PCR template DNA (1-500 ng) in 96-well microplates scanned using the same instrument. Mutation detection assays run include heteroduplex gels (5% polyacrylamide, 2.7 M urea), short tandem repeat (STR) markers, amplified fragment length polymorphisms (AmpFLP), competitive priming PCR, and allele-specific oligotyping. These assays are run using either 1- or 2-color labeling. We detect unlabeled PCR products, such as the AmpFLP marker D1S80 (Perkin-Elmer) by post-staining gels for 10 minutes with SYBR Green 1 (Molecular Probes) and scanning the wet gel. The Fluorimager scans a 20 x 25 cm sample within three minutes, allowing rapid optimization of fluorescent protocols and high sample throughput.

  11. FANCD2 Western blot as a diagnostic tool for Brazilian patients with Fanconi anemia.

    PubMed

    Pilonetto, D V; Pereira, N F; Bitencourt, M A; Magdalena, N I R; Vieira, E R; Veiga, L B A; Cavalli, I J; Ribeiro, R C; Pasquini, R

    2009-03-01

    Fanconi anemia is a rare hereditary disease showing genetic heterogeneity due to a variety of mutations in genes involved in DNA repair pathways, which may lead to different clinical manifestations. Phenotypic variability makes diagnosis difficult based only on clinical manifestations, therefore laboratory tests are necessary. New advances in molecular pathogenesis of this disease led researchers to develop a diagnostic test based on Western blot for FANCD2. The objective of the present study was to determine the efficacy of this method for the diagnosis of 84 Brazilian patients with Fanconi anemia, all of whom tested positive for the diepoxybutane test, and 98 healthy controls. The FANCD2 monoubiquitinated isoform (FANCDS+/FANCD2L-) was not detected in 77 patients (91.7%). In 2 patients (2.4%), there was an absence of both the monoubiquitinated and the non-ubiquitinated proteins (FANCD2S-/FANCD2L-) and 5 patients (5.9%) had both isoforms (FANCD2S+/FANCD2L+). This last phenotype suggests downstream subtypes or mosaicism. All controls were diepoxybutane negative and were also negative on the FANCD2 Western blot. The Western blot for FANCD2 presented a sensitivity of 94% (79/84) and specificity of 100% (98/98). This method was confirmed as an efficient approach to screen Brazilian patients with deleterious mutations on FANCD2 (FANCD2S-/FANCD2L-) or other upstream genes of the FA/BRCA pathway (FANCDS+/FANCD2L-), to confirm the chromosome breakage test and to classify patients according to the level of FA/BRCA pathway defects. However, patients showing both FANCD2 isoforms (FANCD2S+/FANCD2L+) require additional studies to confirm mutations on downstream Fanconi anemia genes or the presence of mosaicism.

  12. Evaluation of SYPRO Ruby total protein stain for the normalization of two-dimensional Western blots.

    PubMed

    Steinberger, Birgit; Brem, Gottfried; Mayrhofer, Corina

    2015-05-01

    Due to post-translational modifications such as phosphorylation, proteins exist as distinct charge variants. Two-dimensional (2D) gel electrophoresis followed by immunoblotting enables the detection of these isoforms. For their accurate relative quantitation in different samples, a loading control is necessary to compensate for technical errors such as imprecise sample loading or transfer. The study reveals that the combinatory approach of SYPRO Ruby and chemiluminescence-based 2D Western blot analysis exhibits high linearity and excellent reproducibility and is applicable for limited sample amounts. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. A Western Blot Protocol for Detection of Proteins Heterologously Expressed in Xenopus laevis Oocytes.

    PubMed

    Jørgensen, Morten Egevang; Nour-Eldin, Hussam Hassan; Halkier, Barbara Ann

    2016-01-01

    Oocytes of the African clawed frog, Xenopus laevis, are often used for expression and biochemical characterization of transporter proteins as the oocytes are particularly suitable for uptake assays and electrophysiological recordings. Assessment of the expression level of expressed transporters at the individual oocyte level is often desirable when comparing properties of wild type and mutant transporters. However, a large content of yolk platelets in the oocyte cytoplasm makes this a challenging task. Here we report a method for fast and easy, semiquantitative Western blot analysis of proteins heterologously expressed in Xenopus oocytes.

  14. Blotting Assisted by Heating and Solvent Extraction for DESI-MS Imaging

    NASA Astrophysics Data System (ADS)

    Cabral, Elaine C.; Mirabelli, Mario F.; Perez, Consuelo J.; Ifa, Demian R.

    2013-06-01

    Imprints of potato sprout ( Solanum tuberosum L.), gingko leaves (Gingko biloba L. ) and strawberries (Fragaria x ananassa Duch. ) were successfully imaged by desorption electrospray ionization mass spectrometry (DESI-MS) on TLC plates through blotting assisted by heating and/or solvent extraction. Ion images showing the distribution of significant compounds such as glycoalkaloid toxins in potato sprout, ginkgolic acids and flavonoids in ginkgo leaves, and sugars and anthocyanidin in strawberry were obtained. Practical implications of this work include analysis of a wide range of irregular or soft materials by different imprinting conditions without requiring the addition of matrices or use of specific kinds of surfaces.

  15. Selection of DNA aptamers against VEGF165 using a protein competitor and the aptamer blotting method.

    PubMed

    Hasegawa, Hijiri; Sode, Koji; Ikebukuro, Kazunori

    2008-05-01

    Two DNA aptamers against a tumor marker protein, human vascular endothelial growth factor (VEGF(165)) were identified. In the screening process, another protein was used as the competitor to isolate those aptamers that have high specificity for the target. In addition, we evaluated the affinities of the enriched library by means of aptamer blotting. The isolated aptamers bound to VEGF(165) with a K(d) value in the range of a few hundred nanomoles, and did not bind to the competitor. This selection method enabled us to efficiently select the specific aptamers against the target protein. These specific aptamers would be useful sensor elements for cancer diagnosis.

  16. Western Blotting Is an Efficient Tool for Differential Diagnosis of Paracoccidioidomycosis and Pulmonary Tuberculosis

    PubMed Central

    Bertoni, Thâmara Aline; Perenha-Viana, Maysa Cláudia Zolin; Patussi, Eliana Valéria; Cardoso, Rosilene Fressatti

    2012-01-01

    Sputum and sera from 134 patients screened for tuberculosis (TB) were analyzed to investigate TB and paracoccidioidomycosis (PCM). Of these patients, 11 (8.2%) were confirmed to have TB, but six (4.5%) were positive only for PCM. All patients with PCM presented anti-43-kDa-component antibodies in Western blotting (WB) assays, while in the TB-positive patients these antibodies did not appear. This preliminary study suggests WB as a potential tool for differential laboratory diagnosis between TB and PCM. PMID:22971781

  17. Neurofilament dot blot assays: novel means of assessing axon viability in culture.

    PubMed

    Hares, Kelly; Kemp, Kevin; Gray, Elizabeth; Scolding, Neil; Wilkins, Alastair

    2011-06-15

    Axonal structure and integrity are vital to overall neuronal maintenance and action potential propagation. Neurofilaments (NFs) are one of the main cytoskeletal components of axons and phosphorylation of NF subunits regulates speed of NF transport through axons and determines optimal axonal calibre required for signal propagation. Many previous studies of neuroprotective agents have focussed on neuronal viability in models of neurodegenerative disease, without specifically considering axon function as an indicator of neuronal damage. In this study, we have focused on developing novel assays for determining axon viability by measuring levels of neurofilament phosphorylation in cultured cortical neurons. The nitric oxide donor DETANONOate (NO) was used as an inflammatory insult and glial cell line-derived neurotrophic factor (GDNF) and superoxide dismutase (SOD) were tested as potential axonal protective agents. Using 'dot blot' methodologies, we show a decrease in NF phosphorylation in cortical neurons exposed to NO-mediated cell toxicity and an attenuation of NO-mediated changes in NF phosphorylation associated with GDNF and SOD treatment. These results correlated well with immunocytochemical counts. We propose therefore that the dot blot assay is a novel method for assessing axonal integrity in vitro and may play a useful role in the future for testing the effects of agents on axonal viability, providing a reliable and reproducible screening method for potential therapeutics for neurodegenerative diseases. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. A simple DNA recombination screening method by RT-PCR as an alternative to Southern blot.

    PubMed

    Albers, Eliene; Sbroggiò, Mauro; Martin-Gonzalez, Javier; Avram, Alexandra; Munk, Stephanie; Lopez-Contreras, Andres J

    2017-01-19

    The generation of genetically engineered mouse models (GEMMs), including knock-out (KO) and knock-in (KI) models, often requires genomic screening of many mouse ES cell (mESC) clones by Southern blot. The use of large targeting constructs facilitates the recombination of exogenous DNA in a specific genomic locus, but limits the detection of its correct genomic integration by standard PCR methods. Genomic Long Range PCR (LR-PCR), using primers adjacent to the homology arms, has been used as an alternative to radioactive-based Southern blot screenings. However, LR-PCRs are often difficult and render many false positive and false negative results. Here, we propose an alternative screening method based on the detection of a genetic modification at the mRNA level, which we successfully optimized in two mouse models. This screening method consists of a reverse-transcription PCR (RT-PCR) using primers that match exons flanking the targeting construct. The detection of the expected modification in this PCR product confirms the integration at the correct genomic location and shows that the mutant mRNA is expressed. This is a simple and sensitive strategy to screen locus-specific recombination of targeting constructs which can also be useful to screen KO and KI mutant mice or cell lines including those generated by CRISPR/Cas9.

  19. Analysis of sperm antigens by sodium dodecyl sulfate gel/protein blot radioimmunobinding method

    SciTech Connect

    Lee, C.Y.G.; Huang, Y.S.; Hu, P.C.; Gomel, V.; Menge, A.C.

    1982-06-01

    A radioimmunobinding method based on the blotting of renatured proteins from sodium dodecyl sulfate gels on to nitrocellulose filter papers was developed to analyze the sperm antigens that elicit serum anti-sperm antibodies. In rabbits, serum anti-sperm antibodies were raised by immunization with homologous epididymal spermatozoa mixed with complete Freund's adjuvant. The raised antisera from either male or female rabbits were shown to react with three major sperm protein bands on sodium dodecyl sulfate gels with the corresponding molecular weights of about 70,000 +/- 5000, 14,000, and 13,000, respectively. In humans, the monoclonal antibodies against human sperm were raised by a hybridoma technique. Out of six independent hybrid cell lines that were generated, three of them were shown to secrete immunoglobulins that react with the same two protein bands on sodium dodecyl sulfate gels, which have the approximate molecular weight of 10,000. The same procedure was also used to analyze human serum samples that were shown to contain anti-sperm antibodies by the known techniques. Unique sperm antigens that elicit anti-sperm antibodies in humans were identified and correlated. The results of this study suggest that sodium dodecyl sulfate gel/protein blot radioimmunobinding method may be a sensitive and useful tool for the study of sperm antigens that elicit autoimmune responses and their association with human infertility.

  20. Validation of endothelin B receptor antibodies reveals two distinct receptor-related bands on Western blot.

    PubMed

    Barr, Travis P; Kornberg, Daniel; Montmayeur, Jean-Pierre; Long, Melinda; Reichheld, Stephen; Strichartz, Gary R

    2015-01-01

    Antibodies are important tools for the study of protein expression but are often used without full validation. In this study, we used Western blots to characterize antibodies targeted to the N or C terminal (NT or CT, respectively) and the second or third intracellular loop (IL2 or IL3, respectively) of the endothelin B receptor (ETB). The IL2-targeted antibody accurately detected endogenous ETB expression in rat brain and cultured rat astrocytes by labeling a 50-kDa band, the expected weight of full-length ETB. However, this antibody failed to detect transfected ETB in HEK293 cultures. In contrast, the NT-targeted antibody accurately detected endogenous ETB in rat astrocyte cultures and transfected ETB in HEK293 cultures by labeling a 37-kDa band but failed to detect endogenous ETB in rat brain. Bands detected by the CT- or IL3-targeted antibody were found to be unrelated to ETB. Our findings show that functional ETB can be detected at 50 or 37kDa on Western blot, with drastic differences in antibody affinity for these bands. The 37-kDa band likely reflects ETB processing, which appears to be dependent on cell type and/or culture condition. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Quantitative dot blot analysis (QDB), a versatile high throughput immunoblot method.

    PubMed

    Tian, Geng; Tang, Fangrong; Yang, Chunhua; Zhang, Wenfeng; Bergquist, Jonas; Wang, Bin; Mi, Jia; Zhang, Jiandi

    2017-08-29

    Lacking access to an affordable method of high throughput immunoblot analysis for daily use remains a big challenge for scientists worldwide. We proposed here Quantitative Dot Blot analysis (QDB) to meet this demand. With the defined linear range, QDB analysis fundamentally transforms traditional immunoblot method into a true quantitative assay. Its convenience in analyzing large number of samples also enables bench scientists to examine protein expression levels from multiple parameters. In addition, the small amount of sample lysates needed for analysis means significant saving in research sources and efforts. This method was evaluated at both cellular and tissue levels with unexpected observations otherwise would be hard to achieve using conventional immunoblot methods like Western blot analysis. Using QDB technique, we were able to observed an age-dependent significant alteration of CAPG protein expression level in TRAMP mice. We believe that the adoption of QDB analysis would have immediate impact on biological and biomedical research to provide much needed high-throughput information at protein level in this "Big Data" era.

  2. Silver and gold nanoparticle coated membranes applied to protein dot blots

    NASA Astrophysics Data System (ADS)

    Xie, F.; Drozdowicz-Tomsia, K.; Shtoyko, T.; Goldys, E. M.

    2011-02-01

    Detection and identification of low abundance biomarker proteins is frequently based on various types of membrane-based devices. Lowering of the protein detection limits is vital in commercial applications such as lateral flow assays and in Western blots widely used in proteomics. These currently suffer from insufficient detection sensitivity and low retention for small 2-5 kDa proteins. In this study, we report the deposition of two types of metal nanoparticles: gold colloids (50-95 nm diameter) and silver fractals onto a range of commonly used types of membranes including polyvinylidene fluoride (PVDF). Due to strong affinity of proteins to noble metals, such modified membranes have the potential to effectively capture trace proteins preventing their loss. The membranes modified by metal particles were characterized optically and by SEM. The membrane performance in protein dot blots was evaluated using the protein—fluorophore conjugates Deep Purple-bovine serum albumin and fluorescein—human serum albumin. We found that the metal nanoparticles increase light extinction by metals, which is balanced by increased fluorescence, so that the effective fluorescence signal is unchanged. This feature combined with the capture of proteins by the nanoparticles embedded in the membrane increases the detection limit of membrane assays.

  3. Development and applications of simultaneous immunochemical staining and serial detection of overlapping proteins in blotting procedures.

    PubMed

    Kuczius, Thorsten; Hummel, Marlene; Böhler, Olga; Humpf, Hans-Ulrich

    2012-12-14

    Immunoblotting techniques are widely used for specific protein identifications and characterizations. The specific bindings of antibodies to epitopes in a protein sequence permits determination of antigens and gives detailed information about protein compositions and expression levels in complex suspensions. However the techniques are mostly restricted to one specific antibody determination. Overlaying proteins are detected using numerous repeated gel runs. For multiple but specific protein determinations on one immunoblot, here we describe the detection of several antigens by simultaneous incubation of antibodies originated from different species followed by sequential addition of secondary antibodies labelled with horseradish peroxidase (HRP) and binding to analogous primary antibodies. Particular signals were visualized step by step using a HRP chemiluminescence substrate while enzymatic HRP reactions were meanwhile inactivated irreversibly by hydrogen peroxide incubation. We demonstrate flexible applications of multiple antigen detections using the Western blotting technique with determination of the CNS protein markers neuron specific enolase, glial fibrillary acid protein and the physiological prion protein (PrP(C)) in brains and in meats as food contaminations and with glycotyping of PrP(C) using antibodies binding to different epitopes. We showed the use of the dot blotting technique with serial determination of different antigens in complex protein suspensions. The method is easy to handle and is flexible and applicable in the fields of diagnostics and public health for detection of overlaying proteins on one immunoblot. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Gel electrophoresis of polyphenol oxidase with instant identification by in situ blotting.

    PubMed

    Cheng, Tsai-Mu; Huang, Pei-Chen; Pan, Ju-Pin; Lin, Kuan-Yu; Mao, Simon J T

    2007-04-15

    Polyphenol oxidase (PPO) or tyrosinase is an important and ubiquitous enzyme responsible for browning in plants and melanization in animals. The molecular size of the plant PPO is varied among the species and its activity can be enhanced by a variety of anionic detergents. In the present study, we developed a simple method for the first-step identification of PPO in fruit and vegetable extracts. First, 3mm chromatographic paper was immersed in 0.5% (w/v) catechol solution as an immobilized PPO substrate. After running the extract with 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), one side of the glass plate was removed. The plate was immediately laid on top of the dried catechol-paper. A dark-brown band corresponding to PPO was visualized within 1 min and was further confirmed by a conventional Western blot using an antibody prepared against mushroom PPO. It also reveals that some vegetation (such as tomato, radish, and oriental melon) with low or no detectable activity in a conventional enzyme assay actually possessed marked levels of PPO activity when assessed by PAGE-blot. We propose that an inhibitor is associated with PPO in some plants; the inhibitor, however, is dissociated during the electrophoresis. Therefore, in addition to identify the molecular form of PPO, the present technique may explore the existence of PPO inhibitor(s) in plants. The detail of the method with respect to its relevance for searching a natural PPO inhibitor is described and discussed.

  5. An overview of technical considerations for Western blotting applications to physiological research.

    PubMed

    Bass, J J; Wilkinson, D J; Rankin, D; Phillips, B E; Szewczyk, N J; Smith, K; Atherton, P J

    2017-01-01

    The applications of Western/immunoblotting (WB) techniques have reached multiple layers of the scientific community and are now considered routine procedures in the field of physiology. This is none more so than in relation to skeletal muscle physiology (i.e., resolving the mechanisms underpinning adaptations to exercise). Indeed, the inclusion of WB data is now considered an essential aspect of many such physiological publications to provide mechanistic insight into regulatory processes. Despite this popularity, and due to the ubiquitous and relatively inexpensive availability of WB equipment, the quality of WB in publications and subsequent analysis and interpretation of the data can be variable, perhaps resulting in spurious conclusions. This may be due to poor laboratory technique and/or lack of comprehension of the critical steps involved in WB and what quality control procedures should be in place to ensure robust data generation. The present review aims to provide a detailed description and critique of WB procedures and technicalities, from sample collection through preparation, blotting and detection, to analysis of the data collected. We aim to provide the reader with improved expertise to critically conduct, evaluate, and troubleshoot the WB process, to produce reproducible and reliable blots.

  6. Capillary blotting of glycosaminoglycans on nitrocellulose membranes after agarose-gel electrophoresis separation.

    PubMed

    Volpi, Nicola; Maccari, Francesca

    2009-01-01

    A method for the blotting and immobilizing of several nonsulfated and sulfated complex polysaccharides on membranes made hydrophilic and positively charged by cationic detergent after their separation by conventional agarose gel electrophoresis is illustrated. This new approach to the study of glycosaminoglycans (GAGs) utilizes the capacity of agarose gel electrophoresis to separate single species of polysaccharides from mixtures and the membrane technology for further preparative and analytical uses.Nitrocellulose membranes are derivatized with the cationic detergent cetylpyridinium chloride and mixtures of GAGs are capillary blotted after their separation in agarose gel electrophoresis. Single purified species of variously sulfated polysaccharides are transferred on derivatized membranes with an efficiency of 100% and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining). This enables a lower amount limit of detection of 0.1 microg. Nonsulfated polyanions, for example hyaluronic acid, may also be transferred to membranes with a limit of detection of approximately 0.1-0.5 microg after irreversible or reversible staining. The membranes may be stained with reversible staining and the same lanes are used for immunological detection or other applications.

  7. More specific bands in the IgG western blot in sera from Scottish patients with suspected Lyme borreliosis.

    PubMed

    Evans, Roger; Mavin, Sally; McDonagh, Susan; Chatterton, Jean M W; Milner, Rachel; Ho-Yen, Darrel O

    2010-08-01

    To identify further Western blot bands that may be specific in the diagnosis of Lyme borreliosis. The Borrelia burgdorferi antibody profiles of 270 western blot positive patients and 241 western blot negative patients from 2008 were examined. 27 different non-specific bands were detected in both groups. Six of 27 (22%) of the non-specific bands were detected significantly more in the western blot positive patients compared to the western blot negative patients (20 kDa, p<0.0001; 28 kDa, p<0.002; 36 kDa, p<0.002; 37 kDa, p<0.007; 48 kDa, p<0.023; 56 kDa, p<0.028; two-tailed F test). Results suggest that the 20, 28 and 48 kDa bands should be regarded as specific.

  8. Highly specific confirmatory western blot test for African swine fever virus antibody detection using the recombinant virus protein p54.

    PubMed

    Alcaraz, C; Rodriguez, F; Oviedo, J M; Eiras, A; De Diego, M; Alonso, C; Escribano, J M

    1995-03-01

    A Western blot technique using a recombinant protein has been developed to confirm positive results obtained in African swine fever (ASF)-specific antibody detection by ELISA. The new confirmatory Western blot is based on the use of protein p54, one of the most antigenic ASF virus structural proteins, expressed in Escherichia coli fused to the N-terminus of MS2 polymerase. The recombinant Western blot assay was highly specific and equally sensitive for ASF virus-infected pigs detection as the conventional Western blot, which uses virus-induced proteins ranging in molecular weight between 23 and 35 kDa. The novel Western blot assay provides a simpler interpretation of the test, eliminates the possibility of false-positive reactions produced by cellular compounds that contaminate the antigen employed in the conventional technique, and avoids the use of live virus in antigen production.

  9. The diagnostic value of Western blot method in patients with cystic echinococcosis.

    PubMed

    Aslan, Mustafa; Yüksel, Pelin; Polat, Erdal; Cakan, Huseyin; Ergin, Sevgi; Öner, Y Ali; Zengin, Kagan; Arıkan, Soykan; Saribas, Suat; Torun, Muzeyyen Mamal; Kocazeybek, Bekir

    2011-04-01

    Cystic echinococcosis (CE) is the larval cystic stage (called echinococcal cysts) of a small taeniid-type tapeworm (Echinococcus granulosus). Carnivores such as dogs are usually definitive hosts. Intermediate hosts are typically herbivores such as sheep and cattle. CE can be detected using various imaging techniques such as ultrasonography or radiology. Moreover the primary diagnosis has to be confirmed by serological tests since the clinical signs of the disease are non-specific. This study examined the antigenic band patterns useful for serologic diagnosis of hydatidosis. We also report on the post-operative evolution of patients treated for this disease and also determined the diagnostic performance of Western blot IgG kit. Twenty-five (16 females and 9 males) non-operated patients with hydatid cysts (NOP) and 33 (21 females and 12 males) operated patients with hydatid cysts (OP) were included as study group and 22 healthy individuals (14 females and 8 males) with no known chronic diseases were included as a control group. The ages of the patients and control group individuals were between 16-83 years. Patient and control groups were matched for age and sex. Cyst hydatid IgG antibodies were detected in the sera from all patient groups but no antibodies were found in the sera from the control group using ELISA IgG method. Twenty-three (92%) non-operated patients and 18 (54.5%) operated patients exhibited positive results when Western blot IgG kit was used. The P7 band pattern was detected in the sera from all operated and non-operated patients. Twenty-seven of these positive cases had p7 and (p7+p16/18), (p7+p24/26) or (p7+p16/18+p24/26). No antibodies against p7, p16/18 ve p24/26 band patterns were seen in sera from the control group A statistically significant difference was detected between operated and nonoperated patients for Western blot positivity.(p<0.01). p: 0.018- X2=5,604- OR: 0.176- 95% CI: 0.037- 0.841. The sensitivity, specificity, positive

  10. Application of a Dot Blot Hybridization Platform to Assess Streptococcus uberis Population Structure in Dairy Herds

    PubMed Central

    Albuquerque, Pedro; Ribeiro, Niza; Almeida, Alexandre; Panschin, Irena; Porfirio, Afonso; Vales, Marta; Diniz, Francisca; Madeira, Helena; Tavares, Fernando

    2017-01-01

    Streptococcus uberis is considered one of the most important pathogens associated with bovine mastitis. While traditionally acknowledged as an environmental pathogen, S. uberis has been shown to adopt a contagious epidemiological pattern in several dairy herds. Since different control strategies are employed depending on the mode of transmission, in-depth studies of S. uberis populations are essential to determine the best practices to control this pathogen. In this work, we optimized and validated a dot blot platform, combined with automatic image analysis, to rapidly assess the population structure of infective S. uberis, and evaluated its efficiency when compared to multilocus sequence analysis (MLSA) genotyping. Two dairy herds with prevalent S. uberis infections were followed in a 6 month period, in order to collect and characterize isolates from cows with persistent infections. These herds, located in Portugal (Barcelos and Maia regions), had similar management practices, with the herd from Barcelos being smaller and having a better milking parlor management, since infected cow segregation was immediate. A total of 54 S. uberis isolates were obtained from 24 different cows from the two herds. To overcome operator-dependent analysis of the dot blots and increase the technique's consistency and reliability, the hybridization signals were converted into probability values, with average probabilities higher than 0.5 being considered positive results. These data allowed to confirm the isolates' identity as S. uberis using taxa-specific markers and to determine the presence of virulence- and antibiotic resistance-related genes. In addition, MLSA allowed to disclose the most prevalent S. uberis clonal lineages in both herds. Seven different clusters were identified, with Barcelos showing a high clonal diversity and Maia a dominant lineage infecting most cows, suggesting distinct epidemiological patterns, with S. uberis displaying an environmental or contagious

  11. Quantitation of proteoglycans as glycosaminoglycans in biological fluids using an alcian blue dot blot analysis.

    PubMed

    Björnsson, S

    1998-02-15

    A method for quantitation of intact proteoglycans as GAGs in biological fluids (blood plasma, synovial fluid) or 4 M guanidine extracts of tissues has been published previously (S. Björnsson, Anal. Biochem. 210, 282-291, 1993). The method is based on the specific interaction between sulfated polymers and the tetravalent cationic dye Alcian blue at pH 1.5 in 0.4 M guanidine-HCl and in the presence of 0.25% Triton. The absorbance assay has a measuring range of 1-20 microgram of glycosaminoglycan (GAG) which is not sensitive enough to measure the low contents of proteoglycans in blood plasma, urine, or wound fluid. A dot blot assay is now described in which the Alcian blue-GAG complexes are collected on a polyvinylidene fluoride membrane, by filtration in a dot blot apparatus, and the stain is quantitated as reflectance by scanning and densitometry. The assay requires 10 microliter of sample and has a measuring range of 10-800 ng of GAG, corresponding to a concentration of 1-80 mg/liter, suitable for proteoglycans in biological fluids. The procedures for chemistry, scanning, densitometry, and curve fitting were each evaluated separately. The error contributed by chemistry accounted for a minor portion of the imprecision. The imprecision contributed by scanning was the most important source of within-run and between-run imprecision, and was caused by inequalities of the charge-coupled device along the scanning arm. Unexpectedly, curve fitting was also a major source of total imprecision in dot blot quantitation and differed with the type of equation used. The between-run imprecision calculated as CV (SD/mean . 100) was 13.0% at 8 mg/liter. The response of the assay was identical for six different commercial preparations of GAGs (chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, and heparin) despite differences in degree of sulfation known to exist. There was no positive or negative interference by blood plasma, apart

  12. Profiling protein expression in circulating tumour cells using microfluidic western blotting

    PubMed Central

    Sinkala, Elly; Sollier-Christen, Elodie; Renier, Corinne; Rosàs-Canyelles, Elisabet; Che, James; Heirich, Kyra; Duncombe, Todd A.; Vlassakis, Julea; Yamauchi, Kevin A.; Huang, Haiyan; Jeffrey, Stefanie S.; Herr, Amy E.

    2017-01-01

    Circulating tumour cells (CTCs) are rare tumour cells found in the circulatory system of certain cancer patients. The clinical and functional significance of CTCs is still under investigation. Protein profiling of CTCs would complement the recent advances in enumeration, transcriptomic and genomic characterization of these rare cells and help define their characteristics. Here we describe a microfluidic western blot for an eight-plex protein panel for individual CTCs derived from estrogen receptor-positive (ER+) breast cancer patients. The precision handling and analysis reveals a capacity to assay sparingly available patient-derived CTCs, a biophysical CTC phenotype more lysis-resistant than breast cancer cell lines, a capacity to report protein expression on a per CTC basis and two statistically distinct GAPDH subpopulations within the patient-derived CTCs. Targeted single-CTC proteomics with the capacity for archivable, multiplexed protein analysis offers a unique, complementary taxonomy for understanding CTC biology and ascertaining clinical impact. PMID:28332571

  13. Detection of multiple cystic fibrosis mutations by reverse dot blot hybridization: a technology for carrier screening.

    PubMed

    Chehab, F F; Wall, J

    1992-05-01

    We describe the implementation of a modified version of the reverse dot blot hybridization technology to detect eight cystic fibrosis mutations. The method is simple, quick, reliable, inexpensive, and nonradioactive and utilizes the sensitivity of the polymerase chain reaction coupled with colored or chemiluminescent substrates for mutation detection. We have used this system in a clinical laboratory to identify the delta F508, G542X, G551D, R553X, 621 + 1G----T, W1282X, N1303K, and 1717G----A mutations. The technique is practical for genotyping individuals at many potential mutation sites, as in cystic fibrosis and beta-thalassemia, in which over 95 mutations can cause disease. This technology appears to be the method of choice for the widespread carrier screening of multiple cystic fibrosis mutations.

  14. Detection of Hypoderma actaeon infestation in Cervus elaphus with ELISA and western blotting.

    PubMed

    Domínguez, Julia; Panadero, Rosario; de la Fuente-López, Concepción

    2010-07-01

    We used enzyme-linked immunosorbent assay (ELISA) and western blotting (WB) to evaluate the reactivity of first-stage larval extracts of Hypoderma lineatum with antibodies in sera from 76 red deer from an endemic area for Hypoderma actaeon. Antibodies in sera from deer infested with H. actaeon recognized hypodermin C from H. lineatum in both ELISA and WB assays. ELISA values were correlated with the epidemiology of the fly infestation in the area where the deer sera were collected. There was no clear relationship between anti-hypodermin C antibody levels and the age of the animals or the number of Hypoderma grubs found at necropsy in the hides of the deer. Our results suggest that first-instar antigens of H. lineatum can be used to diagnose natural infestations by H. actaeon by indicating the presence of first-stage larvae, which can help to accurately describe H. actaeon epidemiology.

  15. Reexamination of alcohol dehydrogenase structural mutants in Drosophila using protein blotting

    SciTech Connect

    Hollocher, H.; Place, A.R.

    1987-06-01

    Using protein blotting and an immuno-overlay procedure, the authors have reexamined the cross-reacting material produced by ADH null-activity mutants generated with ethyl methanesulfonate (EMS). Of the 13 mutants, 11 have an immunodetectable polypeptide of wild-type size. The native and urea denatured isoelectric points (pI) establish that 7 of 13 of the mutations have no effect on protein charge. The electrophoretic mobilities of each variant on increasing percent acrylamide gels (Ferguson analysis), reveal that 9 of the 11 immunodetectable mutations have retained the ability form dimers under native conditions. None of the inactive mutant proteins has the ability to form the adduct-bound isozyme. The authors have found no correlation between protein pI and i vivo stability. The observed frequencies of specific charge class alterations do not dispute the propensity of G:A transitions previously found for EMS mutagenesis.

  16. Western blot analysis of Src kinase assays using peptide substrates ligated to a carrier protein.

    PubMed

    Xu, Jie; Sun, Luo; Ghosh, Inca; Xu, Ming-Qun

    2004-06-01

    We have applied intein-mediated peptide ligation (IPL) to the use of peptide substrates for kinase assays and subsequent Western blot analysis. IPL allows for the efficient ligation of a synthetic peptide with an N-terminal cysteine residue to an intein-generated carrier protein containing a cysteine reactive C-terminal thioester through a native peptide bond. A distinct advantage of this procedure is that each carrier protein molecule ligates only one peptide, ensuring that the ligation product forms a sharp band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We demonstrate the effectiveness of this approach by mutational analysis of peptide substrates derived from human cyclin-dependent kinase, Cdc2, which contains a phosphorylation site of human c-Src protein tyrosine kinase.

  17. Direct identification and characterization of llama (Lama glama L.) whey proteins by microsequencing after western blotting.

    PubMed

    Cantisani, A; Napolitano, L; Giuffrida, M G; Conti, A

    1990-01-01

    Amino acid sequence determination is the most reliable and powerful tool to identify a protein or to classify a new one by comparison of its primary structure with already known sequences. A rapid and simple purification procedure is an essential pre-requisite for routine sequence determination. Structural characterization of llama whey proteins was undertaken for evolutionary as well as economic purposes. N-terminal sequence analyses directly on an immobilon polyvinylidene difluoride (PVDF) membrane, following Western blotting of both native and SDS-denatured llama whey proteins after polyacrylamide gel electrophoresis, revealed three different forms of glycosylated alpha-lactalbumin, and a protein with a high degree of homology with a camel whey protein of unknown function. Furthermore, by immunoblotting techniques, the electrophoretic band corresponding to serum albumin was identified.

  18. Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein.

    PubMed

    Yang, Zhaoshou; Lee, Jihoo; Ahn, Hye-Jin; Chong, Chom-Kyu; Dias, Ronaldo F; Nam, Ho-Woo

    2016-04-01

    Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection.

  19. Western blot assay for quantitative and qualitative antigen detection in vaccine development.

    PubMed

    Kumar, Sanjai; Zheng, Hong; Mahajan, Babita; Kozakai, Yukiko; Morin, Merribeth; Locke, Emily

    2014-05-01

    Immunological methods for quantitative measurement, antigenic characterization, and monitoring the stability of active immunogenic component(s) are a critical need in the vaccine development process. This unit describes an enhanced chemiluminescence-based western blot for quantitative detection of Plasmodium falciparum circumsporozoite protein (PfCSP), a major malaria candidate vaccine antigen. The most salient features of this assay are its high sensitivity and reproducibility; it can reliably detect ∼5 to 10 pg PfCSP expressed on native parasites or recombinantly expressed in Escherichia coli. Although described for a specific vaccine antigen, this assay should be applicable for any antigen-antibody combination for which relevant detection reagents are available. Detailed stepwise experimental procedures and methods for data acquisition and analysis are described. Copyright © 2014 John Wiley & Sons, Inc.

  20. Reverse line blot hybridization used to identify hemoprotozoa in Minorcan cattle.

    PubMed

    Almeria, Sonia; Castella, Joaquim; Ferrer, David; Gutierrez, Juan Francisco; Estrada-Pena, Agustin; Sparagano, Olivier

    2002-10-01

    Piroplasmosis, a tick-borne protozoal disease, is an important disease affecting domestic and wild animals. We performed PCR-based reverse line blot hybridization (RLB) assays on blood samples obtained from 133 cattle exposed to ticks in field conditions in Minorca (Balearic Islands, Spain) in three different seasons. The oligonucleotides used were those for Theileria annulata, T. buffeli, T. taurotragi, T. velifera, Babesia bigemina, B. bovis, B. divergens, and B. major. The RLB technique allowed the simultaneous identification of T. annulata, T. buffeli, B. bigemina, and B. bovis as the piroplasms present in cattle in Minorca. Of the 133 animals, only 4 were not infected by any of the studied parasites. The results indicated endemic piroplasm infection in cattle in Minorca; especially important was the presence of T. annulata. The RLB was highly sensitive and allowed the simultaneous detection and identification of the Theileria and Babesia species in carrier cattle, which cannot be achieved by classical identification methods.

  1. Recombinant Dense Granular Protein (GRA5) for Detection of Human Toxoplasmosis by Western Blot

    PubMed Central

    Ching, Xiao Teng; Lau, Yee Ling; Fong, Mun Yik; Nissapatorn, Veeranoot; Andiappan, Hemah

    2014-01-01

    Toxoplasma gondii infects all warm-blooded animals, including humans, causing serious public health problems and great economic loss for the food industry. Commonly used serological tests require costly and hazardous preparation of whole Toxoplasma lysate antigens from tachyzoites. Here, we have evaluated an alternative method for antigen production, which involved a prokaryotic expression system. Specifically, we expressed T. gondii dense granular protein-5 (GRA5) in Escherichia coli and isolated it by affinity purification. The serodiagnostic potential of the purified recombinant GRA5 (rGRA5) was tested through Western blot analysis against 212 human patient serum samples. We found that rGRA5 protein was 100% specific for analysis of toxoplasmosis-negative human sera. Also, rGRA5 was able to detect acute and chronic T. gondii infections (sensitivities of 46.8% and 61.2%, resp.). PMID:24987700

  2. Restoration of antibody binding to blotted meningococcal outer membrane proteins using various detergents.

    PubMed

    Wedege, E; Bryn, K; Frøholm, L O

    1988-10-04

    Restoration of IgG antibody binding to heat-denatured meningococcal outer membrane proteins has been studied on immunoblots with a series of 14 detergents. Nitrocellulose strips with the blotted proteins were incubated with the detergents and sera from human volunteers vaccinated with meningococcal membrane proteins. Zwitterionic and ionic detergents, containing substituted quarternary ammonium or amino groups with a minimum of 10 C atoms in the alkyl chain, restored the antigenicity of the serotype-specific class 2 porin protein. The concentrations of the Zwittergent detergents necessary for activation decreased with increasing alkyl chain length of the homologues. Only zwitterionic detergents renatured the class 1 protein. Both proteins were weakly antigenic in the presence of the nonionic detergents Triton X-100 and Tween 20. Meningococcal lipopolysaccharide restored antibody binding to the porin, but not to the class 1 protein. Similar concentrations of lipopolysaccharides from two other gram-negative bacteria had no effect.

  3. Contribution of a Comparative Western Blot Method to Early Postnatal Diagnosis of Congenital Syphilis

    PubMed Central

    Foschi, Claudio; Capretti, Maria Grazia; Nardini, Paola; Compri, Monica; Corvaglia, Luigi Tommaso; Faldella, Giacomo; Cevenini, Roberto

    2016-01-01

    Serology has a pivotal role in the diagnosis of congenital syphilis (CS), but problems arise because of the passive transfer of IgG antibodies across the placenta. The aim of this study was to assess the diagnostic value of a comparative Western blot (WB) method finalized to match the IgG immunological profiles of mothers and their own babies at birth in order to differentiate between passively transmitted maternal antibodies and antibodies synthesized by the infants against Treponema pallidum. Thirty infants born to mothers with unknown or inadequate treatment for syphilis were entered in a retrospective study, conducted at St. Orsola-Malpighi Hospital, Bologna, Italy. All of the infants underwent clinical, instrumental, and laboratory examinations, including IgM WB testing. For the retrospective study, an IgG WB assay was performed by blotting T. pallidum antigens onto nitrocellulose sheets and incubating the strips with serum specimens from mother-child pairs. CS was diagnosed in 11 out of the 30 enrolled infants; 9/11 cases received the definitive diagnosis within the first week of life, whereas the remaining two were diagnosed later because of increasing serological test titers. The use of the comparative IgG WB testing performed with serum samples from mother-child pairs allowed a correct CS diagnosis in 10/11 cases. The CS diagnosis was improved by a strategy combining comparative IgG WB results with IgM WB results, leading to a sensitivity of 100%. The comparative IgG WB test is thus a welcome addition to the conventional laboratory methods used for CS diagnosis, allowing identification and adequate treatment of infected infants and avoiding unnecessary therapy of uninfected newborns. PMID:26961856

  4. Rapid detection and differentiation of important Campylobacter spp. in poultry samples by dot blot and PCR.

    PubMed

    Fontanot, Marco; Iacumin, Lucilla; Cecchini, Francesca; Comi, Giuseppe; Manzano, Marisa

    2014-10-01

    The detection of Campylobacter, the most commonly reported cause of foodborne gastroenteritis in the European Union, is very important for human health. The most commonly recognised risk factor for infection is the handling and/or consumption of undercooked poultry meat. The methods typically applied to evaluate the presence/absence of Campylobacter in food samples are direct plating and/or enrichment culture based on the Horizontal Method for Detection and Enumeration of Campylobacter spp. (ISO 10272-1B: 2006) and PCR. Molecular methods also allow for the detection of cells that are viable but cannot be cultivated on agar media and that decrease the time required for species identification. The current study proposes the use of two molecular methods for species identification: dot blot and PCR. The dot blot method had a sensitivity of 25 ng for detection of DNA extracted from a pure culture using a digoxigenin-labelled probe for hybridisation; the target DNA was extracted from the enrichment broth at 24 h. PCR was performed using a pair of sensitive and specific primers for the detection of Campylobacter jejuni and Campylobacter coli after 24 h of enrichment in Preston broth. The initial samples were contaminated by 5 × 10 C. jejuni cells/g and 1.5 × 10(2)C. coli cells/g, thus the number of cells present in the enrichment broth at 0 h was 1 or 3 cell/g, respectively.

  5. Evaluation of the Western blotting method for the diagnosis of congenital toxoplasmosis.

    PubMed

    Capobiango, Jaqueline Dario; Monica, Thaís Cabral; Ferreira, Fernanda Pinto; Mitsuka-Breganó, Regina; Navarro, Italmar Teodorico; Garcia, João Luis; Reiche, Edna Maria Vissoci

    To evaluate the Western blotting method for the detection of IgG anti-Toxoplasma gondii (T. gondii) (IgG-WB) in the serum of children with suspected congenital toxoplasmosis. We accompanied 47 mothers with acquired toxoplasmosis in pregnancy and their children, between June of 2011 and June of 2014. The IgG-WB was done in house and the test was considered positive if the child had antibodies that recognized at least one band on IgG blots different from the mother's or with greater intensity than the corresponding maternal band, during the first three months of life. 15 children (15.1%) met the criteria for congenital toxoplasmosis and 32 (32.3%) had the diagnosis excluded. The symptoms were observed in 12 (80.0%) children and the most frequent were cerebral calcification in 9 (60.0%), chorioretinitis in 8 (53.3%), and hydrocephalus in 4 (26.6%). IgM antibodies anti-T. gondii detected by chemiluminescence (CL) were found in 6 (40.0%) children and the polymerase chain reaction (PCR) for detection of T. gondii DNA was positive in 5 of 7 performed (71.4%). The sensitivity of IgG-WB was of 60.0% [95% confidence interval (CI) 32.3-83.7%] and specificity 43.7% (95% CI 26.7-62.3%). The sensitivity of IgG-WB increased to 76.0 and 89.1% when associated to the research of IgM anti-T. gondii or PCR, respectively. The IgG-WB showed greater sensitivity than the detection of IgM anti-T. gondii; therefore, it can be used for the diagnosis of congenital toxoplasmosis in association with other congenital infection markers. Copyright © 2016 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

  6. Comparative evaluation of western blotting in hepatic and pulmonary cystic echinococcosis.

    PubMed

    Akisu, C; Delibas, S B; Bicmen, C; Ozkoc, S; Aksoy, U; Turgay, N

    2006-12-01

    Many serological tests are widely used in the diagnosis of cystic echinococcosis (CE), caused by the larval stages of Echinococcus granulosus. The present study was carried for differentiation between hepatic and pulmonary cystic echinococcosis by Western Blotting (WB). A total of 121 sera from patients with hepatic CE (37), pulmonary CE (31) and controls (53; consisting of six healthy, seven Hymenolepis nana infection, 20 hepatic and 20 pulmonary diseases other than CE) were examined. In all of the CE patients, E. gronulosus infection was confirmed by surgical intervention. Sera were previously tested using IHA and ELISA to detect the E. gronulosus specific antibodies. Sera from hepatic cases of CE reacted with 16 polypeptides of 6-116 kDa and sera from pulmonary cases of CE reacted with 14 polypeptides of 4-130 kDa by Western Blotting. The WB test enabled the detection of antibodies in the hepatic CE samples for proteins of 24, 32 34, 44-46 and 52-54 kDa in molecular weight in 78.4%, 75.7%, 78.4% and 89.2% of the patients, respectively. In the pulmonary CE samples sera WB test enabled the detection of antibodies 24, 44-46, 100, 110, 116 and 120 124 kDa in molecular weight in 81.3%, 75.0%, 87.5%, 71.9%, 84.4% and 65.6% of the patients, respectively. We indicated that the antigenic components of high molecular weight can be good candidates for differentiation of hepatic CE from pulmonary CE.

  7. Contribution of a Comparative Western Blot Method to Early Postnatal Diagnosis of Congenital Syphilis.

    PubMed

    Marangoni, Antonella; Foschi, Claudio; Capretti, Maria Grazia; Nardini, Paola; Compri, Monica; Corvaglia, Luigi Tommaso; Faldella, Giacomo; Cevenini, Roberto

    2016-05-01

    Serology has a pivotal role in the diagnosis of congenital syphilis (CS), but problems arise because of the passive transfer of IgG antibodies across the placenta. The aim of this study was to assess the diagnostic value of a comparative Western blot (WB) method finalized to match the IgG immunological profiles of mothers and their own babies at birth in order to differentiate between passively transmitted maternal antibodies and antibodies synthesized by the infants against Treponema pallidum Thirty infants born to mothers with unknown or inadequate treatment for syphilis were entered in a retrospective study, conducted at St. Orsola-Malpighi Hospital, Bologna, Italy. All of the infants underwent clinical, instrumental, and laboratory examinations, including IgM WB testing. For the retrospective study, an IgG WB assay was performed by blotting T. pallidum antigens onto nitrocellulose sheets and incubating the strips with serum specimens from mother-child pairs. CS was diagnosed in 11 out of the 30 enrolled infants; 9/11 cases received the definitive diagnosis within the first week of life, whereas the remaining two were diagnosed later because of increasing serological test titers. The use of the comparative IgG WB testing performed with serum samples from mother-child pairs allowed a correct CS diagnosis in 10/11 cases. The CS diagnosis was improved by a strategy combining comparative IgG WB results with IgM WB results, leading to a sensitivity of 100%. The comparative IgG WB test is thus a welcome addition to the conventional laboratory methods used for CS diagnosis, allowing identification and adequate treatment of infected infants and avoiding unnecessary therapy of uninfected newborns. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  8. TSE strain differentiation in mice by immunohistochemical PrP(Sc) profiles and triplex Western blot.

    PubMed

    van Keulen, Lucien J M; Langeveld, Jan P M; Dolstra, Corry H; Jacobs, Jorg; Bossers, Alex; van Zijderveld, Fred G

    2015-10-01

    TSE strains are routinely identified by their incubation period and vacuolation profile in the brain after intracerebral inoculation and serial passaging in inbred mouse lines. There are some major drawbacks to this method that are related to the variation in vacuolation that exists in the brains of mice infected with the same TSE strain and to variation between observers and laboratories in scoring vacuolation and determining the final incubation period. We investigated the potential of PrP(Sc) immunohistochemistry and triplex Western blotting as possible alternative methods to differentiate between TSE strains. TSE reference strains ME7, 87A/87V, 22A/22C, 79A/79V and 301C/301V were intracerebrally inoculated in RIII or VM inbred mice that differ in their PrP genotype. Immunohistochemical PrP(Sc) profiles were drawn up by scanning light microscopy both on coronal and sagittal sections. On the basis of the localization of PrP(Sc) in the cerebral cortex, hippocampus, and cerebellar cortex and the overall type of PrP(Sc) staining, all TSE strains could be well differentiated from each other through their typical strain dependent characteristics. In addition, Western blot showed that the combination of glycosylation profile and 12B2 epitope content of PrP(Sc) allowed to distinguish between all reference strains except for ME7 and 22A in VM mice. TSE strains in mice can be identified on the basis of their PrP(Sc) profile alone. The potential to identify TSE strains in ruminants with these PrP(Sc) profiles after a single primary passage in mice will be the topic of future studies. © 2014 British Neuropathological Society.

  9. Optimization of dot blot method to detect bcr/abl transcripts in chronic myeloid leukemia

    SciTech Connect

    Tharapel, S.A.; Zhao, J.

    1994-09-01

    Detection of abl-bcr fusion transcripts using molecular methodologies is becoming an attractive alternative (or supplement) to traditional cytogenetics in identifying the Philadelphia (Ph) chromosome. Among these methods, RT-PCR technique has provided an extremely powerful tool for improving the detection of bcr/abl translocations through enzymatic amplification of the reverse-transcribed cDNA. The analysis of PCR products can be accomplished by a number of techniques including dot blot following liquid-phase hybridization. In order to render the detection of PCR products more simple, accurate and efficient, and therefore more amenable for the clinical laboratory routine use, we optimized several parameters of the procedure. (1) We discovered that with the starting material of 1 ug of total RNA, the amount of the final PCR amplified products was linear to the PCR cycles between 20 to 30 cycles. Since the dot blot procedure does not separate the amplified products according to their sizes, increased background would increase the false positive rate. (2) If a detection sensitivity of 1 in 10{sup 3} cells is sufficient, then the nested or a second PCR amplification is not necessary. (3) Starting material more than 5 ug of total RNA would decrease the amplification efficiency and therefore compromise the sensitivity. (4) Ten minutes of hybridization gave equal signal intensity as 24 hours. (5) The ionic strength and temperature in the washing step were also tested. Upon optimization of each parameter, the detection procedure was tested on 18 clinical samples. Compared to the procedures that are currently available, our optimized procedure is less time consuming, has higher sensitivity and lower false positive rate. This method has the potential to be automated and therefore can be used as a screening method for Ph chromosome in high volume settings.

  10. Rapid diagnosis of rabies in humans and animals by a dot blot enzyme immunoassay.

    PubMed

    Madhusudana, Shampur Narayan; Paul, Joel Prem Vasanth; Abhilash, Venugopal Karavattu; Suja, Mooriyath Sukumaran

    2004-11-01

    The presently advocated tests for rapid diagnosis of rabies, such as the fluorescent antibody test (FAT) are expensive and require expertise to carry out and interpret the results. In this study, a simple direct dot blot enzyme immunoassay (DIA) has been developed and evaluated to detect the rabies antigen in brain specimens of animals and humans. The utility of this test in the ante-mortem diagnosis of human rabies has also been evaluated. Brain homogenates of suspected rabid animals (n = 250), humans (n = 16) and clinical samples like saliva (n = 12) and cerebrospinal fluid (CSF) (n = 12) were directly spotted on polyvinylidene difluoride membrane (PVDF) and the absorbed rabies nucleoprotein antigen was detected using biotinylated antinucleoprotein antibody followed by treatment with streptavidin peroxidase conjugate and color development with diamino benzedine (DAB). Rabies-infected and normal mouse brain homogenates were used as positive and negative controls, respectively. The results of this test were evaluated with fluorescent antibody technique (for brain samples) and mouse inoculation test (for saliva and CSF samples). A distinct dark brown color was seen in the positive control and all positive samples, while there was no color development with either the negative control or the negative samples. The concordance between the fluorescent antibody test (FAT) and dot immunoassay was 98.4% for brain samples, 83.3% for saliva and 91.6% for CSF samples. The specificity of the test was found to be 100%. The dot blot enzyme immunoassay (DIA) test described here is a sensitive, specific and rapid test for the post-mortem diagnosis of rabies in animals and humans. The utility of this test for the ante-mortem diagnosis of rabies needs to be further evaluated.

  11. Western blot diagnosis of vivax malaria with multiple stage-specific antigens of the parasite

    PubMed Central

    Son, Eui-Sun; Kim, Tong Soo

    2001-01-01

    Western blot analysis was performed to diagnose vivax malaria using stage-specific recombinant antigens. Genomic DNA from the whole blood of a malaria patient was used as templates to amplify the coding regions for the antigenic domains of circumsporozoite protein (CSP-1), merozoite surface protein (MSP-1), apical merozoite antigen (AMA-1), serine repeat antigen (SERA), and exported antigen (EXP-1) of Plasmodium vivax. Each amplified DNA fragment was inserted into a pGEX-4T plasmid to induce the expression of GST fusion protein in Escherichia coli by IPTG. The bacterial cell extracts were separated on 10% SDS-PAGE followed by western blot analysis with patient sera which was confirmed by blood smear examination. When applied with patient sera, 147 (91.9%) out of 160 vivax malaria, 12 (92.3%) out of 13 falciparum malaria, and all 9 vivax/falciparum mixed malaria reacted with at least one antigen, while no reactions occurred with 20 normal uninfected sera. In the case of vivax malaria, CSP-1 reacted with 128 (80.0%) sera, MSP-1 with 102 (63.8%), AMA-1 with 128 (80.0%), SERA with 115 (71.9%), and EXP-1 with 89 (55.6%), respectively. We obtained higher detection rates when using 5 antigens (91.9%) rather than using each antigen solely (55.6-80%), a combination of 2 (76.3-87.5%), 3 (85.6-90.6%), or 4 antigens (89.4-91.3%). This method can be applied to serological diagnosis, mass screening in endemic regions, or safety test in transfusion of prevalent vivax malaria. PMID:11441504

  12. Western blot diagnosis of vivax malaria with multiple stage-specific antigens of the parasite.

    PubMed

    Son, E S; Kim, T S; Nam, H W

    2001-06-01

    Western blot analysis was performed to diagnose vivax malaria using stage-specific recombinant antigens. Genomic DNA from the whole blood of a malaria patient was used as templates to amplify the coding regions for the antigenic domains of circumsporozoite protein (CSP-1), merozoite surface protein (MSP-1), apical merozoite antigen (AMA-1), serine repeat antigen (SERA), and exported antigen (EXP-1) of Plasmodium vivax. Each amplified DNA fragment was inserted into a pGEX-4T plasmid to induce the expression of GST fusion protein in Escherichia coli by IPTG. The bacterial cell extracts were separated on 10% SDS-PAGE followed by western blot analysis with patient sera which was confirmed by blood smear examination. When applied with patient sera, 147 (91.9%) out of 160 vivax malaria, 12 (92.3%) out of 13 falciparum malaria, and all 9 vivax/falciparum mixed malaria reacted with at least one antigen, while no reactions occurred with 20 normal uninfected sera. In the case of vivax malaria, CSP-1 reacted with 128 (80.0%) sera, MSP-1 with 102 (63.8%), AMA-1 with 128 (80.0%), SERA with 115 (71.9%), and EXP-1 with 89 (55.6%), respectively. We obtained higher detection rates when using 5 antigens (91.9%) rather than using each antigen solely (55.6-80%), a combination of 2 (76.3-87.5%), 3 (85.6-90.6%), or 4 antigens (89.4-91.3%). This method can be applied to serological diagnosis, mass screening in endemic regions, or safety test in transfusion of prevalent vivax malaria.

  13. Development of a monoclonal antibody-based colony blot immunoassay for detection of thermotolerant Campylobacter species.

    PubMed

    Huang, Hongsheng; Phipps-Todd, Beverley; McMahon, Tanis; Elmgren, Catherine L; Lutze-Wallace, Cheryl; Todd, Zoe A; Garcia, Manuel M

    2016-11-01

    Campylobacter species, particularly thermotolerant Campylobacter spp., such as C. jejuni, are major human foodborne pathogens. Culture methods have been routinely used for the detection of this organism in various types of samples. An alternative, simple and rapid confirmation test(s) without further tedious biochemical tests would be useful. Meanwhile, Campylobacter-like colonies can be difficult to identify on agar plates overgrown with competitive bacteria, which can lead to false-negative results. This study was to develop a simple colony blot immunoassay using a new monoclonal antibody (Mab) produced in the present study for rapid screening, confirmation and quantification of campylobacters on culture agar plates. The procedure developed in this study was able to specifically detect thermotolerant Campylobacter spp., but not other non-thermotolerant Campylobacter and non-Campylobacter reference strains tested. This assay could detect 10(5) cells in a single dot. This assay showed 100% correlation with the culture method for the blotted membranes from 21 either chicken meat or vegetable samples experimentally inoculated with thermotolerant campylobacters. Among 101 natural samples of chicken meat (n=44), chicken feces (n=20) and vegetables (n=37), this assay also showed positive for 23 chicken meat and 14 fecal samples that were positive for thermotolerant campylobacters by culture method, and identified four additional suspects that were culture negative. Membranes stored at 4°C for at least 4years could also be used for this assay. The assay developed in this study can be used in quantitative study for immediate or archival usage, and for diagnostic test to preliminarily confirm the presence of thermotolerant Campylobacter on agar plates. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

  14. HIV‑2 antibody detection after indeterminate or negative HIV‑1 Western blot in Cuba, 2005-2008.

    PubMed

    Díaz, Dervel F; Ortiz, Eva; Martín, Dayamí; Nibot, Carmen; Rizo, Adis; Silva, Eladio

    2012-01-01

    INTRODUCTION Differentiating between HIV-1 and HIV-2 infection is the first step to understanding HIV transmission, epidemiology and pathogenesis in geographical areas where both viruses circulate. In Cuba, positive results in mixed HIV-1/2 screening assays are confirmed by HIV-1 Western blot. Indeterminate results constitute the main limitation of this test and HIV-2 infection is among their possible causes; hence the importance of second-stage screening and confirmatory tests for HIV-2 infection. OBJECTIVE Investigate the contribution of HIV-2 antibodies to negative or indeterminate HIV-1 Western blot results in serum samples from 2005 through 2008 in Cuba. METHODS HIV-2 reactivity was studied using the ELISA DAVIH-VIH-2 diagnostic kit (Cuba) in 1723 serum samples with negative or indeterminate results for HIV-1 Western blot from January 2005 through December 2008. Duplicate sera reactive by ELISA were confirmed by HIV-2 Western blot, results interpreted according to WHO criteria. The epidemiological interview established by Cuba's National Program for Prevention and Control Sexually-Transmitted Diseases and HIV/AIDS was applied to HIV-2 Western blot-positive patients. RESULTS Among all sera studied, HIV-2 ELISA identified 12 reactive serum samples (0.70%) and 1711 non-reactive (99.30%). Western blot analysis of the 12 ELISA-reactive samples confirmed two positive samples (16.67%), 4 negative (33.33%) and 6 indeterminate (50%). Positive samples reacted against the p16, p26, gp36, p53, p56, p68 and gp105 proteins. All 12 ELISA-reactive samples belonged to the HIV-1 Western blot indeterminate group. The two HIV-2-positive samples showed well defined reactivity to gp160, p53, p55 and p34 of HIV-1. HIV-1 seroconversion was observed in all 10 remaining samples during serological followup. CONCLUSIONS Two new HIV-2 seropositive cases were diagnosed using DAVIH-VIH-2 and HIV-2 Western blot in indeterminate HIV-1 Western blot samples. Results support the recommendation

  15. Double-blotting: a solution to the problem of nonspecific binding of secondary antibodies in immunoblotting procedures.

    PubMed

    Lasne, Françoise

    2009-01-01

    Nonspecific interactions between blotted proteins and unrelated secondary antibodies generate false positives in immunoblotting techniques. Some procedures have been developed to reduce this adsorption but they may work in specific applications and be ineffective in other ones. "Double-blotting" has been developed to overcome this problem. It consists of interpolating a second blotting step between the usual probings of the blot membrane with the primary antibody and the secondary antibodies. This step, by isolating the primary antibody from the interfering proteins, guarantees the specificity of the probing with the secondary antibody. This method has been developed for the study of erythropoietin in concentrated urine since a strong nonspecific binding of biotinylated secondary antibodies to some urinary proteins is observed using classical immunoblotting protocols. However, its concept makes it usable in other applications that come up against this kind of problem. This method is expected to be especially useful for investigating proteins that are present in minute amounts in complex biological media.

  16. Double-blotting: a solution to the problem of nonspecific binding of secondary antibodies in immunoblotting procedures.

    PubMed

    Lasne, Françoise

    2015-01-01

    Nonspecific interactions between blotted proteins and unrelated secondary antibodies generate false positives in immunoblotting techniques. Some procedures have been developed to reduce this adsorption, but they may work in specific applications and be ineffective in others. "Double-blotting" has been developed to overcome this problem. It consists of interpolating a second blotting step between the usual probings of the blot membrane with the primary antibody and the secondary antibodies. This step, by isolating the primary antibody from the interfering proteins, guarantees the specificity of the probing with the secondary antibody. This method has been developed for the study of erythropoietin in concentrated urine since a strong nonspecific binding of biotinylated secondary antibodies to some urinary proteins is observed using classical immunoblotting protocols. However, its concept makes it usable in other applications that come up against this kind of problem. This method is expected to be especially useful for investigating proteins that are present in minute amounts in complex biological media.

  17. β-actin as a loading control for plasma-based Western blot analysis of major depressive disorder patients.

    PubMed

    Zhang, Rufang; Yang, Deyu; Zhou, Chanjuan; Cheng, Ke; Liu, Zhao; Chen, Liang; Fang, Liang; Xie, Peng

    2012-08-15

    Western blot analysis is a commonly used technique for determining specific protein levels in clinical samples. For normalization of protein levels in Western blot, a suitable loading control is required. On account of its relatively high and constant expression, β-actin has been widely employed in Western blot of cell cultures and tissue extracts. However, β-actin's presence in human plasma and this protein's putative role as a plasma-based loading control for Western blot analysis remain unknown. In this study, an enzyme-linked immunosorbent assay was used to determine the concentration of β-actin in human plasma, which is 6.29±0.54 ng/ml. In addition, the linearity of β-actin immunostaining and loaded protein amount was evaluated by Western blot, and a fine linearity (R²=0.974±0.012) was observed. Furthermore, the expression of plasma β-actin in major depressive disorder subjects and healthy controls was compared. The data revealed no statistically significant difference between these two groups. Moreover, the total coefficient of variation for β-actin expression in the two groups was 9.2±1.2%. These findings demonstrate that β-actin is present in human plasma and may possibly be used as a suitable loading control for plasma-based Western blot analysis in major depressive disorder. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. The prevalence and significance of HTLV-I/II seroindeterminate Western blot patterns.

    PubMed

    Abrams, Anna; Akahata, Yoshimi; Jacobson, Steven

    2011-08-01

    Human T-lymphotropic virus type I (HTLV-I) infects an estimated 15-20 million persons worldwide. A number of diseases have been associated with the virus including adult T-cell leukemia (ATL), HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP), HTLV-I uveitis, and HTLV-I-associated infective dermatitis. Once it was shown that there is an increased risk for developing HAM/TSP associated with blood transfusion, screening for HTLV-1 among blood banks was implemented in Japan, United States, France, and the Netherlands. This process includes detection by an enzyme immunoassay (EIA) followed by a confirmatory Western blot (WB) in which recombinant proteins specific for HTLV-I Env glycoproteins are incorporated into WB strips. HTLV-I seropositive results are defined by the presence of antibodies against either gp46 or gp62/68 (both Env protein bands) and either p19, p24, or p53 (one of the gag bands). HTLV-II seropositivity is confirmed by the presence of rgp46-II. However, numerous cases have been documented in which serum samples are reactive by EIA, but an incomplete banding pattern is displayed by subsequent confirmatory WB. Although the significance of these HTLV-I/II seroindeterminates is unclear, it may suggest a much higher incidence of exposure to HTLV-I/II than previously estimated.

  19. Evaluation of an enzyme-linked immunoelectrotransfer blot test for the confirmatory serodiagnosis of human toxocariasis.

    PubMed

    Roldán, William H; Espinoza, Yrma A

    2009-05-01

    To improve the serodiagnosis of human toxocariasis, a sensitive and specific enzyme-linked immunoelectrotransfer blot (EITB-IgG) test was developed and evaluated using Toxocara canislarvae excretory-secretory antigens for detecting anti-Toxocara IgG antibodies. The EITB-IgG profile of toxocariasis was characterized by comparing 27 sera from patients with toxocariasis, 110 sera from healthy subjects and 186 sera from patients with other helminth diseases (ascariasis, ancylostomiasis, trichuriasis, enterobiasis, strongyloidiasis, hymenolepiasis, diphyllobothriasis, taeniasis, cysticercosis, hydatidosis and fascioliasis). Antigenic bands of 24, 28, 30, 35, 56, 117, 136 and 152 kDa were predominantly recognized in sera from all patients with toxocariasis. However, only bands of 24-35 kDa were highly specific for Toxocara infection (98.3%), whereas other antigenic bands observed displayed cross-reactivity. Additionally, when the results of the EITB-IgG test were compared to those of the ELISA-IgG test, a 100% concordance was observed for positive results in human toxocariasis cases. The concordance for negative results between the two tests for healthy subjects and patients with other helminth diseases were 96.3% and 53.7%, respectively, showing that the EITB-IgG test has a higher specificity than ELISA. In conclusion, the EITB-IgG test is a very useful tool to confirm the serological diagnosis of human toxocariasis.

  20. Glyceraldehyde-3-phosphate dehydrogenase: a universal internal control for Western blots in prokaryotic and eukaryotic cells.

    PubMed

    Wu, Yonghong; Wu, Min; He, Guowei; Zhang, Xiao; Li, Weiguang; Gao, Yan; Li, Zhihui; Wang, Zhaoyan; Zhang, Chenggang

    2012-04-01

    In the current study, we examined the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in a number of organisms and the stability of GAPDH under various conditions. Our results revealed that GAPDH is present in multiple Escherichia coli strains, the yeast strain GS115, Caenorhabditis elegans, rat PC12 cells, and both mouse and rat brain. Furthermore, GAPDH was stably expressed under different concentrations of inducer and at different times of induction in E. coli (BL21) cells and yeast GS115 cells. Stable expression of GAPDH protein was also observed in C.elegans and PC12 cells that were treated with different concentrations of paraquat or sodium sulfite, respectively. In addition, we were able to detect and identify the endogenous gapA protein in E.coli via immunoprecipitation and MALDI-TOF-MS analysis. Endogenous gapA protein and exogenously expressed (subcloned) GAPDH proteins were detected in E. coli BL21 but not for gapC. With the exception of gapC in E. coli, the various isoforms of GAPDH possessed enzymatic activity. Finally, sequence analysis revealed that the GAPDH proteins were 76% identical, with the exception of E. coli gapC. Taken together, our results indicate that GAPDH could be universally used as an internal control for the Western blot analysis of prokaryotic and eukaryotic samples. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  1. Western blot analysis of antibody response to pneumococcal protein antigens in a murine model of pneumonia.

    PubMed Central

    Mouneimne, H; Juvin, M; Beretti, J L; Azoulay-Dupuis, E; Vallee, E; Geslin, P; Petitpretz, P; Berche, P; Gaillard, J L

    1997-01-01

    To detect new antigen candidates for serological tests, we studied the antibody response to pneumococcal protein antigens in mice infected intratracheally with various Streptococcus pneumoniae strains. Sera were tested by Western blotting against whole-cell protein extracts. Mice developed a detectable immunoglobulin G-type response against a small number of polypeptides. The antibody response was strain dependent: sera from individuals infected with the same strain gave similar banding patterns on immunoblots. The banding patterns varied with the strain used for infection. However, a band at 36 to 38 kDa was recognized by all reactive sera. This band appeared to correspond to a polypeptide that was antigenically well conserved among the different S. pneumoniae serotypes. An antibody response to this antigen developed in mice irrespective of the capsular type, the virulence, and the susceptibility to penicillin G of the infecting strain. Thus, this 36- to 38-kDa protein antigen may be of value for the development of a serological test for humans. PMID:9384307

  2. Clinical usefulness of Western blotting and ELISA avidity for the diagnosis of human toxocariasis.

    PubMed

    Rudzińska, M; Kowalewska, B; Sikorska, K

    2017-01-01

    The serodiagnosis of human toxocariasis is difficult. Specific IgGs detected routinely with ELISA based on Toxocara excretory-secretory (TES) antigens often persist for years at an elevated level, which does not allow either the differentiation between an active and persistent infection or monitoring of the effect of treatment. Additionally, false-positive results may occur in co-infections with other helminths due to cross-reactions. We evaluated the usefulness of an IgG avidity index (AI) and a Western blotting (WB) IgG in the diagnosis of patients suspected of Toxocara infection. We studied 138 subjects who were submitted to serological testing two or more times. Confirmation of an infection by WB was achieved in 73.2% of patients. A high AI was obtained in 89.1% of patients, and low AI and borderline AI were found in only 10.9%. Low and borderline values of AI remained at similar levels in subsequent studies over 2-3 years. The results showed the necessity of obligatory verification of all ELISA IgG positive and questionable results by WB. The index of IgG avidity may be helpful in excluding recent infection, but its usefulness in detecting an active phase of invasion requires further research.

  3. Identification of Yeast V-ATPase Mutants by Western Blots Analysis of Whole Cell Lysates

    NASA Astrophysics Data System (ADS)

    Parra-Belky, Karlett

    2002-11-01

    A biochemistry laboratory was designed for an undergraduate course to help students better understand the link between molecular engineering and biochemistry. Students identified unknown yeast strains with high specificity using SDS-PAGE and Western blot analysis of whole cell lysates. This problem-solving exercise is a common application of biochemistry in biotechnology research. Three different strains were used: a wild-type and two mutants for the proton pump vacuolar ATPase (V-ATPase). V-ATPases are multisubunit enzymes and the mutants used were deletion mutants; each lacked one structural gene of the complex. After three, three-hour labs, mutant strains were easily identified by the students and distinguished from wild-type cells analyzing the pattern of SDS-PAGE distribution of proteins. Identifying different subunits of one multimeric protein allowed for discussion of the structure and function of this metabolic enzyme, which captured the interest of the students. The experiment can be adapted to other multimeric protein complexes and shows improvement of the described methodology over previous reports, perhaps because the problem and its solution are representative of the type of techniques currently used in research labs.

  4. Serological diagnosis of North American Paragonimiasis by Western blot using Paragonimus kellicotti adult worm antigen.

    PubMed

    Fischer, Peter U; Curtis, Kurt C; Folk, Scott M; Wilkins, Patricia P; Marcos, Luis A; Weil, Gary J

    2013-06-01

    Abstract. We studied the value of an IgG Western blot (WB) with Paragonimus kellicotti (Pk) antigen for diagnosis of North American paragonimiasis. The test was evaluated with sera from patients with Pk and Paragonimus westermani infections, with control sera from patients with other helminth infections, and sera from healthy Americans. All 11 proven Pk infection sera and two samples from suspected cases that were negative by P. westermani WB at the Centers for Disease Control and Prevention (CDC) contained antibodies to antigens at 34 kDa and at 21/23 kDa. Seven of 7 P. westermani sera contained antibodies to the 34 kDa antigen, but only 2 recognized the 21/23 kDa doublet. No control samples were reactive with these antigens. Antibody reactivity declined after praziquantel treatment. Thus, the P. kellicotti WB appears to be superior to P. westermani WB for diagnosing Pk infections, and it may be useful for assessing responses to treatment.

  5. Serological Diagnosis of North American Paragonimiasis by Western Blot Using Paragonimus kellicotti Adult Worm Antigen

    PubMed Central

    Fischer, Peter U.; Curtis, Kurt C.; Folk, Scott M.; Wilkins, Patricia P.; Marcos, Luis A.; Weil, Gary J.

    2013-01-01

    We studied the value of an IgG Western blot (WB) with Paragonimus kellicotti (Pk) antigen for diagnosis of North American paragonimiasis. The test was evaluated with sera from patients with Pk and Paragonimus westermani infections, with control sera from patients with other helminth infections, and sera from healthy Americans. All 11 proven Pk infection sera and two samples from suspected cases that were negative by P. westermani WB at the Centers for Disease Control and Prevention (CDC) contained antibodies to antigens at 34 kDa and at 21/23 kDa. Seven of 7 P. westermani sera contained antibodies to the 34 kDa antigen, but only 2 recognized the 21/23 kDa doublet. No control samples were reactive with these antigens. Antibody reactivity declined after praziquantel treatment. Thus, the P. kellicotti WB appears to be superior to P. westermani WB for diagnosing Pk infections, and it may be useful for assessing responses to treatment. PMID:23589531

  6. HIV-1: absence of infection in subjects with indeterminate western blot.

    PubMed

    Migali, E; Mariotti, D; Lovari, A; Tenani, T; Imperiali, P; Ozzola, G

    1993-01-01

    The search for specific antibodies against the Human Immunodeficiency Virus type-1 (Ab anti HIV 1), using an immunoenzymatic test and a subsequent confirmation test (Western Blot-WB) in patients who were previously positive or borderline at the first test, singled out from about 12500 tested subjects, fifteen patients with indeterminate WB (WBi) for the presence of an abnormal band. The presence of p24 was predominant in those WBi (about 50%); generally p24 was the only band found. In all serum samples with WBi, the viral antigen p24 (Ag p24) was absent. In 3 subjects with WBi, after at least six months from the first test, not only did the same pattern persist but also the search for VIH-1 DNA sequence using the polymerase chain reaction (PCR) gave a negative result. According to our experience and the literature on the subject, we suggest that patients with low risk of infection and whose WBi does not modify with time have a remote possibility of being infected by HIV.

  7. Genotype-specific RNA probes for direct identification of wild polioviruses by blot hybridization.

    PubMed Central

    De, L; Yang, C F; Da Silva, E; Boshell, J; Cáceres, P; Gómez, J R; Pallansch, M; Kew, O

    1997-01-01

    We have developed RNA probes for the direct identification of wild poliovirus isolates by blot hybridization. The probes are complementary to sequences of the first 30 to 32 codons of VP1, which evolve more extensively (approximately 1.5-fold) than the rest of VP1. To illustrate our general approach, we describe the design of probes specific to each of four major genotypes recently endemic (1981 to 1991) to the Americas: Andean type 1, Brazil type 1, Brazil type 3, and Central America-Mexico type 3. A wild isolate of each genotype was selected according to molecular and epidemiologic criteria to be representative of the principal lineages in circulation. Variable VP1 sequences of the representative isolates were amplified by the reverse transcriptase PCR and were inserted into a plasmid vector containing a T7 promoter. The in vitro transcripts, labeled with digoxigenin, served as probes. These formed stable hybrids only with RNAs of isolates of the corresponding genotypes. Hybrids were detected by a sensitive chemiluminescence assay, capable under normal diagnostic conditions of detecting specific wild poliovirus sequences in samples containing up to a 100-fold excess of Sabin vaccine strain-related sequences of the same serotype. PMID:9350743

  8. A new Western blot assay for the detection of porcine cytomegalovirus (PCMV).

    PubMed

    Plotzki, Elena; Keller, Martina; Ivanusic, Daniel; Denner, Joachim

    2016-10-01

    Porcine cytomegalovirus (PCMV) may be harmful for human recipients if xenotransplantation using pig cell, tissue or organ will be performed transmitting the virus from donor pigs to human recipients. PCMV is widespread in pigs and closely related to human pathogenic herpesviruses, however there are no data concerning infection of humans. In contrast, recently it had been shown that transplantation of organs from pigs infected with PCMV into non-human primate recipients resulted in a significant reduction of the survival time compared with the transplantation of organs from uninfected pigs. To prevent transmission of PCMV in future pig to human xenotransplantations, sensitive and specific detection methods should be used. Here a new Western blot assay using recombinant proteins corresponding to two domains of the glycoprotein gB of PCMV is described. With this assay, the presence of PCMV-specific antibodies in different pig breeds was analysed. Antibodies were detected in a high percentage of animals, in one breed up to 85%. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. An alternative strategy to western blot as a confirmatory diagnostic test for HIV infection.

    PubMed

    Feng, Xia; Wang, Jibao; Gao, Zhiyun; Tian, Yu; Zhang, Ling; Chen, Huichao; Zhang, Tong; Xiao, Lin; Yao, Jun; Xing, Wenge; Qiu, Maofeng; Jiang, Yan

    2017-03-01

    In China, western blot (WB) is the recommended procedure for the diagnosis of HIV infection. However, this technique is time consuming and labor intensive, and its complexity restricts wide application in resource-limited regions. The aim of this study was to evaluate the efficacy of a dry blood spots (DBS)-urine paired enzyme-linked immunosorbent assay (ELISA) test, instead of WB, for HIV antibody detection. Plasma, DBS, and urine samples were collected from 1213 subjects from different populations. Two diagnostic testing strategies were conducted in parallel. The equivalence of the paired ELISA and WB strategies was assessed. A diagnosis of HIV was determined in 250 subjects according to the paired ELISA test, and in 249 according to the WB strategy. The discordant case was judged HIV-positive during follow-up. In total, 18 subjects were diagnosed with possible HIV using the paired ELISA test, among whom, 11 subjects tested negative with WB, and one was confirmed to be HIV-positive during follow-up. For the remaining 945 subjects, both strategies indicated a negative result. The kappa test indicated good conformity (kappa=0.954) between the two diagnostic strategies. The DBS-urine paired ELISA could be applied as an alternative to WB in HIV diagnosis, which would be valuable in resource-limited regions owing to the associated affordability and ease of use. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  10. Performance of PCR-reverse blot hybridization assay for detection of rifampicin-resistant Mycobacterium leprae.

    PubMed

    Wang, Hye-young; Kim, Hyunjung; Kim, Yeun; Bang, Hyeeun; Kim, Jong-Pill; Hwang, Joo Hwan; Cho, Sang-Nae; Kim, Tae Ue; Lee, Hyeyoung

    2015-10-01

    Drug resistance in Mycobacterium leprae is a significant problem in countries where leprosy is endemic. A sensitive, specific, and high-throughput reverse blot hybridization assay (REBA) for the detection of genotypic resistance to rifampicin (RIF) was designed and evaluated. It has been shown that resistance to RIF in M. leprae involves mutations in the rpoB gene encoding the -subunit of the RNA polymerase. The PCR-REBA simultaneously detects both 6 wild-type regions and 5 different mutations (507 AGC, 513 GTG, 516 TAT, 531 ATG, and 531 TTC) including the most prevalent mutations at positions 507 and 531. Thirty-one clinical isolates provided by Korea Institute of Hansen-s Disease were analyzed by PCR-REBA with RIF resistance of rpoB gene. As a result, missense mutations at codons 507 AGC and 531 ATG with 2-nucleotide substitutions were found in one sample, and a missense mutation at codon 516 TAT and ΔWT6 (deletion of 530-534) was found in another sample. These cases were confirmed by DNA sequence analysis. This rapid, simple, and highly sensitive assay provides a practical alternative to sequencing for genotypic evaluation of RIF resistance in M. leprae.

  11. [Rapid diagnosis of non-deletion alpha-thalassemias by reverse dot blot].

    PubMed

    Li, Li-yan; Mo, Qiu-hua; Xu, Xiang-min

    2003-08-01

    To establish a rapid and convenient method of reverse dot blot (RDB) analysis for detecting the point mutations of non-deletion alpha-thalassemia in Chinese. Label biotin to primers and amplify human alpha2 globin gene selectively, then hybridize PCR products with a set of oligonucleotide probes immobilized on strips, and develop colour to detect non-deletion alpha-thalassemia defects. The PCR system using biotin-labeled primers could specifically amplify a 1085 bp fragment of human alpha2 globin gene which encompasses all six alpha-thalassemia mutations. After being hybridized with sequence-specific oligonucleotide probes and colour development, it could simultaneously identify all six types of non-deletion alpha-thalassemias encountered in Chinese. This method does not need semi-nested PCR, and the products amplified by biotinylated primers can be used directly to hybridize with the probes on strips under the identical conditions of hybridization. So, it is a specific and multiplex detection assay for screening non-deletion alpha-thalassemia defects in Chinese.

  12. Digital chemiluminescence imaging of DNA sequencing blots using a charge-coupled device camera.

    PubMed Central

    Karger, A E; Weiss, R; Gesteland, R F

    1992-01-01

    Digital chemiluminescence imaging with a cryogenically cooled charge-coupled device (CCD) camera is used to visualize DNA sequencing fragments covalently bound to a blotting membrane. The detection is based on DNA hybridization with an alkaline phosphatase(AP) labeled oligodeoxyribonucleotide probe and AP triggered chemiluminescence of the substrate 3-(2'-spiro-adamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD). The detection using a direct AP-oligonucleotide conjugate is compared to the secondary detection of biotinylated oligonucleotides with respect to their sensitivity and nonspecific binding to the nylon membrane by quantitative imaging. Using the direct oligonucleotide-AP conjugate as a hybridization probe, sub-attomol (0.5 pg of 2.7 kb pUC plasmid DNA) quantities of membrane bound DNA are detectable with 30 min CCD exposures. Detection using the biotinylated probe in combination with streptavidin-AP was found to be background limited by nonspecific binding of streptavidin-AP and the oligo(biotin-11-dUTP) label in equal proportions. In contrast, the nonspecific background of AP-labeled oligonucleotide is indistinguishable from that seen with 5'-32P-label, in that respect making AP an ideal enzymatic label. The effect of hybridization time, probe concentration, and presence of luminescence enhancers on the detection of plasmid DNA were investigated. Images PMID:1480487

  13. Conservation of repetitive DNA sequences in deer species studied by southern blot transfer.

    PubMed

    Lima-de-Faria, A; Arnason, U; Widegren, B; Essen-Möller, J; Isaksson, M; Olsson, E; Jaworska, H

    1984-01-01

    The Cervidae show one of the largest variations in chromosome number found within a mammalian family. The five species of the deer family which are the subject of this study vary in chromosome number from 2n = 70 to 2n = 6. Digestion with the restriction enzymes EcoRI, HpaII, HaeIII and MspI reveals that there is a series of highly repetitive sequences forming similar band patterns in the different species. To obtain information on the degree of homology among these conserved sequences we isolated a HpaII restriction fragment of approximately 990 base pairs from reindeer DNA. This DNA sequence was 32P-labelled and hybridized by the Southern blot technique to DNAs cleaved with HpaII and HaeIII from the reindeer and four other Cervidae species. Hybridization to specific restriction fragments was recorded in all species. The patterns of hybridization showed a higher degree of similarity between reindeer, elk and roe deer than between reindeer and the Asiatic species (fallow deer and muntjac). Homologies are still present between the highly repetitive sequences of the five species despite the drastic reorganization that led to a change in chromosome number from 6 to 70.

  14. A simple dot-blot-Sirius red-based assay for collagen quantification.

    PubMed

    Rodríguez-Rodríguez, Pilar; Arribas, Silvia M; de Pablo, Angel Luis López; González, M Carmen; Abderrahim, Fatima; Condezo-Hoyos, Luis

    2013-08-01

    The assessment of collagen content in tissues is important in biomedical research, since this protein is altered in numerous diseases. Hydroxyproline and Sirius red based assays are the most common methods for collagen quantification. However, these procedures have some pitfalls, such as the requirement of oxygen-free medium or expensive equipment and large sample size or being unsuitable for hydrolyzed collagen, respectively. Our objective was to develop a specific, versatile, and user-friendly quantitative method applicable to small tissue samples and extracts obtained from elastin purification, therefore, suitable for simultaneous quantification of elastin. This method is based on the binding of Sirius red to collagen present in a sample immobilized on a PVDF membrane, as in the dot-blot technique, and quantified by a scanner and image analysis software. Sample loading, Sirius red concentration, temperature and incubation time, type of standard substance, albumin interference, and quantification time are optimized. The method enabled the quantification of (1) intact collagen in several rat tissue homogenates, including small resistance-sized arteries, (2) partially hydrolyzed collagen obtained from NaOH extracts, compatible with elastin purification, and (3) for the detection of differences in collagen content between hypertensive and normotensive rats. We conclude that the developed technique can be widely used since it is versatile (quantifies intact and hydrolyzed collagen), requires small sample volumes, is user-friendly (low-cost, easy to use, minimum toxic materials, and reduced time of test), and is specific (minimal interference with serum albumin).

  15. Diagnosis of cutaneous tuberculosis in biopsy specimens by PCR and southern blotting.

    PubMed Central

    Quirós, E; Maroto, M C; Bettinardi, A; González, I; Piédrola, G

    1996-01-01

    AIMS: To evaluate the use of a gene amplification and hybridisation method for detecting mycobacterial nucleic acid as a possible diagnostic method for cutaneous tuberculosis infection. METHODS: Biopsy specimens from 20 patients with various skin conditions of possible tuberculous aetiology were studied. Six patients had ulcerative nodules, seven lupiform lesions, two non-necrotic granulomas, one scrofulous lichen, one impetigo, one erythematosus lesions, one warty lesions, and one suspected tuberculous lipoma. Biopsy specimens were stained using Ziehl-Neelsen stain and cultured in Lowenstein-Jensen medium. DNA was extracted and then amplified by PCR using primers specific for the Mycobacterium tuberculosis complex. Specificity was confirmed by Southern blotting. RESULTS: Of the specimens, 30% were positive for mycobacteria on staining with Ziehl-Neelsen stain, 60% were culture positive and 85% PCR positive. Only 35.2% of specimens were positive with all three techniques. A further 32.5% were both culture and PCR positive. All PCR negative samples were also negative when cultured or stained with Ziehl-Neelsen stain. Of the PCR positive specimens, 29.4% were negative when cultured or stained. CONCLUSIONS: PCR, using suitable primers, is an efficient and sensitive method for the diagnosis of cutaneous tuberculosis. PMID:8944606

  16. Characterization of excretory-secretory antigens of adult Toxocara canis by western blotting.

    PubMed

    Sudhakar, N R; Samanta, S; Sahu, Shivani; Raina, O K; Gupta, S C; Goswami, T K; Lokesh, K M; Kumar, Ashok

    2014-06-01

    Toxocara canis is one of the most common helminth worm of dogs which continues to stimulate both public health concern alongside the higher scientific interest. It may cause visceral and ocular damage in humans especially in children. The identification of specific antigens of T. canis is important so as to develop better diagnostic techniques. Excretory-secretory (ES) antigens were prepared by culturing the adult T. canis worms in RPMI 1640 medium without serum supplementation followed by ammonium sulphate precipitation. These antigens were separated using sodium dodecyl sulphate-electrophoresis (SDS-PAGE). Recovered proteins ranged from 30 to 384 kDa. The specific reactivity of the T. canis excretory-secretory (TC-ES) proteins was checked by western blotting. The immuno-reactivity of the naturally infected dog sera with the TC-ES antigens showed five bands at 43, 57,105, 139 and 175 kDa. The immuno-reactivity of the hyper immune serum raised in rabbits against TC-ES antigens was observed with ten polypeptides of 21, 25, 30, 37, 45, 50, 57, 69, 77 and 105 kDa. Common antigens band were observed at 57 and 105 KDa. These antigens merit further evaluation as candidate for use in diagnosis of toxocariasis in humans and adult dogs.

  17. Renaturation of blotted allergens increases the sensitivity of specific IgE detection.

    PubMed

    Muro, M D; Fernández, C; Moneo, I

    1996-01-01

    Several authors have demonstrated that renaturation is an essential step for the appropriate recognition of blotted proteins. The use of nonionic detergents has been described as a useful alternative to enhance the antigenicity in immunoblotting, although elution from proteins by detergents has been observed. To measure the influence of different factors on the sensitivity of specific IgE by immunoblotting, we used twenty human sera from atopic patients who were allergic or nonallergic to a common, reliable allergen (grass pollen mixture). The use of Nonidet-P40 was found to be a useful alternative for the renaturation of the allergens. No elution from the membrane was found when employing this detergent, even at high concentrations (3%), and its use gave better sensitivity than methanol. On the other hand, we detected that methanol possessed renaturing properties. A transfer method using diffusion instead of electric transfer gave the best results and two membranes could be obtained from each gel. Using this method, we found that after NP-40 incubation of the membrane, the use of bovine albumin could be omitted as blocking agent and that its use had even deleterious effects.

  18. High-selectivity cytology via lab-on-a-disc western blotting of individual cells.

    PubMed

    Kim, John J; Sinkala, Elly; Herr, Amy E

    2017-02-28

    Cytology of sparingly available cell samples from both clinical and experimental settings would benefit from high-selectivity protein tools. To minimize cell handling losses in sparse samples, we design a multi-stage assay using a lab-on-a-disc that integrates cell handling and subsequent single-cell western blotting (scWestern). As the two-layer microfluidic device rotates, the induced centrifugal force directs dissociated cells to dams, which in turn localize the cells over microwells. Cells then sediment into the microwells, where the cells are lysed and subjected to scWestern. Taking into account cell losses from loading, centrifugation, and lysis-buffer exchange, our lab-on-a-disc device handles cell samples with as few as 200 cells with 75% cell settling efficiencies. Over 70% of microwells contain single cells after the centrifugation. In addition to cell settling efficiency, cell-size filtration from a mixed population of two cell lines is also realized by tuning the cell time-of-flight during centrifugation (58.4% settling efficiency with 6.4% impurity). Following the upstream cell handling, scWestern analysis detects four proteins (GFP, β-TUB, GAPDH, and STAT3) in a glioblastoma cell line. By integrating the lab-on-a-disc cell preparation and scWestern analysis, our platform measures proteins from sparse cell samples at single-cell resolution.

  19. Trypanosoma cruzi: variability of stocks from Colombia determined by molecular karyotype and minicircle Southern blot analysis.

    PubMed

    Triana, Omar; Ortiz, Sylvia; Dujardin, Jean-Claude; Solari, Aldo

    2006-05-01

    Nineteen Trypanosoma cruzi stocks, most of them of wild origin, and four Trypanosoma rangeli stocks from Colombia were analysed by molecular karyotype analysis with cloned DNA cruzipain as the probe. Another 27 cloned stocks of T. cruzi from different geographic areas of South America were used as reference for T. cruzi lineages. Phenetic analysis of chromosome size polymorphism demonstrated a great variability of Colombian T. cruzi stocks, suggesting that most belong to lineage I, although two of them belong to lineage II. The 2 lineage II T. cruzi, 17 T. cruzi lineage I, and 3 T. rangeli stocks from Colombia were studied further by Southern blot analysis with a panel of kinetoplast DNA minicircle probes. Hybridisation results indicate that the two T. cruzi II stocks are genetically distant from each other and from T. cruzi lineages IIb, IId, and IIe from Chile. Finally, T. cruzi minicircle probes do not cross-hybridise in any stringency condition tested with T. rangeli minicircles, a clear indication that these parasites can be easily distinguished by this method.

  20. Validity of the Enzyme-linked Immunoelectrotransfer Blot (EITB) for naturally acquired porcine cysticercosis

    PubMed Central

    Jayashi, César M.; Gonzalez, Armando E.; Neyra, Ricardo Castillo; Rodríguez, Silvia; García, Hector H.; Lightowlers, Marshall W.

    2017-01-01

    The Enzyme-linked Immunoelectrotransfer Blot (EITB) has been used widely as a screening test for Taenia solium cysticercosis in swine. However, the relation between seropositivity and infection in pig populations from endemic areas has not been well defined. The aim of this study is to relate EITB seropositivity with infection and infection burden, analyse the trade-off between sensitivity and specificity with various cut-off points for the EITB assay, and finally describe the serology changes in a cohort of rural pigs raised under natural conditions. A group of 107 pigs that were used as controls during a vaccination field trial in Peru was our study population. The prevalence of porcine cysticercosis determined by necropsy examination was 16.82% (18/107) in these animals. Using EITB reactivity to ≥ 1 band as a cut-off point for the assay, the sensitivity was 88.89% (65.29-98.62, 95% CI) and the specificity was 48.31% (37.59-59.16, 95% CI). Comparing other cut-off points, involving up to as many as 7 reactive bands, a reactivity of ≥ 3 bands provided the best trade-offs in sensitivity and specificity. Using this cut-off point for the assay, the sensitivity was 77.77% (52.36 - 93.59, 95% CI) and the specificity was 76.40% (66.22 - 84.76, 95% CI). A significant association was found between cyst counts over 100 cysts and reactivity to ≥ 3 bands in the EITB assay (Fisher’s exact test, p<0.05). The results of this study suggest that the use of the EITB assay to study porcine cysticercosis may require setting different cut-offs under field and experimental conditions, and depending upon the objective of the screening process. PMID:24183647

  1. Antibody responses to Borrelia burgdorferi detected by western blot vary geographically in Canada

    PubMed Central

    Ogden, Nicholas H.; Arsenault, Julie; Hatchette, Todd F.; Mechai, Samir; Lindsay, L. Robbin

    2017-01-01

    Lyme disease is emerging in eastern and central Canada, and most cases are diagnosed using the two-tier serological test (Enzyme Immuno Assay [EIA] followed by Western blot [WB]). Simplification of this algorithm would be advantageous unless it impacts test performance. In this study, accuracy of individual proteins of the IgG WB algorithm in predicting the overall test result in samples from Canadians was assessed. Because Borrelia burgdorferi strains vary geographically in Canada, geographic variations in serological responses were also explored. Metrics of relative sensitivity, specificity and the kappa statistic measure of concordance were used to assess the capacity of responses to individual proteins to predict the overall IgG WB result of 2524 EIA (C6)-positive samples from across Canada. Geographic and interannual variations in proportions of samples testing positive were explored by logistic regression. No one protein was highly concordant with the IgG WB result. Significant variations were found amongst years and geographic regions in the prevalence of samples testing positive using the overall IgG WB algorithm, and for individual proteins of the algorithm. In most cases the prevalence of samples testing positive were highest in Nova Scotia, and lower in samples from Manitoba westwards. These findings suggest that the current two tier test may not be simplified and continued use of the current two-tier test method and interpretation is recommended. Geographic and interannual variations in the prevalence of samples testing positive may be consistent with B. burgdorferi strain variation in Canada, and further studies are needed to explore this. PMID:28182723

  2. Epidemiology of Babesia, Anaplasma and Trypanosoma species using a new expanded reverse line blot hybridization assay.

    PubMed

    Paoletta, Martina Soledad; López Arias, Ludmila; de la Fournière, Sofía; Guillemi, Eliana Carolina; Luciani, Carlos; Sarmiento, Néstor Fabián; Mosqueda, Juan; Farber, Marisa Diana; Wilkowsky, Silvina Elizabeth

    2017-09-01

    Vector-borne hemoparasitic infections are a major problem that affects livestock industries worldwide, particularly in tropical and subtropical regions. In this work, a reverse line blot (RLB) hybridization assay was developed for the simultaneous detection and identification of Anaplasma, Babesia and bovine trypanosomes, encompassing in this way the most relevant hemoparasites that affect cattle. A total of 186 bovine blood samples collected from two different ecoepidemiological regions of northeast Argentina, with and without tick control, were analyzed with this new RLB. High diversity of parasites, such as Babesia bovis, B. bigemina, Anaplasma marginale and three different Trypanosoma species, was found. High rates of coinfections were also detected, and significant differences were observed not only in the prevalence of parasites but also in the level of coinfections between the two analyzed areas. Regarding the Trypanosoma genus, we provide molecular evidence of the presence of T. vivax and T. theileri for the first time in Argentina. Besides, since the RLB is a prospective tool, it allowed the identification of a yet unknown bovine trypanosome which could not be assigned to any of the bovine species known so far. In the present study we provide new insights on the prevalence of several pathogens that directly impact on livestock production in Argentina. The RLB assay developed here allows to identify simultaneously numerous pathogenic species which can also be easily expanded to detect other blood borne pathogens. These characteristics make the RLB hybridization assay an essential tool for epidemiological survey of all vector-borne pathogens. Copyright © 2017 Elsevier GmbH. All rights reserved.

  3. Evaluation of immunodominant proteins of Mycobacterium avium paratuberculosis cell wall by Western blot analysis.

    PubMed

    Hashemi, Maryam; Madani, Rasool; Razmi, Nematollah

    2014-04-01

    Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a slow growing mycobactin, whose dependence on mycobacterial species is known to be the causative agent of Johne's disease (paratuberculosis) in all species of domestic ruminants worldwide. The organism is transmitted via close contact, ingestion, or transplacentally from mother to fetus and occurs commonly in grazing domestic animals. Johne's disease (JD) is characterized by gradual weight loss, decreased milk production, and diarrhea due to the chronic, progressive, granulomatous enteritis and lymphadenitis. The disease can cause serious economic damage to the dairy industry due to the loss of milk production and early culling of infected animals. In recent years, researchers have focused on the identification of a specific antigen of M. paratuberculosis to use in diagnosis test and preparation of effective vaccine. The goal of this study is evaluation of the immunodominant proteins of M. paratuberculosis cell wall. The amount of protein was determined with a Lowry assay (22.68 μg/100 μL). For production of polyclonal antibody against proteins of M. paratuberculosis cell wall, a New Zealand white rabbit was immunized with antigen and Freund's adjuvant. After immunization, the rabbit was bled to produce enriched serum. Antibodies were purified from serum with ion exchange chromatography. In the Ouchterlony test, the reactions between antigen and antibodies were seen in dilutions of one quarter for serum, one quarter for Ig, and one half for IgG by clear precipitation lines due to the well immunization of the rabbit. Electrophoresis and Western blot analysis were used and subsequently a sharp band appeared in nitrocellulose paper; these bands were about 25, 37, 50, 75, and 150 kDa molecular weight, which indicated immunodominant proteins.

  4. Evaluation of the Aspergillus Western Blot IgG Kit for Diagnosis of Chronic Aspergillosis

    PubMed Central

    Oliva, A.; Flori, P.; Hennequin, C.; Dubus, J.-C.; Reynaud-Gaubert, M.; Charpin, D.; Vergnon, J. M.; Gay, P.; Colly, A.; Piarroux, R.; Pelloux, H.

    2014-01-01

    Immunoprecipitin detection (IPD) is the current reference confirmatory technique for anti-Aspergillus antibody detection; however, the lack of standardization is a critical drawback of this assay. In this study, we evaluated the performance of the Aspergillus Western blot (Asp-WB) IgG kit (LDBio Diagnostics, Lyon, France), a recently commercialized immunoblot assay for the diagnosis of various clinical presentations of chronic aspergillosis. Three hundred eight serum samples from 158 patients with aspergillosis sensu lato (s.l.) were analyzed. More specifically, 267 serum samples were derived from patients with Aspergillus disease, including 89 cases of chronic pulmonary aspergillosis, 10 of aspergilloma, and 32 of allergic bronchopulmonary aspergillosis, while 41 samples were from patients with Aspergillus colonization, including 15 cystic fibrosis (CF) and 12 non-CF patients. For blood donor controls, the Asp-WB specificity was 94%, while the kit displayed a sensitivity for the aspergillosis s.l. diagnosis of 88.6%, with a diagnostic odds ratio (DOR) of 119 (95% confidence interval [CI], 57 to 251). The DOR values were 185.22 (95% CI,78.79 to 435.45) and 43.74 (95% CI, 15.65 to 122.20) for the diagnosis of Aspergillus disease and Aspergillus colonization, respectively. Among the patients, the sensitivities of the Asp-WB in the diagnosis of Aspergillus colonization were 100% and 41.7% in CF and non-CF patients, respectively. The Asp-WB yielded fewer false-negative results than did IPD. In conclusion, the Asp-WB kit performed well for the diagnosis of various clinical presentations of aspergillosis in nonimmunocompromised patients, with an enhanced standardization and a higher sensitivity than with IPD, which is the current reference method. PMID:25392351

  5. Evaluation of a Western Blot Test in an Outbreak of Acute Pulmonary Histoplasmosis

    PubMed Central

    Pizzini, Claudia V.; Zancopé-Oliveira, Rosely M.; Reiss, Errol; Hajjeh, Rana; Kaufman, Leo; Peralta, José Mauro

    1999-01-01

    A western blot (WB) test was evaluated for detection of antibodies against native glycosylated and chemically deglycosylated M and H antigens of Histoplasma capsulatum in serum obtained from patients during the acute phase of pulmonary histoplasmosis that occurred during an outbreak. Of 275 serum samples tested by immunodiffusion and complement fixation (CF) samples from 40 patients affected during this outbreak and from 37 negative controls were tested by WB test. A group of patients whose sera were negative for CF antibodies and precipitins early in the acute stage of histoplasmosis but who all seroconverted during convalescence 6 weeks later were tested with the WB test. Antibodies against untreated H and M antigens were detected at a 1:100 dilution by WB test in 45% of the 20 acute-phase serum samples and in all 20 of the convalescent-phase specimens. The WB test’s sensitivity for acute-phase specimens increased to 90% (18 of 20 specimens) when H and M antigens were treated by periodate oxidation to inactivate susceptible carbohydrate epitopes. When native glycosylated antigens were used in the WB test, positive reactions were observed in negative control serum specimens (3 of 37 specimens; 8%) and in serum specimens obtained from asymptomatic persons screened as part of the outbreak investigation (13 of 20 specimens; 65%). These positive reactions were also attributed to glycosidic epitopes since the specificity of the WB test increased from 78 to 100% when periodate-treated H and M antigens were used. WB test with deglycosylated H and M antigens of histoplasmin provides a rapid, sensitive, and specific test to diagnose acute pulmonary histoplasmosis before precipitins can be detected. PMID:9874658

  6. Two-Phase Contiguous Supported Lipid Bilayer Model for Membrane Rafts via Polymer Blotting and Stenciling.

    PubMed

    Richards, Mark J; Daniel, Susan

    2017-02-07

    The supported lipid bilayer has been portrayed as a useful model of the cell membrane compatible with many biophysical tools and techniques that demonstrate its appeal in learning about the basic features of the plasma membrane. However, some of its potential has yet to be realized, particularly in the area of bilayer patterning and phase/composition heterogeneity. In this work, we generate contiguous bilayer patterns as a model system that captures the general features of membrane domains and lipid rafts. Micropatterned polymer templates of two types are investigated for generating patterned bilayer formation: polymer blotting and polymer lift-off stenciling. While these approaches have been used previously to create bilayer arrays by corralling bilayers patches with various types of boundaries impenetrable to bilayer diffusion, unique to the methods presented here, there are no physical barriers to diffusion. In this work, interfaces between contiguous lipid phases define the pattern shapes, with continuity between them allowing transfer of membrane-bound biomolecules between the phases. We examine effectors of membrane domain stability including temperature and cholesterol content to investigate domain dynamics. Contiguous patterning of supported bilayers as a model of lipid rafts expands the application of the SLB to an area with current appeal and brings with it a useful toolset for characterization and analysis. These combined tools should be helpful to researchers investigating lipid raft dynamics and function and biomolecule partitioning studies. Additionally, this patterning technique may be useful for applications such as bioseparations that exploit differences in lipid phase partitioning or creation of membranes that bind species like viruses preferentially at lipid phase boundaries, to name a few.

  7. Western blot analysis of sera from dogs with suspected food allergy.

    PubMed

    Favrot, Claude; Linek, Monika; Fontaine, Jacques; Beco, Luc; Rostaher, Ana; Fischer, Nina; Couturier, Nicolas; Jacquenet, Sandrine; Bihain, Bernard E

    2017-04-01

    Food allergy is often suspected in dogs with clinical signs of atopic dermatitis. This diagnosis is confirmed with an elimination diet and a subsequent challenge with regular food. Laboratory tests for the diagnosis of food allergy in dogs are unreliable and/or technically difficult. Cyno-DIAL(®) is a Western blot method that might assist with the selection of an appropriate elimination diet. To evaluate the performance of Cyno-DIAL(®) for the selection of an elimination diet and diagnosis of food allergy. Thirty eight dogs with atopic dermatitis completed an elimination diet. Combining the results of the diet trials and the challenges, 14 dogs were classified as food allergic (FA), 22 as nonfood-allergic and two as ambiguous cases. Amongst all dogs and amongst dogs with a clinical diagnosis of FA, 3% and 7% (respectively) were positive to Royal Canin Anallergenic(®) , Vet-Concept Kanguru(®) or Vet-Concept Dog Sana(®) ; 8% and 7% to Hill's d/d Duck and Rice(®) ; 8% and 21% to Hill's z/d Ultra Allergen Free(®) ; 53% and 64% to Eukanuba Dermatosis FP(®) ; and 32% and 43% to a home-cooked diet of horse meat, potatoes and zucchini. The specificity and sensitivity of Cyno-DIAL(®) for diagnosing food allergy were 73% and 71%, respectively. Although Cyno-DIAL(®) was considered potentially useful for identifying appropriate foods for elimination diet trials, it cannot be recommended for the diagnosis of food allergy. The Cyno-DIAL(®) test performed better than some previously evaluated ELISA-based tests. © 2017 ESVD and ACVD.

  8. Development of a dot blot assay using gene probes for the detection of enteroviruses in water

    SciTech Connect

    Margolin, A.B.

    1986-01-01

    Enteric viruses are viruses which replicate in the intestinal tract of man and animals. One mode of transmission for enteric viruses is the fecal-oral route. Drinking water which has been contaminated with sewage or sewage effluent has been implicated as a means for the spread of enteric viruses. Current methods for the detection of enteric viruses in water requires the use of animal cell culture. This technique has several drawbacks. More rapid techniques, such as fluorescent antibody or radioimmunoassay do not have the needed sensitivity to detect the low levels of virus found in contaminated water. An alternative technique for the detection of viruses in water was sought. Recent advances in recombinant DNA technology now makes it possible to detect viruses without the use of cell culture or antibodies. Gene probes that hybridize to the RNA of poliovirus and hepatitis A virus were tested for their ability to detect different enteric viruses. The probes were labeled with /sup 32/P dCTP and /sup 32/P dATP to a specific activity greater then 1.0 x 10/sup 9/ cpm/ug DNA. One infectious unit of poliovirus and hepatitis A virus was detected using labeled cDNA probes. Upon comparison, the dot blot assay was as sensitive as tissue culture for the detection of poliovirus in beef extract, secondary effluent, and tap water. Environmental samples, such as secondary effluent, reclaimed wastewater and unchlorinated drinking water were also assayed for poliovirus and hepatitis A virus with the use of gene probes. The results presented here offer an alternative method for screening water samples for the presence of enteric viruses.

  9. Antibody responses to Borrelia burgdorferi detected by western blot vary geographically in Canada.

    PubMed

    Ogden, Nicholas H; Arsenault, Julie; Hatchette, Todd F; Mechai, Samir; Lindsay, L Robbin

    2017-01-01

    Lyme disease is emerging in eastern and central Canada, and most cases are diagnosed using the two-tier serological test (Enzyme Immuno Assay [EIA] followed by Western blot [WB]). Simplification of this algorithm would be advantageous unless it impacts test performance. In this study, accuracy of individual proteins of the IgG WB algorithm in predicting the overall test result in samples from Canadians was assessed. Because Borrelia burgdorferi strains vary geographically in Canada, geographic variations in serological responses were also explored. Metrics of relative sensitivity, specificity and the kappa statistic measure of concordance were used to assess the capacity of responses to individual proteins to predict the overall IgG WB result of 2524 EIA (C6)-positive samples from across Canada. Geographic and interannual variations in proportions of samples testing positive were explored by logistic regression. No one protein was highly concordant with the IgG WB result. Significant variations were found amongst years and geographic regions in the prevalence of samples testing positive using the overall IgG WB algorithm, and for individual proteins of the algorithm. In most cases the prevalence of samples testing positive were highest in Nova Scotia, and lower in samples from Manitoba westwards. These findings suggest that the current two tier test may not be simplified and continued use of the current two-tier test method and interpretation is recommended. Geographic and interannual variations in the prevalence of samples testing positive may be consistent with B. burgdorferi strain variation in Canada, and further studies are needed to explore this.

  10. Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies.

    PubMed

    Burghi, Valeria; Fernández, Natalia Cristina; Gándola, Yamila Belén; Piazza, Verónica Gabriela; Quiroga, Diego Tomás; Guilhen Mario, Érica; Felix Braga, Janaína; Bader, Michael; Santos, Robson Augusto Souza; Dominici, Fernando Pablo; Muñoz, Marina Cecilia

    2017-01-01

    Mas receptor (MasR) is a G protein-coupled receptor proposed as a candidate for mediating the angiotensin (Ang)-converting enzyme 2-Ang (1-7) protective axis of renin-angiotensin system. Because the role of this receptor is not definitively clarified, determination of MasR tissue distribution and expression levels constitutes a critical knowledge to fully understanding its function. Commercially available antibodies have been widely employed for MasR protein localization and quantification, but they have not been adequately validated. In this study, we carried on an exhaustive evaluation of four commercial MasR antibodies, following previously established criteria. Western Blotting (WB) and immunohistochemistry studies starting from hearts and kidneys from wild type (WT) mice revealed that antibodies raised against different MasR domains yielded different patterns of reactivity. Furthermore, staining patterns appeared identical in samples from MasR knockout (MasR-KO) mice. We verified by polymerase chain reaction analysis that the MasR-KO mice used were truly deficient in this receptor as MAS transcripts were undetectable in either heart or kidney from this animal model. In addition, we evaluated the ability of the antibodies to detect the human c-myc-tagged MasR overexpressed in human embryonic kidney cells. Three antibodies were capable of detecting the MasR either by WB or by immunofluorescence, reproducing the patterns obtained with an anti c-myc antibody. In conclusion, although three of the selected antibodies were able to detect MasR protein at high expression levels observed in a transfected cell line, they failed to detect this receptor in mice tissues at physiological expression levels. As a consequence, validated antibodies that can recognize and detect the MasR at physiological levels are still lacking.

  11. Evaluation of the Aspergillus Western blot IgG kit for diagnosis of chronic aspergillosis.

    PubMed

    Oliva, A; Flori, P; Hennequin, C; Dubus, J-C; Reynaud-Gaubert, M; Charpin, D; Vergnon, J M; Gay, P; Colly, A; Piarroux, R; Pelloux, H; Ranque, S

    2015-01-01

    Immunoprecipitin detection (IPD) is the current reference confirmatory technique for anti-Aspergillus antibody detection; however, the lack of standardization is a critical drawback of this assay. In this study, we evaluated the performance of the Aspergillus Western blot (Asp-WB) IgG kit (LDBio Diagnostics, Lyon, France), a recently commercialized immunoblot assay for the diagnosis of various clinical presentations of chronic aspergillosis. Three hundred eight serum samples from 158 patients with aspergillosis sensu lato (s.l.) were analyzed. More specifically, 267 serum samples were derived from patients with Aspergillus disease, including 89 cases of chronic pulmonary aspergillosis, 10 of aspergilloma, and 32 of allergic bronchopulmonary aspergillosis, while 41 samples were from patients with Aspergillus colonization, including 15 cystic fibrosis (CF) and 12 non-CF patients. For blood donor controls, the Asp-WB specificity was 94%, while the kit displayed a sensitivity for the aspergillosis s.l. diagnosis of 88.6%, with a diagnostic odds ratio (DOR) of 119 (95% confidence interval [CI], 57 to 251). The DOR values were 185.22 (95% CI,78.79 to 435.45) and 43.74 (95% CI, 15.65 to 122.20) for the diagnosis of Aspergillus disease and Aspergillus colonization, respectively. Among the patients, the sensitivities of the Asp-WB in the diagnosis of Aspergillus colonization were 100% and 41.7% in CF and non-CF patients, respectively. The Asp-WB yielded fewer false-negative results than did IPD. In conclusion, the Asp-WB kit performed well for the diagnosis of various clinical presentations of aspergillosis in nonimmunocompromised patients, with an enhanced standardization and a higher sensitivity than with IPD, which is the current reference method.

  12. Validity of the Enzyme-linked Immunoelectrotransfer Blot (EITB) for naturally acquired porcine cysticercosis.

    PubMed

    Jayashi, César M; Gonzalez, Armando E; Castillo Neyra, Ricardo; Rodríguez, Silvia; García, Hector H; Lightowlers, Marshall W

    2014-01-17

    The Enzyme-linked Immunoelectrotransfer Blot (EITB) has been used widely as a screening test for Taenia solium cysticercosis in swine. However, the relation between seropositivity and infection in pig populations from endemic areas has not been well defined. The aim of this study is to relate EITB seropositivity with infection and infection burden, analyse the trade-off between sensitivity and specificity with various cut-off points for the EITB assay, and finally describe the serology changes in a cohort of rural pigs raised under natural conditions. A group of 107 pigs that were used as controls during a vaccination field trial in Peru was our study population. The prevalence of porcine cysticercosis determined by necropsy examination was 16.82% (18/107) in these animals. Using EITB reactivity to ≥ 1 band as a cut-off point for the assay, the sensitivity was 88.89% (65.29-98.62, 95% CI) and the specificity was 48.31% (37.59-59.16, 95% CI). Comparing other cut-off points, involving up to as many as 7 reactive bands, a reactivity of ≥ 3 bands provided the best trade-offs in sensitivity and specificity. Using this cut-off point for the assay, the sensitivity was 77.77% (52.36-93.59, 95% CI) and the specificity was 76.40% (66.22-84.76, 95% CI). A significant association was found between cyst counts over 100 cysts and reactivity to ≥ 3 bands in the EITB assay (Fisher's exact test, p<0.05). The results of this study suggest that the use of the EITB assay to study porcine cysticercosis may require setting different cut-offs under field and experimental conditions, and depending upon the objective of the screening process. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Electrostatic protein immobilization using charged polyacrylamide gels and cationic detergent microfluidic Western blotting.

    PubMed

    Kim, Dohyun; Karns, Kelly; Tia, Samuel Q; He, Mei; Herr, Amy E

    2012-03-06

    We report a novel protein immobilization matrix for fully integrated microfluidic Western blotting (WB). The electrostatic immobilization gel (EIG) enables immobilization of all proteins sized using cetyl trimethylammonium bromide polyacrylamide gel electrophoresis (CTAB-PAGE), for subsequent electrophoretic probing with detection affinity reagents (e.g., labeled antibodies). The "pan-analyte" capture strategy introduced here uses polyacrylamide gel grafted with concentrated point charges (zwitterionic macromolecules), in contrast to existing microfluidic WB strategies that rely on a sandwich immunoassay format for analyte immobilization and detection. Sandwich approaches limit analyte immobilization to capture of only a priori known targets. A charge interaction mechanism study supports the hypothesis that electrostatic interaction plays a major role in analyte immobilization on the EIG. We note that protein capture efficiency depends on both the concentration of copolymerized charges and ionic strength of the gel buffer. We demonstrate pan-analyte immobilization of sized CTAB-laden model proteins (protein G, ovalbumin, bovine serum albumin, β-galactosidase, lactoferrin) on the EIG with initial capture efficiencies ranging from 21 to 100%. Target proteins fixed on the EIG (protein G, lactoferrin) are detected using antibody probes with signal-to-noise ratios of 34 to 275. The approach advances protein immunoblotting performance through 200× reduction on sample consumption, 12× reduction in assay duration, and automated assay operation, compared to slab-gel WB. Using the microfluidic WB assay, assessment of lactoferrin in human tear fluid is demonstrated with a goal of advancing toward nonbiopsy-based diagnosis of Sjögren's Syndrome, an autoimmune disease.

  14. A rapid chemiluminescent slot blot immunoassay for the detection and quantification of Clostridium botulinum neurotoxin type E, in cultures.

    PubMed

    Cadieux, Brigitte; Blanchfield, Burke; Smith, James P; Austin, John W

    2005-05-01

    A simple, rapid, cost-effective in vitro slot blot immunoassay was developed for the detection and quantification of botulinum neurotoxin type E (BoNT/E) in cultures. Culture supernatants of 36 strains of clostridia, including 12 strains of Clostridium botulinum type E, 12 strains of other C. botulinum neurotoxin serotypes, and 12 strains of other clostridial species were tested. Samples containing BoNT/E were detected using affinity-purified polyclonal rabbit antisera prepared against BoNT/E with subsequent detection of secondary antibodies using chemiluminescence. All strains of C. botulinum type E tested positive, while all non C. botulinum type E strains tested negative. The sensitivity of the slot blot immunoassay for detection of BoNT/E was approximately four mouse lethal doses (MLD). The intensity of chemiluminescence was directly correlated with the concentration of BoNT/E up to 128 MLD, allowing quantification of BoNT/E between 4 and 128 MLD. The slot blot immunoassay was compared to the mouse bioassay for detection of BoNT/E using cultures derived from fish samples inoculated with C. botulinum type E, and cultures derived from naturally contaminated environmental samples. A total of 120 primary enrichment cultures derived from fish samples, of which 103 were inoculated with C. botulinum type E, and 17 were uninoculated controls, were assayed. Of the 103 primary enrichment cultures derived from inoculated fish samples, all were positive by mouse bioassay, while 94 were also positive by slot blot immunoassay, resulting in a 7.5% false-negative rate. All 17 primary enrichment cultures derived from the uninoculated fish samples were negative by both mouse bioassay and slot blot immunoassay. A total of twenty-six primary enrichment cultures derived from environmental samples were tested by mouse bioassay and slot blot immunoassay. Of 13 primary enrichment cultures positive by mouse bioassay, 12 were also positive by slot blot immunoassay, resulting in a 3

  15. Qualitative and quantitative evaluation of Simon™, a new CE-based automated Western blot system as applied to vaccine development.

    PubMed

    Rustandi, Richard R; Loughney, John W; Hamm, Melissa; Hamm, Christopher; Lancaster, Catherine; Mach, Anna; Ha, Sha

    2012-09-01

    Many CE-based technologies such as imaged capillary IEF, CE-SDS, CZE, and MEKC are well established for analyzing proteins, viruses, or other biomolecules such as polysaccharides. For example, imaged capillary isoelectric focusing (charge-based protein separation) and CE-SDS (size-based protein separation) are standard replacement methods in biopharmaceutical industries for tedious and labor intensive IEF and SDS-PAGE methods, respectively. Another important analytical tool for protein characterization is a Western blot, where after size-based separation in SDS-PAGE the proteins are transferred to a membrane and blotted with specific monoclonal or polyclonal antibodies. Western blotting analysis is applied in many areas such as biomarker research, therapeutic target identification, and vaccine development. Currently, the procedure is very manual, laborious, and time consuming. Here, we evaluate a new technology called Simple Western™ (or Simon™) for performing automated Western analysis. This new technology is based on CE-SDS where the separated proteins are attached to the wall of capillary by a proprietary photo activated chemical crosslink. Subsequent blotting is done automatically by incubating and washing the capillary with primary and secondary antibodies conjugated with horseradish peroxidase and detected with chemiluminescence. Typically, Western blots are not quantitative, hence we also evaluated the quantitative aspect of this new technology. We demonstrate that Simon™ can quantitate specific components in one of our vaccine candidates and it provides good reproducibility and intermediate precision with CV <10%. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Identification of α1-Antitrypsin as a Potential Candidate for Internal Control for Human Synovial Fluid in Western Blot.

    PubMed

    Wang, Shaowei; Zhou, Jingming; Wei, Xiaochun; Li, Pengcui; Li, Kai; Wang, Dongming; Wei, Fangyuan; Zhang, Jianzhong; Wei, Lei

    Western blot of synovial fluid has been widely used for osteoarthritis (OA) research and diagnosis, but there is no ideal loading control for this purpose. Although β-actin is extensively used as loading control in western blot, it is not suitable for synovial fluid because it is not required in synovial fluid as a cytoskeletal protein. A good loading control for synovial fluid in OA studies should have unchanged content in synovial fluids from normal and OA groups, because synovial fluid protein content can vary with changes in synovial vascular permeability with OA onset. In this study, we explore the potential of using α1-antitripsin (A1AT) as loading control for OA synovial fluid in western blot. A1AT level is elevated in inflammatory conditions such as rheumatoid arthritis (RA). Unlike RA, OA is a non-inflammation disease, which does not induce A1AT. In this study, we identified A1AT as an abundant component of synovial fluid by Mass Spectrometry and confirmed that the level of A1AT is relative constant between human OA and normal synovial fluid by western blot and ELISA. Hence, we proposed that A1AT may be a good loading control for western blot in human OA synovial fluid studies provided that pathological conditions such as RA or A1AT deficiency associated liver or lung diseases are excluded.

  17. Enrichment of PrPSc in formalin-fixed, paraffin-embedded tissues prior to analysis by Western blot.

    PubMed

    Nicholson, Eric M

    2011-07-01

    Diagnosis of prion disease is primarily through immunodetection of the infectious agent. Typically, 2 distinct procedures are recommended for a definitive diagnosis, with immunohistochemistry and Western blot providing the most information as to the specific isolate in question. In the past, these approaches required formalin-fixed, paraffin-embedded tissue and fresh or frozen tissue, respectively; however, methods have been developed that allow for use of fixed tissue for Western blot. The present study describes a method of enriching PrP(Sc) in formalin-fixed, paraffin-embedded tissues prior to Western blot analysis for the detection of PrP(Sc). With this modified procedure, 5 times the previously reported sample size may be used for analysis, greatly enhancing the sensitivity of this procedure.

  18. Double-blotting: a solution to the problem of non-specific binding of secondary antibodies in immunoblotting procedures.

    PubMed

    Lasne, F

    2001-07-01

    "Double-blotting" (DB) was developed to overcome the problem of non-specific binding of secondary antibodies in immunoblotting (IB). After it had been probed by the primary antibody, the membrane with the blotted proteins was assembled with a second blank membrane and submitted to a second blotting under acidic conditions. The primary antibody molecules were thus desorbed from their corresponding antigen and transferred onto the second membrane, whereas the antigen and the interfering proteins remained bound to the first one. The second membrane could then be probed by the secondary antibodies without the risk of non-specific binding. This method was developed for the study of erythropoietin (EPO) in concentrated urine since a strong non-specific binding of biotinylated secondary antibodies to some urinary proteins had been observed using classical IB protocols.

  19. Double-blotting: a solution to the problem of nonspecific binding of secondary antibodies in immunoblotting procedures.

    PubMed

    Lasne, Françoise

    2003-05-01

    "Double-blotting" (DB) has been developed to overcome the problem of nonspecific binding of secondary antibodies in immunoblotting (IB). After it has been probed by the primary antibody, the membrane with the blotted proteins is assembled with a second blank membrane and submitted to a second blotting under acidic conditions. The primary antibody molecules are thus desorbed from their corresponding antigen and transferred onto the second membrane, whereas the antigen and the interfering proteins remain bound to the first one. The second membrane can then be probed by the secondary antibodies without the risk of nonspecific binding. This method has been developed for the study of erythropoietin (EPO) in concentrated urine since a strong nonspecific binding of biotinylated secondary antibodies to some urinary proteins is observed using classical IB protocols. However, its concept makes it usable in other applications that come up against this kind of problem.

  20. Detection of enterotoxigenic Clostridium perfringens in spices used in Mexico by dot blotting using a DNA probe.

    PubMed

    Rodríguez-Romo, L A; Heredia, N L; Labbé, R G; García-Alvarado, J S

    1998-02-01

    Several reports on the microbiology of spices and herbs indicate the presence of Clostridium perfringens, a spore-forming foodborne pathogen responsible for gastrointestinal disease. In the present study, a total of 380 samples of spices and herbs (cumin seed, black pepper, oregano, garlic powder, and bay leaves) widely used in Mexico were analyzed for the presence of C. perfringens, and the enterotoxigenicity of the isolates was determined by a dot-blot technique using an enterotoxin degoxigenin-labeled DNA probe. C. perfringens counts varied from <100 to 433 CFU/g in garlic powder, from <100 to 200 CFU/g in black pepper, from <100 to 433 CFU/g in cumin seed, from <100 to 340 CFU/g in oregano, and from < 100 to 450 CFU/g in bay leaves. The dot-blot technique detected the enterotoxin gene in 8 (4.25%) of 188 confirmed isolates of C. perfringens. dot-blot.

  1. Product-selective blot: a technique for measuring enzyme activities in large numbers of samples and in native electrophoresis gels

    SciTech Connect

    Thompson, G.A.; Davies, H.M.; McDonald, N.

    1985-08-01

    A method termed product-selective blotting has been developed for screening large numbers of samples for enzyme activity. The technique is particularly well suited to detection of enzymes in native electrophoresis gels. The principle of the method was demonstrated by blotting samples from glutaminase or glutamate synthase reactions into an agarose gel embedded with ion-exchange resin under conditions favoring binding of product (glutamate) over substrates and other substances in the reaction mixture. After washes to remove these unbound substances, the product was measured using either fluorometric staining or radiometric techniques. Glutaminase activity in native electrophoresis gels was visualized by a related procedure in which substrates and products from reactions run in the electrophoresis gel were blotted directly into a resin-containing image gel. Considering the selective-binding materials available for use in the image gel, along with the possible detection systems, this method has potentially broad application.

  2. Fertilization of Northern Hardwoods

    Treesearch

    R. Lea; D.G. Brockway

    1986-01-01

    Northern hardwoods grow over a considerable range of climatic and edaphic conditions and exhibit a wide range in productivity.Many northern hardwood forests are capable of high production relative to other forest types, but are often slow to reach maximum productivity because of low nutrient availability.Altering the patterns of biomass accumulation so that managers...

  3. What are northern hardwoods?

    Treesearch

    Richard M. Godman

    1992-01-01

    The term "northern hardwoods" was used in the early 1900's to separate the hardwoods of the northern region from those growing in the South. With continued usage in the North the term now represents all dense hardwood species both in the Lake States and Northeast. Unfortunately, this has complicated describing and applying silvicultural practices for...

  4. Stain-free detection as loading control alternative to Ponceau and housekeeping protein immunodetection in Western blotting.

    PubMed

    Rivero-Gutiérrez, B; Anzola, A; Martínez-Augustin, O; de Medina, F Sánchez

    2014-12-15

    It is currently a routine practice to require a measurement of a housekeeping reference, including actin, glyceraldehyde-3-phosphate dehydrogenase, β-tubulin, among others, in Western blots, as it is the rule in RNA blots. Reversible Ponceau staining has been applied successfully to check equal loading of gels. Here we test a new technique, with the Stain-Free gels from Bio-Rad, against both Ponceau staining and housekeeping protein immunodetection under different conditions. Our results show that Stain-Free gels outperform Ponceau staining and that both are more consistent than housekeeping proteins as a loading control.

  5. Stain-Free total protein staining is a superior loading control to β-actin for Western blots.

    PubMed

    Gilda, Jennifer E; Gomes, Aldrin V

    2013-09-15

    Semi-quantification of proteins using Western blots typically involves normalization against housekeeping genes such as β-actin. More recently, Ponceau S and Coomassie blue staining have both been shown to be suitable alternatives to housekeeping genes as loading controls. Stain-Free total protein staining offers the advantage of no staining or destaining steps. Evaluation of the use of Stain-Free staining as an alternative to β-actin or the protein stain Ponceau S showed that Stain-Free staining was superior to β-actin and as good as or better than Ponceau S staining as a loading control for Western blots.

  6. Enrichment of PrPSc in Formalin Fixed Paraffin Embedded Tissues Prior to Analysis by Western Blot

    USDA-ARS?s Scientific Manuscript database

    Diagnosis of prion disease is primarily through immunodetection of the infectious agent. Typically, 2 distinct procedures are recommended for a definitive diagnosis with immunohistochemistry and Western blot providing the most information as to the specific isolate in question. In the past these app...

  7. Novel chemiluminescent Western blot blocking and antibody incubation solution for enhanced antibody-antigen interaction and increased specificity.

    PubMed

    Schwartz, Kimberly; Bochkariov, Dmitry

    2017-07-13

    Western blotting is a ubiquitous tool used in protein and molecular biology research, providing information about the presence, size, relative abundance, and state of a protein in a mixture. First, the proteins in a sample are separated by size using SDS-PAGE then transferred onto a membrane for detection with a set of primary and secondary antibodies. High-quality Western data requires high signal-to-noise ratios, which depend upon reduction of nonspecific antibody interactions. Blocking is a critical step in the Western blot method as it prevents the antibodies from binding nonspecifically to the membrane and irrelevant proteins. A solution of nonfat dry milk (NFDM) in physiological buffer is commonly used for this purpose, but does not perform well with every type of antibody and is not optimal for low-abundance proteins. We present a novel blocking solution for chemiluminescent Western blots, AdvanBlock™-chemi, which outperforms NFDM in experiments with 20 unique antibodies by increasing signal-to-noise ratios and minimizing nonspecific binding. This solution enhances protein detection by Western blot and provides consistent results for detection of low abundant and modified proteins. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Western blot assay using recombinant p26 antigen for detection of equine infectious anemia virus-specific antibodies.

    PubMed

    Alvarez, I; Gutierrez, G; Ostlund, E; Barrandeguy, M; Trono, K

    2007-12-01

    We analyzed the performance of a single-band Western blot (WB) test using recombinant p26 (rp26) capsid protein of equine infectious anemia virus. According to the results obtained, the rp26 WB test is a reliable confirmatory diagnostic tool to be used as a complementary test after an enzyme-linked immunosorbent assay or agar gel immunodiffusion test yielding doubtful results.

  9. Proteomic analysis of human biopsy samples by single two-dimensional electrophoresis: Coomassie, silver, mass spectrometry, and Western blotting.

    PubMed

    McDonough, Jason L; Neverova, Irina; Van Eyk, Jennifer E

    2002-08-01

    Proteomic analysis of myocardial tissue from patient populations is critical to our understanding of cardiac disease, but has been limited until now by the small size of biopsies (approximately 20-50 microg), and complicated by the difference in relative abundance of contractile proteins over other cellular components. Here we describe an approach to analysis of myocardial biopsies from patients undergoing coronary artery bypass surgery. First, individual biopsies are selectively extracted, producing subfractions that correspond to the contractile proteins and the cytosolic proteins. Two-dimensional electrophoresis separated proteins are detected by first staining with Coomassie blue then silver, to permit a wider range of accurate quantification. Western blotting of two-dimensional separated samples, to validate peptide mass fingerprinting data, previously required additional gel separations for transfer since staining protocols are not compatible with transfer to membranes or immunoblotting. An existing silver destaining protocol was adapted to allow removal of silver from a whole gel, followed by transfer and Western blotting. An existing Coomassie blue removal protocol was also adapted to permit Western blotting of gels stained with Coomassie blue and silver. Together, these techniques permit peptide mass fingerprinting concurrent with Western blotting of a single protein spot from a single biopsy, eliminating the need for repeated gel separations, and improving spot alignment between immunoblots and stained gels. In the end, this approach may allow a more complete characterization of protein changes in small human biopsies, and also reduce the number of repeated gel separations necessary for a standard proteomic analysis.

  10. Detection of X chromosome aneuploidy using Southern blot analysis during routine population-based screening for fragile X syndrome.

    PubMed

    Adir, Vardit; Shahak, Elena; Dar, Hanna; Borochowitz, Zvi U

    2003-01-01

    We report herein two cases where detection of X chromosome aneuploidy (cytogenetically proved 45,X/46XX and 47,XXX) was made possible by molecular diagnosis during population-based carrier screening for Fragile X syndrome, using Southern blot analysis. This study emphasizes the value of molecular analysis for gene dosage to suggest chromosomal aneuploidy.

  11. Northern Trust Mines

    EPA Pesticide Factsheets

    The United States and the Navajo Nation entered into settlement agreements that provide funds to conduct investigations and any needed cleanup at 16 of the 46 priority mines, including six mines in the Northern Abandoned Uranium Mine Region.

  12. Northern Arizona Volcanoes

    NASA Image and Video Library

    2006-05-01

    Northern Arizona is best known for the Grand Canyon. Less widely known are the hundreds of geologically young volcanoes, at least one of which buried the homes of local residents. This image was acquired by NASA Terra spacecraft.

  13. Spotting Saturn Northern Storm

    NASA Image and Video Library

    2011-07-06

    NASA Cassini spacecraft captures a composite near-true-color view of the largest and most intense storm observed on Saturn. The storm is seen churning through the atmosphere in Saturn northern hemisphere.

  14. Standard loading controls are not reliable for Western blot quantification across brain development or in pathological conditions.

    PubMed

    Goasdoue, Kate; Awabdy, Doreen; Bjorkman, Stella Tracey; Miller, Stephanie

    2016-02-01

    A frequently utilized method of data quantification in Western blot analysis is comparison of the protein of interest with a house keeping gene or control protein. Commonly used proteins include β-actin, glyceraldehyde 3 phosphate dehydrogenase (GAPDH), and α-tubulin. Various reliability issues have been raised when using this technique for data analysis-particularly when investigating protein expression changes during development and in disease states. In this study, we have demonstrated that β-actin, GAPDH, and α-tubulin are not appropriate controls in the study of development and hypoxic-ischemic induced damage in the piglet brain. We have also shown that using an in-house pooled standard, loaded on all blots is a reliable method for controlling interassay variability and data normalization in protein expression analysis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Rapid Screening of the Epidermal Growth Factor Receptor Phosphosignaling Pathway via Microplate-Based Dot Blot Assays

    PubMed Central

    Cappione, Amedeo; Smith, Janet; Mabuchi, Masaharu; Nadler, Timothy

    2012-01-01

    Expression profiling on a large scale, as is the case in drug discovery, is often accomplished through use of sophisticated solid-phase protein microarrays or multiplex bead technologies. While offering both high-throughput and high-content analysis, these platforms are often too cost prohibitive or technically challenging for many research settings. Capitalizing on the favorable attributes of the standard ELISA and slot blotting techniques, we developed a modified dot blot assay that provides a simple cost-effective alternative for semiquantitative expression analysis of multiple proteins across multiple samples. Similar in protocol to an ELISA, but based in a membrane bound 96-well microplate, the assay takes advantage of vacuum filtration to expedite the tedious process of washing in between binding steps. We report on the optimization of the assay and demonstrate its use in profiling temporal changes in phosphorylation events in the well-characterized EGF-induced signaling cascade of A431 cells. PMID:22934183

  16. Detection of La/SS-B by western blot using nanogold-tagged antibodies and silver enhancement.

    PubMed

    Maier, Shannon; Hubbell, Sherry; Scofield, R Hal

    2009-01-01

    Immunogold staining with silver enhancement is a versatile, sensitive and specific method for immunodetection of diverse protein antigens separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose or polyvinylidene difluoride membranes. "Next-generation" antibodies tagged with nanogold particles have a wide scope of use including but not limited to immunohistochemistry, western blotting, electron microscopy, fluorescent activated cell sorting procedures, and cell isolation and migration studies. Herein, we describe the use of a nanogold-tagged anti-mouse IgG secondary antibody and silver enhancement methodologies coupled with antigen-specific unlabeled primary antibodies for the detection of the La/SS-B autoantigen by western blotting as a useful alternative to chemiluminescent and enzymatic detection methods.

  17. Analysis of Gene and Protein Expression in Atherosclerotic Mouse Aorta by Western Blot and Quantitative Real-Time PCR.

    PubMed

    Rivera-Torres, José

    2015-01-01

    Atherosclerosis involves changes in gene and protein expression patterns in affected arteries. Quantification of these alterations is essential for understanding the molecular mechanisms underlying this pathology. Western blot and real-time PCR-used to quantify protein and messenger RNA levels, respectively-are invaluable molecular biology tools, particularly when material is limited. The availability of many genetically modified mouse models of atherosclerosis makes the mouse aorta an ideal tissue in which to carry out these expression pattern analyses. In this chapter, protocols are presented for mRNA and protein extraction from mouse aorta and for the accurate quantification of mRNA expression by RT-PCR and of proteins by western blot.

  18. Snapshot blotting: transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to grids for electron microscopy.

    PubMed Central

    Jett, S D; Bear, D G

    1994-01-01

    We present a technique, "snapshot blotting," for the electrophoretic transfer of nucleic acids and nucleoprotein complexes in gel electrophoresis bands onto highly stable carbon film-coated grids for imaging by electron microscopy. The method permits structural analysis of macromolecular species that have been resolved by a gel mobility-shift assay. To demonstrate the efficiency and integrity of the transfer process for a multiprotein-DNA assembly, we have imaged various species of a prokaryotic transcription complex, using the cleavage-defective EcoRI(Q111) protein as an orientation marker and as a blockade of transcription elongation. Snapshot blotting should be of great utility in the structural characterization of nucleic acids and protein-nucleic acid interactions. Images PMID:8041711

  19. A single-step simultaneous protein staining procedure for polyacrylamide gels and nitrocellulose membranes by Alta during western blot analysis.

    PubMed

    Pal, Jayanta K; Berwal, Sunil K; Soni, Rupali N

    2012-01-01

    A simple method for staining of proteins simultaneously on sodium dodecyl sulfate (SDS) polyacrylamide gels and nitrocellulose membranes by Alta during western blot analysis is described. A 5% solution of Alta, a commercially available cosmetic preparation, is added in the upper tank buffer during electrophoresis. On completion of electrophoresis, the gel is washed in distilled water and viewed on a white light plate and a transilluminator to photograph the protein profiles. The gel is processed for western blot transfer of proteins onto a nitrocellulose membrane, and upon completion, the protein profiles on the membrane are viewed and photographed as stated above. The membrane can then be processed for immunostaining as per the standard procedure. Thus, the staining procedure using Alta is simple, rapid (without any need of destaining), and cost-effective.

  20. Differentiation of Enterococcus faecium from Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains by PCR and dot-blot hybridisation.

    PubMed

    Langa, S; Fernández, A; Martín, R; Reviriego, C; Marín, M L; Fernández, L; Rodríguez, J M

    2003-12-01

    Variations in length and sequence of the 16S/23S spacer region of Enterococcus faecium provided the basis for development of simple PCR and dot-blot hybridisation assays that enabled the differentiation of potentially probiotic Enterococcus faecium strains from Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. Such assays may be useful for differentiation of yoghurt starter cultures and enterococcal strains when they are simultaneously present in probiotic food products.

  1. Investigation of telomerase activity and apoptosis on invasive ductal carcinoma of the breast using immunohistochemical and Western blot methods.

    PubMed

    Simsek, B C; Turk, B A; Ozen, F; Tuzcu, M; Kanter, M

    2015-08-01

    Invasive ductal carcinoma (IDC) comprises the largest group of breast cancers. This study aimed to investigate telomerase activity and apoptosis using immunohistochemical and Western blot methods. In total, 75 cases that had been diagnosed as IDC and 20 cases that had undergone a freezing procedure were included. The histological sections were stained with Bax, Bcl-2, hTERT and BNIP3. The ages of the patients, as well as their hormonal status and tumour sizes and grades were evaluated, as well as the staining characteristics of the antibodies in question. A decrease in Bcl-2 positivity and an increase in Bax positivity were found immunohistochemically with increasing tumour grades. The data obtained by western blot method showed that Bcl-2 was highest in grade 1 tumours although these results were not statistically significant. The relationship between estrogen and progesterone receptor positivity and Bcl-2 was statistically significant, suggesting there is hormonal control through apoptosis. BNIP3 was found to be decreased with increasing tumour grades. Similarly, BNIP3 was found to be having the lowest value in grade 3 tumours by western blot method. Furthermore, hTERT was found to be increased with increasing tumour grades. In the western blot method, hTERT increased nearly four-fold compared to the control. In addition, hTERT, which was seen in very high levels in tumours, may be a helpful cancer marker. Both hTERT and BNIP3 are important markers that can provide information about prognosis. Big improvements can be achieved in tumour progression control with new treatment modalities that stop telomerase activity and hypoxic cell death.

  2. Stabilization of hypoxia-inducible factor-1α in buffer containing cobalt chloride for Western blot analysis.

    PubMed

    Srinivasan, Sathyanarayanan; Dunn, Jeff F

    2011-09-01

    Hypoxia-inducible factor-1α (HIF-1α) is a widely studied protein with significant biomedical impact. Care is needed to stabilize HIF-1α protein during sample preparation for Western blot analysis due to its rapid degradation in the presence of oxygen. Enzyme inhibitor cocktails can be complex and expensive. We present a protease inhibitor-free buffer, containing cobalt chloride, which is effective at stabilizing HIF-1α, while being inexpensive, straightforward, and convenient, and has potential for widespread application.

  3. Western Blot Assay Using Recombinant p26 Antigen for Detection of Equine Infectious Anemia Virus-Specific Antibodies▿

    PubMed Central

    Alvarez, I.; Gutierrez, G.; Ostlund, E.; Barrandeguy, M.; Trono, K.

    2007-01-01

    We analyzed the performance of a single-band Western blot (WB) test using recombinant p26 (rp26) capsid protein of equine infectious anemia virus. According to the results obtained, the rp26 WB test is a reliable confirmatory diagnostic tool to be used as a complementary test after an enzyme-linked immunosorbent assay or agar gel immunodiffusion test yielding doubtful results. PMID:17959820

  4. Investigation of Anti-Toxocara Antibodies in Epileptic Patients and Comparison of Two Methods: ELISA and Western Blotting

    PubMed Central

    Zibaei, Mohammad; Firoozeh, Farzaneh; Bahrami, Parviz; Sadjjadi, Seyed Mahmoud

    2013-01-01

    The relationship between Toxocara infection and epilepsy was previously demonstrated by several case-control studies and case reports. These previous studies were often based on the enzyme-linked immunosorbent assay (ELISA) using Toxocara excretory-secretory antigens, which are not specific due to cross-reactivity with other parasitic infections such as ascariasis, trichuriasis, and anisakiasis. An immunoblot analysis is highly specific and can detect low levels of Toxocara antibodies. Therefore, this assay may be useful in the identification of toxocariasis in epileptic patients. We examined patients who had epilepsy and healthy subjects for seropositivity for Toxocara infection by ELISA and Western blotting. Out of 85 epileptic patients, 10 (11.8%) and 3 (3.5%) persons exhibited Toxocara immunoglobulin G (IgG) antibodies responses by ELISA and by both techniques, respectively. Moreover, in the healthy group (n = 85), 3 (3.5%) persons were positive by ELISA, but none was detected by Western blotting. This study indicates that Toxocara infection is a risk factor for epilepsy in Iran. These findings strongly suggest the need to perform Western blotting immunodiagnosis, as well as the ELISA using Toxocara excretory-secretory antigens, to improve diagnosis of human toxocariasis in patients with epilepsy. PMID:23710354

  5. Western blot confirmation of the H+/K+-ATPase proton pump in the human larynx and submandibular gland.

    PubMed

    Altman, Kenneth W; Kinoshita, Yayoi; Tan, Melin; Burstein, David; Radosevich, James A

    2011-11-01

    The authors have previously demonstrated the H(+)/K(+)-ATPase (proton pump) in human larynx and lung glands via immunohistochemistry (IHC). The present hypothesis is that the proton pump is expressed in other seromucinous glands of the digestive tract that can be confirmed by IHC and Western blot analysis. Prospective controlled tissue analysis study. Academic medical institution. Ten anonymous fresh-frozen donor specimens were obtained, comprising 3 submandibular glands, 4 larynges, and 3 normal stomach specimens for control. Submandibular gland sections were immunostained with 2 monoclonal antibodies selectively reactive with α or β subunits of the H(+)/K(+)-ATPase. Western blot analysis was performed on all specimens. Consistent IHC staining was observed in the submandibular gland specimens for both α and β subunits. Western blot analysis revealed very strong expression for the stomach at 100 kDa, corresponding to the α protein, and weak but notable banding for all larynx and submandibular gland specimens. Similar findings were noted for the 60- to 80-kDa glycosylated β subunit protein, as well as the 52-kDa β subunit precursor for all specimens. The H(+)/K(+)-ATPase (proton) pump is present in the human larynx and submandibular gland although in much lower concentrations than in the stomach. Proton pump involvement in human aerodigestive seromucinous glands may have a role in protecting mucosa from acid environments (local or systemic), explain heightened laryngeal sensitivity in those patients with laryngopharyngeal reflux, and be a site of action for proton pump inhibitor pharmacotherapy.

  6. Development of PCR/dot blot assay for specific detection and differentiation of taeniid cestode eggs in canids.

    PubMed

    Armua-Fernandez, Maria Teresa; Nonaka, Nariaki; Sakurai, Tatsuya; Nakamura, Seita; Gottstein, Bruno; Deplazes, Peter; Phiri, Isaac G K; Katakura, Ken; Oku, Yuzaburo

    2011-01-01

    We report the development of a colourimetric PCR/dot blot assay targeting the mitochondrial gene NADH dehydrogenase subunit 1 (nad1) for differential diagnosis of taeniid eggs. Partial sequences of the cestode nad1 gene were aligned and new primers were designed based on conserved regions. Species-specific oligonucleotide probes (S-SONP) for canine taeniid cestodes were then designed manually based on the variable region between the conserved primers. Specifically, S-SONP were designed for the Taenia crassiceps, T. hydatigena, T. multiceps, T. ovis, T. taeniaeformis, Echinococcus granulosus (genotype 1), E. multilocularis and E. vogeli. Each probe showed high specificity as no cross-hybridisation with any amplified nad1 fragment was observed. We evaluated the assay using 49 taeniid egg-positive samples collected from dogs in Zambia. DNA from 5 to 10 eggs was extracted in each sample. Using the PCR/dot blot assay, the probes successfully detected PCR products from T. hydatigena in 42 samples, T. multiceps in 3 samples, and both species (mixed infection) in the remaining 4 samples. The results indicate that the PCR/dot blot assay is a reliable alternative for differential diagnosis of taeniid eggs in faecal samples.

  7. Differential detection of cytoplasmic Wilms tumor 1 expression by immunohistochemistry, western blotting and mRNA quantification.

    PubMed

    Maki, Takehiro; Ikeda, Hiroaki; Kuroda, Aki; Kyogoku, Noriaki; Yamamura, Yoshiyuki; Tabata, Yukiko; Abiko, Takehiro; Tsuchikawa, Takahiro; Hida, Yasuhiro; Shichinohe, Toshiaki; Tanaka, Eiichi; Kaga, Kichizo; Hatanaka, Kanako; Matsuno, Yoshihiro; Imai, Naoko; Hirano, Satoshi

    2017-01-01

    Wilms tumor 1 (WT1) is considered to be a promising target of cancer treatment because it has been reported to be frequently expressed at high levels in various malignancies. Although WT1-targeted cancer treatment has been initiated, conclusive detection methods for WT1 are not established. The present study aimed to consolidate immunohistochemistry for WT1 with statistical basis. Transfected cells with forced WT1 expression yielded specific western blot bands and nuclear immunostaining; cytoplasmic immunostaining was not specifically recognized. Immunohistochemistry, western blotting, and quantitative reverse transcriptase-polymerase chain reaction were performed in 35 human cell lines using multiple WT1 antibodies and their results were quantified. Relationships among the quantified results were statistically analyzed; the nuclear immunostaining positively correlated with western blot bands and mRNA expression levels, whereas cytoplasmic immunostaining did not. These results indicate that nuclear immunostaining reflects WT1 expression but cytoplasmic immunostaining does not. The nuclear immunostaining was barely (3/541) observed in primary cancer of esophagus, bile duct, pancreas and lung. Although the present study has some limitations, the results indicate that the cytoplasmic immunostaining does not correlate with actual WT1 expression and prompts researchers to carefully evaluate target molecule expression in treatment of cancer.

  8. Definition of antigen specificity for antimitochondrial proteins detected by Western blotting using native mitochondrial proteins in primary biliary cirrhosis.

    PubMed

    Miyakawa, H; Kawaguchi, N; Kikuchi, K; Fujikawa, H; Kitazawa, E; Matsushita, M

    2001-10-01

    The major autoantigens to anti-mitochondrial antibody (AMA) in primary biliary cirrhosis (PBC) have previously been identified to be PDC-E2, BCOADC-E2, and OGDC-E2. However, analysis of these autoantigens to AMA cannot be examined using the two routine assays; immmunofluorescence and ELISA. Moreover, there are some problems in specificity and sensitivity in these routine assays. So, analysis with Western blotting using native mitochondrial protein as the antigen is required; it allows the identification of the molecular weights for the proteins which react with AMA in patients' sera. However, since the antigen-proteins used are not unified, molecular weights of AMA corresponding proteins vary among laboratories. In the present study, as the first step to help address this issue, we investigated the antigen specificity of protein bands detected by Western blotting using our in-house bovine and porcine heart mitochondrial proteins. Three major recombinant mitochondrial proteins were prepared. The antigen specificity was examined by the absorption tests preincubated with the three recombinant mitochondrial proteins. The molecular weights of developing our bovine and porcine heart mitochondrial proteins using SDS-PAGE were multiple protein bands including 74, 52, 50, and 43 kDa protein bands. Of them, the 74, 50, and 43 kDa protein bands were absorbed with preincubations of recombinant PDC-E2, BCOADC-E2, and OGDC-E2 protein, respectively. AMA specificity of these three major proteins with our Western blotting was confirmed.

  9. Northern Plains of Mars

    NASA Technical Reports Server (NTRS)

    2004-01-01

    22 November 2004 This Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) image shows a typical view of the martian northern plains. Thousands of square kilometers of the northern middle and polar latitudes of Mars look similar to the scene in this image. In late spring and in summer, dust devils crisscross the northern plains, leaving a variety of dark streaks. The streaks do not survive from year to year, indicating their ephemeral nature. The circular features in this image, including the prominent bright circular feature near the bottom, are the locations of buried meteor impact craters. This image is located near 58.1oN, 207.6oW, and covers an area approximately 3 km (1.9 mi) wide. Sunlight illuminates the scene from the lower left.

  10. Northern Plains 'Crater'

    NASA Technical Reports Server (NTRS)

    2004-01-01

    10 December 2004 The lower left (southwest) corner of this Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) image shows the location of a somewhat filled and buried meteor impact crater on the northern plains of Mars. The dark dots are boulders. A portion of a similar feature is seen in the upper right (northeast) corner of the image. This picture, showing landforms (including the odd mound north/northeast of the crater) that are typical of the martian northern lowland plains, was obtained as part of the MGS MOC effort to support the search for a landing site for the Phoenix Mars Scout lander. Phoenix will launch in 2007 and land on the northern plains in 2008. This image is located near 68.0oN, 227.4oW, and covers an area approximately 3 km (1.9 mi) wide. The scene is illuminated by sunlight from the lower left.

  11. Diapause-specific gene expression in the northern house mosquito, Culex pipiens L., identified by suppressive subtractive hybridization

    USDA-ARS?s Scientific Manuscript database

    In this study we probe the molecular events underpinning diapause observed in overwintering females of Culex pipiens. Using suppressive subtractive hybridization (SSH) we have identified 40 genes that are either upregulated or downregulated during this seasonal period of dormancy. Northern blot hybr...

  12. FACING NORTHEAST ACROSS NORTHERN END OF PARK TOWARDS ITS NORTHERN ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    FACING NORTHEAST ACROSS NORTHERN END OF PARK TOWARDS ITS NORTHERN CORNER - Candler Park Historic District, Roughly bounded by Moreland, Dekalb, McLendon & Harold Avenues, Matthews Street & Clifton Terrace, Atlanta, Fulton County, GA

  13. Blot-based detection of dehydroalanine-containing glutathione peroxidase with the use of biotin-conjugated cysteamine.

    PubMed

    Rhee, Sue Goo; Cho, Chun-Seok

    2010-01-01

    Dehydroalanine (DHA), alpha,beta-unsaturated amino acid, is found in the position corresponding to the serine, cysteine, and selenocysteine (Sec) residues of various proteins. Proteinaceous Sec is readily oxidized and subsequently undergoes beta-elimination to produce DHA. Glutathione peroxidase (GPx), which contains a Sec at the active site, is irreversibly inactivated by its own substrate as the result of the oxidation of selenium atom followed by the conversion of oxidized Sec to DHA. We developed a convenient method for estimation of the amount of DHA-GPx1 in cell homogenates. This blot-based method depends on specific addition of biotin-conjugated cysteamine to the DHA residue followed by detection of biotinylated protein based on its interaction with streptavidin. The method required an immunoprecipitation of GPx1 before labeling with the cysteamine derivative because many other proteins contain DHA. With the use of this method, we found that conversion of the Sec residue at the active site of GPx1 to DHA occurred during aging of red blood cells (RBCs) in vivo as well as in RBCs exposed to H(2)O(2) generated either externally by glucose oxidase or internally as a result of aniline-induced Hb autoxidation. Accordingly, the content of DHA-GPx1 in each RBC likely reflects total oxidative stress experienced by the cell during its lifetime of 120 days. Previous studies suggested that the activity of GPx1 in RBCs is most influenced by lifestyle and environmental factors such as the use of dietary supplements and smoking habit. Therefore, DHA-GPx1 in RBCs might be a suitable surrogate marker for evaluation of oxidative stress in the body. Our blot-based method for the detection of DHA-GPx1 will be very useful for evaluation of such stress. In addition, similar blot detection method can be devised for other proteins for which immunoprecipitating antibodies are available.

  14. Western blot patterns of serum autoantibodies against optic nerve antigens in dogs with goniodysgenesis-related glaucoma

    PubMed Central

    Pumphrey, Stephanie A.; Pizzirani, Stefano; Pirie, Christopher G.; Anwer, M. Sawkat; Logvinenko, Tanya

    2014-01-01

    Objective To investigate whether differences existed between clinically normal dogs and dogs with goniodysgenesis-related glaucoma (GDRG) in serum autoantibodies against optic nerve antigens. Animals 16 dogs with GDRG, 17 healthy dogs with unremarkable pectinate ligament and iridocorneal angle morphology, and 13 euthanized dogs with no major ocular abnormalities or underlying diseases. Procedures Western blotting was performed with optic nerve extracts from the euthanized dogs as an antigen source and serum from clinically normal dogs and dogs with GDRG as a primary antibody (autoantibody) source. Blots were evaluated for presence and density of bands. Results Multiple bands were identified on western blots from all dogs with GDRG and all clinically normal dogs, with a high degree of variability among individual dogs. Dogs with GDRG were significantly more likely than healthy dogs to have bands present at 38, 40, and 68 kDa. Dogs with GDRG had significant increases in autoreactivity at 40 and 53 kDa and a significant decrease in autoreactivity at 48 kDa. Conclusions and Clinical Relevance Significant differences in serum autoantibodies against optic nerve antigens were found in dogs with versus without GDRG. Although it remains unclear whether these differences were part of the pathogenesis of disease or were sequelae to glaucomatous changes, these findings provide support for the hypothesis that immune-mediated mechanisms play a role in the development or progression of GDRG. However, the high degree of variability among individual dogs and the considerable overlap between groups suggest that the clinical usefulness of this technique for distinguishing dogs with GDRG from clinically normal dogs is likely limited. PMID:23531071

  15. Affinity and Western blotting reveal homologies between ovine intervertebral disc serine proteinase inhibitory proteins and bovine pancreatic trypsin inhibitor.

    PubMed

    Melrose, J; Shen, B; Ghosh, P

    2001-12-01

    The objective of this study was to assess any similarities between ovine intervertebral disc (IVD) serine proteinase inhibitory proteins (SPIs) and known mammalian IVD SPIs. Ovine IVDs were dissected into the annulus fibrosus and nucleus pulposus and the tissue finely diced then extracted with 4 M guanidine hydrochloride. The tissue extracts were subjected to caesium chloride density gradient ultracentrifugation to separate the large high buoyant density (rho > 1.5 g/mL) proteoglycans from the SPI proteins of low buoyant density (rho < 1.33 g/mL). The top two ultracentrifuge fractions containing the SPIs of interest were subjected to enzyme linked immunosorbent analysis (ELISA) and also examined by Western and Affinity blotting using an antibody to bovine pancreatic trypsin inhibitor and biotinylated trypsin respectively for detection and an alkaline phosphatase 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium system for visualisation. The major SPI proteins present in the Western and Affinity blots were 34-36 kDa species, minor 12 and 16, and 85 and 120 kDa species were also present. Qualitatively similar results were obtained for each respective tissue zone of the lumbar and lumbosacral disc specimens examined. Densitometric analysis of the major 34-36 kDa SPI bands visualised on Western and Affinity blots using NIH 1.61.1 image analysis software indicated that lumbar IVD samples contained higher levels of this SPI species than lumbosacral IVD samples. ELISA confirmed that lumbar IVD extracts contained quantitatively higher levels of BPTI equivalents per g of tissue extracted than lumbosacral IVDs. This study therefore has demonstrated that the ovine disc contains a range of SPI species which share some homology with bovine pancreatic trypsin inhibitor and in this respect are similar to SPIs previously demonstrated in canine IVDs.

  16. Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis

    SciTech Connect

    Aravalli, Rajagopal N.; Park, Chang W.; Steer, Clifford J.

    2016-08-26

    The Sleeping Beauty transposon (SB-Tn) system is being used widely as a DNA vector for the delivery of therapeutic transgenes, as well as a tool for the insertional mutagenesis in animal models. In order to accurately assess the insertional potential and properties related to the integration of SB it is essential to determine the copy number of SB-Tn in the host genome. Recently developed SB100X transposase has demonstrated an integration rate that was much higher than the original SB10 and that of other versions of hyperactive SB transposases, such as HSB3 or HSB17. In this study, we have constructed a series of SB vectors carrying either a DsRed or a human β-globin transgene that was encompassed by cHS4 insulator elements, and containing the SB100X transposase gene outside the SB-Tn unit within the same vector in cis configuration. These SB-Tn constructs were introduced into the K-562 erythroid cell line, and their presence in the genomes of host cells was analyzed by Southern blot analysis using non-radioactive probes. Many copies of SB-Tn insertions were detected in host cells regardless of transgene sequences or the presence of cHS4 insulator elements. Interestingly, the size difference of 2.4 kb between insulated SB and non-insulated controls did not reflect the proportional difference in copy numbers of inserted SB-Tns. We then attempted methylation-sensitive Southern blots to assess the potential influence of cHS4 insulator elements on the epigenetic modification of SB-Tn. Our results indicated that SB100X was able to integrate at multiple sites with the number of SB-Tn copies larger than 6 kb in size. In addition, the non-radioactive Southern blot protocols developed here will be useful to detect integrated SB-Tn copies in any mammalian cell type.

  17. Northern Arizona University

    ERIC Educational Resources Information Center

    Butcher, Michael F.; Saltonstall, Margot; Bickel, Sarah; Brandel, Rick

    2009-01-01

    Northern Arizona University (NAU) is a public university nestled below the San Francisco Peaks in Flagstaff, Arizona. It enrolls more than 21,000 undergraduate and graduate students at its main campus in Flagstaff, through its 35 statewide sites, and via online program offerings. Within the university organizational system, Student Affairs has a…

  18. Northern Arizona University

    ERIC Educational Resources Information Center

    Butcher, Michael F.; Saltonstall, Margot; Bickel, Sarah; Brandel, Rick

    2009-01-01

    Northern Arizona University (NAU) is a public university nestled below the San Francisco Peaks in Flagstaff, Arizona. It enrolls more than 21,000 undergraduate and graduate students at its main campus in Flagstaff, through its 35 statewide sites, and via online program offerings. Within the university organizational system, Student Affairs has a…

  19. Northern goshawk (Accipiter gentilis)

    Treesearch

    John R. Squires; Richard T. Reynolds

    1997-01-01

    The Northern Goshawk (hereafter referred to as Goshawk) is a large forest raptor, occupying boreal and temperate forests throughout the Holarctic. In North America, it breeds from Alaska to Newfoundland and south (Fig. 1). This partial migrant winters throughout its breeding range including occasionally the Great Plains and southeastern states; some...

  20. Western blot analysis of virus-specific antibody responses for capripox and contagious pustular dermatitis viral infections in sheep.

    PubMed

    Chand, P; Kitching, R P; Black, D N

    1994-10-01

    This paper reports the development and evaluation of serological tests for the differentiation of antibodies in animals infected with capripox and parapox viruses. Agar-gel immunodiffusion tests using sera from sheep with naturally-acquired infections and from sheep experimentally inoculated with orf or capripox viruses showed cross reactions. Virus-specific antibody responses to structural proteins of the viruses were analysed by Western-blot analysis. This analysis readily differentiated the infections as either capripox or contagious pustular dermatitis. The antibody responses to the 32 kDa and 26 kDa proteins of capripoxvirus provided a firm basis for differentiation.

  1. Transcription Factors Downstream of IL-4 and TGF-β Signals: Analysis by Quantitative PCR, Western Blot, and Flow Cytometry.

    PubMed

    Sugimoto, Atsushi; Kawakami, Ryoji; Mikami, Norihisa

    2017-01-01

    IL-9-producing Th9 cell is a novel Th cell subset involved in type II allergic inflammations such as asthma. Th9 cells can be induced from naïve Th cells in the presence of IL-4 and TGF-β. It is also well established that downstream signals of IL-4 and TGF-β, including STAT6, IRF4, Smad, and PU.1, directly mediate IL-9 production in Th9 cells. In this chapter we describe the methods of flow cytometry, qPCR and western blot analysis to determine the expression or activation of these transcription factors downstream of IL-4 and TGF-β.

  2. Simultaneous detection of fourteen Italian cystic fibrosis mutations in seven exons by reverse dot-blot analysis.

    PubMed

    Rady, M; D'Alcamo, E; Seia, M; Iapichino, L; Ferrari, M; Russo, S; Romeo, G; Maggio, A

    1995-10-01

    The increasing number of cystic fibrosis (CF) mutations is a great obstacle to the use of DNA procedures in the detection of gene defects. We describe a fast, cheap and non-radioactive procedure, Reverse dot-blot analysis (RDB), for the simultaneous detection of CF mutations in the Italian population. We used this approach to study seven exons of the CF gene for 14 CF gene defects and were able to characterize 222 of 272 CF chromosomes (80%). The cost of the procedure was $25 per sample analysed.

  3. How to Distinguish Between the Activity of HDAC1-3 and HDAC6 with Western Blot.

    PubMed

    Beyer, Mandy; Kiweler, Nicole; Mahboobi, Siavosh; Krämer, Oliver H

    2017-01-01

    Histone deacetylases (HDACs) catalyze the deacetylation of lysine residues in their target proteins. This biochemical modification can have profound effects on the functions of these proteins and a dysregulation of HDAC activity contributes to severe diseases, including neoplastic transformation. In the following chapter, we present a strategy that allows to distinguish between the inhibition of the class I HDACs HDAC1, 2, and 3 and of the class IIb HDAC HDAC6. This method is based on Western blot and relies on the detection of hyperacetylated substrates of class I or class IIb HDACs in lysates from cells that were treated with histone deacetylase inhibitors (HDACi).

  4. Detection of Rickettsia in Rhipicephalus sanguineus Ticks and Ctenocephalides felis Fleas from Southeastern Tunisia by Reverse Line Blot Assay

    PubMed Central

    Khrouf, Fatma; M'Ghirbi, Youmna; Znazen, Abir; Ben Jemaa, Mounir; Hammami, Adnene

    2014-01-01

    Ticks (n = 663) and fleas (n = 470) collected from domestic animals from southeastern Tunisia were screened for Rickettsia infection using reverse line blot assay. Evidence of spotted fever group Rickettsia was obtained. We detected Rickettsia felis in fleas, Rickettsia massiliae Bar 29 and the Rickettsia conorii Israeli spotted fever strain in ticks, and Rickettsia conorii subsp. conorii and Rickettsia spp. in both arthropods. The sensitivity of the adopted technique allowed the identification of a new association between fleas and R. conorii subsp. conorii species. The presence of these vector-borne Rickettsia infections should be considered when diagnosing this disease in humans in Tunisia. PMID:24226919

  5. Northern Pintail (Anas acuta)

    USGS Publications Warehouse

    Austin, J.E.; Miller, M.R.; Poole, A.; Gill, F.

    1995-01-01

    The Northern Pintail is a medium-sized dabbling duck of slender, elegant lines and conservative plumage coloration. It is circumpolar in distribution and abundant in North America, with core nesting habitat in Alaska and the Prairie Pothole Region of southern Canada and the northern Great Plains. Breeders favor shallow wetlands interspersed throughout prairie grasslands or arctic tundra. An early fall migrant, the species arrives on wintering areas beginning in August, after wing molt, often forming large roosting and feeding flocks on open, shallow wetlands and flooded agricultural fields. The birds consume grains, marsh plant seeds, and aquatic invertebrates throughout the fall and winter. Northern Pintails are among the earliest nesting ducks in North America, beginning shortly after ice-out in many northern areas. Individuals form new pair bonds each winter but are highly promiscuous during the nesting season, with mated and unmated males often involved in vigorous, acrobatic Pursuit Flights. Annual nest success and productivity vary with water conditions, predation, and weather. Females build nests on the ground, often long distances from water. Only the female incubates; her mate leaves shortly after incubation begins. Ducklings hatch together in one day, follow the female to water after a day in the nest, and fledge by July or August. Adults and ducklings consume mainly aquatic invertebrates during the breeding season. Predators and farming operations destroy many thousands of Northern Pintail nests annually; farming has also greatly reduced the amount of quality nesting cover available. Winter habitats are threatened by water shortages, agricultural development, contamination, and urbanization. Periods of extended drought in prairie nesting regions have caused dramatic population declines, usually followed by periods of recovery. Over the long term, however, the continental population of Northern Pintails has declined significantly from 6 million birds in

  6. Detection of Mycobacterium avium subsp. paratuberculosis by buoyant density centrifugation, sequence capture PCR and dot blot hybridisation.

    PubMed

    Halldórsdóttir, Stefania; Englund, Stina; Nilsen, Sigrun Fredsvold; Olsaker, Ingrid

    2002-07-22

    Detection of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) by polymerase chain reaction (PCR) is often hampered by the lack of efficient methods for sample treatment. We report a protocol for analysis of faecal samples based on buoyant density centrifugation in Percoll and IS900 sequence capture PCR combined with a dot blot assay for detection of low-grade infection of M. paratuberculosis. Serial dilutions of M. paratuberculosis genomic DNA and M. paratuberculosis bacteria were used to assess the sensitivity of the method. The final evaluation was performed with spiked faecal samples, which also were analysed by culture. The presence of PCR inhibitory substances in processed faecal samples was evaluated by including a PCR internal control. By using buoyant density centrifugation, sequence capture PCR, and dot blot hybridisation, we achieved a sensitivity of 10(3)CFU (colony forming units)/g of faeces. The detection limit by culture was assessed to 10(2)CFU/g of faeces. We conclude that the described protocol is a fast and sensitive alternative to bacterial culture of faecal samples.

  7. Identification of Glioblastoma Phosphotyrosine-Containing Proteins with Two-Dimensional Western Blotting and Tandem Mass Spectrometry

    PubMed Central

    Guo, Tianyao; Wang, Xiaowei; Li, Maoyu; Yang, Haiyan; Li, Ling; Peng, Fang

    2015-01-01

    To investigate the presence of, and the potential biological roles of, protein tyrosine phosphorylation in the glioblastoma pathogenesis, two-dimensional gel electrophoresis- (2DGE-) based Western blotting coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis was used to detect and identify the phosphotyrosine immunoreaction-positive proteins in a glioblastoma tissue. MS/MS and Mascot analyses were used to determine the phosphotyrosine sites of each phosphopeptide. Protein domain and motif analysis and systems pathway analysis were used to determine the protein domains/motifs that contained phosphotyrosine residue and signal pathway networks to clarify the potential biological functions of protein tyrosine phosphorylation. A total of 24 phosphotyrosine-containing proteins were identified. Each phosphotyrosine-containing protein contained at least one tyrosine kinase phosphorylation motif and a certain structural and functional domains. Those phosphotyrosine-containing proteins were involved in the multiple signal pathway systems such as oxidative stress, stress response, and cell migration. Those data show 2DGE-based Western blotting, MS/MS, and bioinformatics are a set of effective approaches to detect and identify glioblastoma tyrosine-phosphorylated proteome and to effectively rationalize the biological roles of tyrosine phosphorylation in the glioblastoma biological systems. It provides novel insights regarding tyrosine phosphorylation and its potential role in the molecular mechanism of a glioblastoma. PMID:26090378

  8. [A case of LGMD2A identified with both western blot analysis and immunostaining of calpain 3 in biopsied muscle].

    PubMed

    Ibi, T; Jing, L; Nakao, N; Minami, N; Sahashi, K

    2000-10-01

    A 45-year-old housewife had proximal dominant limb muscle weakness from around 25 years of age. Her parents were cousins. None of family members was affected. Progressive muscle weakness and atrophy were prominent at the posterior compartments of legs and trunk. Serum CK was moderately elevated. Muscle pathology revealed variation in fiber size, moderate increase in numbers of internal nuclei and abundant lobulated fibers. On immunostaining using by monoclonal antibody against human calpain 3 (NCL-CALP-2 C4; Novocastra) to the biopsied muscle, calpain 3 was completely absent in the sarcoplasm, while granular debris and in part positive striation were noted in control muscle. By Western blot analysis, a band corresponding to 94 kDa of calpain 3 was not detected. A genetic analysis of calpain 3 revealed homozygous C-565-G mutation (Leu189Val). From the present study. Western blot analysis and immunostaining by using calpain 3 antibody were suggested to be useful to diagnose LGMD2A in LGMD patients.

  9. Laboratory diagnosis of hymenoptera venom allergy: comparative study between specific IgE, western blot and allergen leukocyte stimulation (CAST).

    PubMed

    Santos, M C Pereira; Carlos, M L Palma; Pedro, E; Carlos, A G Palma

    2002-01-01

    Allergy to hymenoptera venom is a classical IgE mediated disease with a potentially fatal course. Specific venom immunotherapy (SIT) is the most effective mean of treating this serious condition, after the diagnosis has been clearly established by a clinical history, in-vivo and in vitro tests. We have evaluated the usefulness of a cellular test (CAST) which is a recently developed ELISA method based on the evaluation of sulfidoleukotriene secretion by leukocytes stimulated with specific antigen. We also evaluated the correlation of CAST with skin tests, specific IgE (sIgE) and western blot for sIgE for hymenoptera venom sIgE. We have included in this study 14 patients, with a clinical history suggestive of hymenoptera venom allergy. None of them had previously been subjected to immunotherapy. A good correlation was obtained between skin tests, sIgE and western blot. However, there was no correlation between these methods and CAST. We conclude that the positivity of CAST method raises some questions about other mechanisms, which maybe non-IgE dependent. Although the number of patients in this study is quite small, the immunoblot analysis may be a valuable additional method in insect venom allergy.

  10. Simple and Sensitive Detection of HBsAg by Using a Quantum Dots Nanobeads Based Dot-Blot Immunoassay

    PubMed Central

    Zhang, Pengfei; Lu, Huiqi; Chen, Jia; Han, Huanxing; Ma, Wei

    2014-01-01

    Simple and sensitive detection of infectious disease at an affordable cost is urgently needed in developing nations. In this regard, the dot blot immunoassay has been used as a common protein detection method for detection of disease markers. However, the traditional signal reporting systems, such as those using enzymes or gold nanoparticles lack sensitivity and thus restrict the application of these methods for disease detection. In this study, we report a simple and sensitive detection method for the detection of infectious disease markers that couples the dot-blot immunoassay with quantum dots nanobeads (QDNBs) as a reporter. First, the QDNBs were prepared by an oil-in-water emulsion-evaporation technique. Because of the encapsulation of several QDs in one particle, the fluorescent signal of reporter can be amplified with QDNBs in a one-step test and be read using a UV lamp obviating the need for complicated instruments. Detection of disease-associated markers in complex mixture is possible, which demonstrates the potential of developing QDNBs into a sensitive diagnostic kit. PMID:24505238

  11. Mutation analysis of fragile X syndrome by Southern blot, radioactive PCR, silver-stained polyacrylamide gel and DIG DNA

    SciTech Connect

    Lee, Sook-Hwan; Kim, Un-Kyung; Chung-Woong, M.S.

    1994-09-01

    Fragile X syndrome is the most common inherited form of mental retardation. In fragile X syndrome, the underlying mutation is caused by an expansion of the CTG triplet in the 5{prime} untranslated region of the FMR-1 gene located at Xq27.3 and diagnosed by methylation of the associated CpG island. This disorder becomes clinically manifested when the mutation is caused by an expansion of (CGG)n reaching a threshold of about 600bp (200 repeats). The number of inserted repeats increases through the generation. We have analyzed fragile X syndrome by 4 different methods: Southern blot, radioactive PCR, polyacrylamide gel and DIG DNA labeling/detection techniques. Southern blot and DIG DNA labeling/detection by double DNA digestion with EcoRI and EagI reveals both the presence of the mutation and the methylation status. Radioactive PCR and silver-stained polyacrylamide gel is a rapid and sensitive technique to define the unaffected carriers and NTMs, but it is difficult to amplify such a highly GC-rich sequence. Further testing in other fragile X patients is currently in progress.

  12. [Western blot technique standardization for specific diagnosis of Chagas disease using excretory-secretory antigens of Trypanosoma cruzi epimastigotes].

    PubMed

    Escalante, Hermes; Jara, César; Davelois, Kelly; Iglesias, Miguel; Benites, Adderly; Espinoza, Renzo

    2014-01-01

    Evaluate the effectiveness of Western Blot for the specific diagnosis of Chagas disease using excretory-secretory antigens of Trypanosoma cruzi epimastigotes. Antigens were obtained after twenty hours of incubation in Eagle’s Minimum Essential Medium, which were prepared at a protein concentration of 0.2 ug/uL to be faced with 10 mL pool of serum from patients with Chagas disease and a conjugated anti-IgG labeled with peroxidase. The presence of the following antigens was observed: 10, 12, 14, 15, 19, 20, 23, 26, 30, 33, 36, 40, 42, 46, 58, 63, 69, 91, 100, and 112 kDa; of which antigens of 10, 12, 14, 15, 19, 20, 23, and 26 kDa were considered to be specific using pools of serum from patients with other parasitosis and serum from people with no parasites. The sensitivity of the technique was assessed using individual serum from 65 patients with Chagas disease; and the specificity with serum from 40 patients with other parasitosis, and serums from five people who did not have parasites. The technique has a sensitivity of 95.4% in the detection of one to eight specific bands, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 93.7%. Western Blot technique with excretory-secretory antigens of T. cruzi epimastigotes is effective in the diagnosis of Chagas disease in Peru; therefore, it can be used as a confirmatory test.

  13. Applications of an Automated and Quantitative CE-Based Size and Charge Western Blot for Therapeutic Proteins and Vaccines.

    PubMed

    Rustandi, Richard R; Hamm, Melissa; Lancaster, Catherine; Loughney, John W

    2016-01-01

    Capillary Electrophoresis (CE) is a versatile and indispensable analytical tool that can be applied to characterize proteins. In recent years, labor-intensive SDS-PAGE and IEF slab gels have been replaced with CE-SDS (CGE) and CE-IEF methods, respectively, in the biopharmaceutical industry. These two CE-based methods are now an industry standard and are an expectation of the regulatory agencies for biologics characterization. Another important and traditional slab gel technique is the western blot, which detects proteins using immuno-specific reagents after SDS-PAGE separation. This technique is widely used across industrial and academic laboratories, but it is very laborious, manual, time-consuming, and only semi-quantitative. Here, we describe the applications of a relatively new CE-based western blot technology which is automated, fast, and quantitative. We have used this technology for both charge- and size-based CE westerns to analyze biotherapeutic and vaccine products. The size-based capillary western can be used for fast antibody screening, clone selection, product titer, identity, and degradation while the charge-based capillary western can be used to study product charge heterogeneity. Examples using this technology for monoclonal antibody (mAb), Enbrel, CRM197, and Clostridium difficile (C. difficile) vaccine proteins are presented here to demonstrate the utility of the capillary western techniques. Details of sample preparation and experimental conditions for each capillary western mode are described in this chapter.

  14. Quantum dot bio-conjugate: as a western blot probe for highly sensitive detection of cellular proteins

    NASA Astrophysics Data System (ADS)

    Kale, Sonia; Kale, Anup; Gholap, Haribhau; Rana, Abhimanyu; Desai, Rama; Banpurkar, Arun; Ogale, Satishchandra; Shastry, Padma

    2012-03-01

    In the present study, we report a quantum dot (QD)-tailored western blot analysis for a sensitive, rapid and flexible detection of the nuclear and cytoplasmic proteins. Highly luminescent CdTe and (CdTe)ZnS QDs are synthesized by aqueous method. High resolution transmission electron microscopy, Raman spectroscopy, fourier transform infrared spectroscopy, fluorescence spectroscopy and X-ray diffraction are used to characterize the properties of the quantum dots. The QDs are functionalized with antibodies of prostate apoptosis response-4 (Par-4), poly(ADP-ribose) polymerases and β actin to specifically bind with the proteins localized in the nucleus and cytoplasm of the cells, respectively. The QD-conjugated antibodies are used to overcome the limitations of conventional western blot technique. The sensitivity and rapidity of protein detection in QD-based approach is very high, with detection limits up to 10 pg of protein. In addition, these labels provide the capability of enhanced identification and localization of marker proteins in intact cells by confocal laser scanning microscopy.

  15. Label-free DNA quantification via a 'pipette, aggregate and blot' (PAB) approach with magnetic silica particles on filter paper.

    PubMed

    Li, Jingyi; Liu, Qian; Alsamarri, Hussein; Lounsbury, Jenny A; Haversitick, Doris M; Landers, James P

    2013-03-07

    Reliable measurement of DNA concentration is essential for a broad range of applications in biology and molecular biology, and for many of these, quantifying the nucleic acid content is inextricably linked to obtaining optimal results. In its most simplistic form, quantitative analysis of nucleic acids can be accomplished by UV-Vis absorbance and, in more sophisticated format, by fluorimetry. A recently reported new concept, the 'pinwheel assay', involves a label-free approach for quantifying DNA through aggregation of paramagnetic beads in a rotating magnetic field. Here, we describe a simplified version of that assay adapted for execution using only a pipet and filter paper. The 'pipette, aggregate, and blot' (PAB) approach allows DNA to induce bead aggregation in a pipette tip through exposure to a magnetic field, followed by dispensing (blotting) onto filter paper. The filter paper immortalises the extent of aggregation, and digital images of the immortalized bead conformation, acquired with either a document scanner or a cell phone camera, allows for DNA quantification using a noncomplex algorithm. Human genomic DNA samples extracted from blood are quantified with the PAB approach and the results utilized to define the volume of sample used in a PCR reaction that is sensitive to input mass of template DNA. Integrating the PAB assay with paper-based DNA extraction and detection modalities has the potential to yield 'DNA quant-on-paper' devices that may be useful for point-of-care testing.

  16. Phylogenetic distribution of apolipoproteins A-I and E in vertebrates as determined by Western blot analysis.

    PubMed

    Duggan, A E; Callard, I P

    2001-08-01

    A putative apolipoprotein E (apoE) has been identified in the HDL and VHDL fractions of the turtle. This observation is of particular interest considering apoE has been reported absent in the domestic hen (Hermier et al., '95; Biochim Biophys Acta: 105-118, 1995) and thus presumed absent in nonmammalian vertebrates altogether. As a result, partial amino acid sequencing of this protein was performed and revealed that one fragment shared 41% sequence identity to human apoE. Western blot analysis using antisera to apoE demonstrated cross-reactivity to a 34-kDa protein (putative apoE) in turtle plasma. Further investigation using anti-apoE antibody in Western blot analysis detected immunoreactive apoE in the plasma of lamprey, spiny dogfish, skate, and alligator, but not in flounder, newt or python; its absence in several species of birds was confirmed. Using anti-apoA-I antibody, apoA-I was detected in all vertebrate groups except a representative teleost (flounder). Apo-A-I antibody cross-reacted weakly with some putative apoE proteins (chicken, spiny dogfish and skate) and the reverse was true for anti-apoE, which cross-reacted with putative apoA-I in birds, reptiles, and elasmobranchs, confirming the molecular similarity and phylogenetic relatedness of these two proteins.

  17. A solid-phase immunoassay of protease-resistant prion protein with filtration blotting involving sodium dodecyl sulfate.

    PubMed

    Kobayashi, Yoshiteru; Kohno, Naoyuki; Wanibe, Shoko; Hirayasu, Kazunari; Uemori, Hitoshi; Tagawa, Yuichi; Yokoyama, Takashi; Shinagawa, Morikazu

    2006-02-15

    The precise diagnosis for bovine spongiform encephalopathy (BSE) is crucial for preventing new transmission to humans. Several testing procedures are reported for determining protease-resistant prion protein in various tissues as a major hallmark of prion diseases such as BSE, scrapie, and Creutzfeldt-Jakob disease. However, contamination of materials from tissues or degradation of the specimens sometimes disturbs the accuracy of the assay. Here, we have developed a novel method for solid-phase immunoassay of the disease-specific conformational isoform, PrP(Sc), using filtration blotting of protein in the presence of sodium dodecyl sulfate (SDS) followed by a filtration-based immunoassay with a single anti-prion protein antibody, together with the improved fractionation procedure involving high concentrations of surfactant/detergent. The SDS/heat treatment renders unfolded PrP(Sc) quantitative retention on a polyvinylidene difluoride filter and allows enhancement of the analyte signal with immunodetection; thus, all of the tested specimens are determined with 100% accuracy. In addition, the immunoassay is completed in approximately 1h, indicating its usefulness not only for the screening of BSE specimens but probably also for the postmortem BSE diagnosis of fallen stock as the antibody recognizes the core part of PrP(Sc). The solid-phase immunoassay method, including the filtration blotting with SDS, would be applicable to determining even more sensitively proteins other than PrP(Sc), especially those having rigid conformations.

  18. Nitrotyrosine Density of Rabbit Urinary Bladder Muscle and Mucosa Measured via Western Blotting and 96-Well Plate Analysis.

    PubMed

    Fitzpatrick, Brittany; Schuler, Catherine; Leggett, Robert E; Levin, Robert M

    2012-01-01

    Purpose. Nitrotyrosine was quantitated in rabbit bladder muscle and mucosa using two analytical systems: Western blotting analyses and a 96-well plate quantitative analysis kit. Materials and Methods. Rabbit bladder muscle and mucosa were obtained from control rabbits. For the Western analysis, the samples were loaded into a SDS page gel and then transferred to a PVDF membrane. The optical density was measured using a Kodak Scanner. Using the 96-well plate, the samples and standards were loaded, incubated with primary and secondary antibody, washed and vacuumed with 10x wash buffer three times between each incubation period. Stop buffer was added to the plate and the results were quantified via the plate reader. Results. For both muscle and mucosa tissue, the optical density readings were linear with tissue concentration; the concentration of nitrotyrosine in the mucosa was significantly higher than in the muscle. However, whereas the Western blot analysis is based on relative optical densities, the 96-well plate kit provides a truly quantitative analysis. Discussion. Mucosa tissue displayed a higher density of nitrotyrosine than did detrusor muscle tissue. This may well be due to the significantly higher metabolic activity of the mucosa compared to the muscle.

  19. The application of a photon-counting camera in very sensitive, bioluminescence-enhanced detection systems for protein blotting. Ultrasensitive detection systems for protein blotting and DNA hybridization, II.

    PubMed

    Hauber, R; Miska, W; Schleinkofer, L; Geiger, R

    1988-03-01

    A relatively simple, very sensitive bioluminescence-enhanced detection system for protein blots was described recently. This method utilizes antibodies conjugated with alkaline phosphatase. Alkaline phosphatase releases D-luciferin (Photinus pyralis) from D-luciferin-O-phosphate. Liberated D-luciferin reacts with luciferase, ATP and oxygen with light emission. The light produced is measured with a very sensitive photon counting camera (Argus 100), permitting visualization and localization of the alkaline phosphatase-conjugated antibodies on nitrocellulose sheets. Under non-optimized conditions the limit of detection is at present 5 to 500 fg of protein (rabbit immunoglobulin G), corresponding to 30 to 3 amol. The method is therefore 10(5) times more sensitive than other used at present.

  20. Dunes in Northern Summer

    NASA Image and Video Library

    2017-02-01

    This dune field formed near the base of the North Polar cap. Dunes require a source of loose particulate material to form. The source of the northern dune fields around the polar cap may be from the layers of dusty ice that are eroded by strong polar winds. This image was taken during the Martian northern summer, so there is no frost present on the dunes. The dunes closest to the base of the polar cap are long and parallel, indicating strong winds from the direction of the cap. As they get farther away from the polar cap, they start to form more crescent shaped dunes, called barchan dunes. Repeated observations by HiRISE of dunes like these show measurable changes in some locations. This discovery adds to the growing evidence that there are active processes happening all over the surface of Mars today. http://photojournal.jpl.nasa.gov/catalog/PIA11181

  1. Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis.

    PubMed

    Aravalli, Rajagopal N; Park, Chang W; Steer, Clifford J

    2016-08-26

    The Sleeping Beauty transposon (SB-Tn) system is being used widely as a DNA vector for the delivery of therapeutic transgenes, as well as a tool for the insertional mutagenesis in animal models. In order to accurately assess the insertional potential and properties related to the integration of SB it is essential to determine the copy number of SB-Tn in the host genome. Recently developed SB100X transposase has demonstrated an integration rate that was much higher than the original SB10 and that of other versions of hyperactive SB transposases, such as HSB3 or HSB17. In this study, we have constructed a series of SB vectors carrying either a DsRed or a human β-globin transgene that was encompassed by cHS4 insulator elements, and containing the SB100X transposase gene outside the SB-Tn unit within the same vector in cis configuration. These SB-Tn constructs were introduced into the K-562 erythroid cell line, and their presence in the genomes of host cells was analyzed by Southern blot analysis using non-radioactive probes. Many copies of SB-Tn insertions were detected in host cells regardless of transgene sequences or the presence of cHS4 insulator elements. Interestingly, the size difference of 2.4 kb between insulated SB and non-insulated controls did not reflect the proportional difference in copy numbers of inserted SB-Tns. We then attempted methylation-sensitive Southern blots to assess the potential influence of cHS4 insulator elements on the epigenetic modification of SB-Tn. Our results indicated that SB100X was able to integrate at multiple sites with the number of SB-Tn copies larger than 6 kb in size. In addition, the non-radioactive Southern blot protocols developed here will be useful to detect integrated SB-Tn copies in any mammalian cell type. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Dermatophytosis in northern Africa.

    PubMed

    Nweze, E I; Eke, I

    2016-03-01

    Infections caused by dermatophytes are a global problem and a major public health burden in the world today. In Africa, especially in the northern geographical zone, dermatophytic infections are being reported at an alarming rate. This is mostly because of some local but unique cultural practices, socioeconomic and environmental conditions, lack of reliable diagnostic personnel and facilities and ineffective treatment. Interestingly, the pathogen spectrum and the clinical manifestation are most times different from what is seen in other continents. Several epidemiological studies have been performed on the incidence and aetiology of dermatophytoses in northern Africa. However, there is currently no review article with up-to-date information on the relevant findings reported so far in this region. This information is necessary for clinicians who treat dermatophytic infections all over the world since agents of dermatophytes are no longer restricted because of the rapid mobility of humans from one part of the world to another. Moreover, the epidemiology of dermatophytoses is known to change over time, thus requiring the update of information from time to time. A review of relevant studies published on dermatophytoses in northern Africa is presented. This covers all of old Sudan, Algeria, Egypt, Libya, Tunisia and Morocco.

  3. Northern Plains Patterns

    NASA Technical Reports Server (NTRS)

    2003-01-01

    MGS MOC Release No. MOC2-513, 14 October 2003

    Patterns are common on the northern plains of Mars. Like their terrestrial counterparts in places like Siberia, Alaska, and northern Canada, patterned ground on Mars might be an indicator of the presence of ground ice. Whether it is true that the patterns on Mars are related to ground ice and whether the ice is still present beneath the martian surface are unknown. This Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) picture shows an example of patterned ground on the martian northern plains near 72.4oN, 252.6oW. The dark dots and lines are low mounds and chains of mounds. The circular feature near the center of the image is the location of a buried meteor impact crater; its presence today is marked only by the dark boulders on its rim and ejecta blanket that have managed to remain uncovered at the martian surface. The area shown is 3 km (1.9 mi) wide and illuminated by sunlight from the lower left.

  4. Western blot analysis of virus-specific antibody responses for capripox and contagious pustular dermatitis viral infections in sheep.

    PubMed Central

    Chand, P.; Kitching, R. P.; Black, D. N.

    1994-01-01

    This paper reports the development and evaluation of serological tests for the differentiation of antibodies in animals infected with capripox and parapox viruses. Agar-gel immunodiffusion tests using sera from sheep with naturally-acquired infections and from sheep experimentally inoculated with orf or capripox viruses showed cross reactions. Virus-specific antibody responses to structural proteins of the viruses were analysed by Western-blot analysis. This analysis readily differentiated the infections as either capripox or contagious pustular dermatitis. The antibody responses to the 32 kDa and 26 kDa proteins of capripoxvirus provided a firm basis for differentiation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7925674

  5. Quantitative analysis of a biopharmaceutical protein in cell culture samples using automated capillary electrophoresis (CE) western blot.

    PubMed

    Xu, Dong; Marchionni, Kentaro; Hu, Yunli; Zhang, Wei; Sosic, Zoran

    2017-10-25

    An effective control strategy is critical to ensure the safety, purity and potency of biopharmaceuticals. Appropriate analytical tools are needed to realize such goals by providing information on product quality at an early stage to help understanding and control of the manufacturing process. In this work, a fully automated, multi-capillary instrument is utilized for size-based separation and western blot analysis to provide an early readout on product quality in order to enable a more consistent manufacturing process. This approach aims at measuring two important qualities of a biopharmaceutical protein, titer and isoform distribution, in cell culture harvest samples. The acquired data for isoform distribution can then be used to predict the corresponding values of the final drug substance, and potentially provide information for remedy through timely adjustment of the downstream purification process, should the expected values fall out of the accepted range. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  6. Detection of human cytomegalovirus by slot-blot hybridization assay employing oligo-primed /sup 32/P-labelled probe

    SciTech Connect

    Agha, S.A.; Coleman, J.C.; Selwyn, S.; Mahmound, L.A.; Abd-Elaal, A.M.; Archard, L.C.

    1988-12-01

    A /sup 32/P-labelled Hind III-0 DNA fragment (nine Kilobases; Kb) from human cytomegalovirus AD-169 (HCMV) was used in slot-blot hybridization assay for the detection of HCMV in clinical samples. The results obtained with DNA hybridization assay (DNA HA) were compared with virus isolation using conventional tube cell culture (CTC) and centrifugation vial culture (CVC), immunofluorescence (IF), and complement fixation test (CFT). Of 15 CTC-positive samples, 13 were positive with DNA HA (sensitivity 86.7%). Also, 14 additional samples were DNA HA-positive but CTC-negative. CVC and/or IF confirmed the diagnosis in nine of 14; the remaining five samples were from three patients who showed fourfold rising antibody titre by CFT. Although DNA HA using /sup 32/P-labelled probes is relatively cumbersome and expensive, it is a valuable test for quantitation of viral shedding in patients with HCMV infections who may benefit from antiviral therapy.

  7. Detection of haemoparasites in cattle by reverse line blot hybridisation with a note on the distribution of ticks in Sicily.

    PubMed

    Georges, K; Loria, G R; Riili, S; Greco, A; Caracappa, S; Jongejan, F; Sparagano, O

    2001-08-31

    A reverse line blot hybridisation (RLB) of 21 oligonucleotides with polymerase chain reaction (PCR) amplified regions of 16S rRNA (Ehrlichia/Anaplasma group) or 18S rRNA (Babesia/Theileria group) genes of haemoparasites detected Theileria annulata, T. buffeli/orientalis, Babesia bovis, B. bigemina, B. divergens, Ehrlichia bovis, Anaplasma marginale, A. centrale and unknown species within the Rickettsia tribe.A very high prevalence of mixed infections was detected, which indicated that animals infected with Babesia spp. were also infected with Theileria spp. and/or Anaplasma spp. The tick distribution appeared to be seasonal with Hyalomma marginatum as the most frequently observed tick and Boophilus annulatus and Ixodes ricinus as the least frequently observed ticks. Other species identified in the 818 ticks collected during the five sampling periods between April 1998 and November 1999 included H. lusitanicum, Rhipicephalus sanguineus group, R. bursa, Dermacentor marginatus, Haemaphysalis punctata, B. annulatus and I. ricinus.

  8. Lectin staining and Western blot data showing differential sialylation of nutrient-deprived cancer cells to sialic acid supplementation.

    PubMed

    Badr, Haitham A; AlSadek, Dina M M; Mathew, Mohit P; Li, Chen-Zhong; Djansugurova, Leyla B; Yarema, Kevin J; Ahmed, Hafiz

    2015-12-01

    This report provides data that are specifically related to the differential sialylation of nutrient deprived breast cancer cells to sialic acid supplementation in support of the research article entitled, "Nutrient-deprived cancer cells preferentially use sialic acid to maintain cell surface glycosylation" [1]. Particularly, breast cancer cells, when supplemented with sialic acid under nutrient deprivation, display sialylated glycans at the cell surface, but non-malignant mammary cells show sialylated glycans intracellularly. The impact of sialic acid supplementation under nutrient deprivation was demonstrated by measuring levels of expression and sialylation of two markers, EGFR1 and MUC1. This Data in Brief article complements the main manuscript by providing detailed instructions and representative results for cell-level imaging and Western blot analyses of changes in sialylation during nutrient deprivation and sialic acid supplementation. These methods can be readily generalized for the study of many types of glycosylation and various glycoprotein markers through the appropriate selection of fluorescently-labeled lectins.

  9. Eighteen Years of Molecular Genotyping the Hemophilia Inversion Hotspot: From Southern Blot to Inverse Shifting-PCR

    PubMed Central

    Rossetti, Liliana C.; Radic, Claudia P.; Abelleyro, Miguel M.; Larripa, Irene B.; De Brasi, Carlos D.

    2011-01-01

    The factor VIII gene (F8) intron 22 inversion (Inv22) is a paradigmatic duplicon-mediated rearrangement, found in about one half of patients with severe hemophilia A worldwide. The identification of this prevalent cause of hemophilia was delayed for nine years after the F8 characterization in 1984. The aim of this review is to present the wide diversity of practical approaches that have been developed for genotyping the Inv22 (and related int22h rearrangements) since discovery in 1993. The sequence— Southern blot, long distance-PCR and inverse shifting-PCR—for Inv22 genotyping is an interesting example of scientific ingenuity and evolution in order to resolve challenging molecular diagnostic problems. PMID:22072947

  10. Standardisation of Western blotting to detect HTLV-1 antibodies synthesised in the central nervous system of HAM/TSP patients.

    PubMed

    Ribeiro, Luiz Claudio Pereira; Gonçalves, Cassia Cristina Alves; Slater, Carla Maria Sena Andrade; Carvalho, Silvia Maia Farias de; Puccioni-Sohler, Marzia

    2013-09-01

    Intrathecal synthesis of human T-lymphotropic virus type 1 (HTLV-1) antibodies (Abs) represents conclusive evidence of a specific immune response in the central nervous system of HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients. Western blotting (WB) for HTLV Abs in serum is a confirmatory test for HTLV-1 infection. The aim of this study was to standardise the Western blot to demonstrate the intrathecal pattern of Abs against HTLV-1 proteins in HAM/TSP patients. Paired cerebrospinal fluid (CSF) and serum samples were selected from 20 patients with definite HAM/TSP, 19 HTLV-1 seronegative patients and two HTLV-1 patients without definite HAM/TSP. The presence of reactive bands of greater intensity in the CSF compared to serum (or bands in only the CSF) indicated the intrathecal synthesis of anti-HTLV-1 Abs. All definite HAM/TSP patients presented with an intrathecal synthesis of anti-HTLV-1 Abs; these Abs were not detected in the control patients. The most frequent intrathecal targets of anti-HTLV-1 Abs were GD21, rgp46-I and p24 and, to a lesser extent, p19, p26, p28, p32, p36, p53 gp21 and gp46. The intrathecal immune response against env (GD21 and rgp46-I) and gag (p24) proteins represents the most important humoral pattern in HAM/TSP. This response may be used as a diagnostic marker, considering the frequent association of intrathecal anti-HTLV-1 Ab synthesis with HAM/TSP and the pathogenesis of this neurological disease.

  11. Imprinting mutations in Angelman syndrome detected by Southern blotting using a probe containing exon {alpha} of SNRPN

    SciTech Connect

    Beuten, J.; Sutcliffe, J.S.; Nakao, M.

    1994-09-01

    Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are associated with paternal and maternal deficiencies respectively, of gene expression within human chromosome 15q11-q13, and are caused by deletion, uniparental disomy (UPD), or other mutations. The SNRPN gene maps in this region, is paternally expressed, and is a candidate gene for PWS. Southern blotting using methylation-sensitive enzymes and a genomic DNA probe from the CpG island containing exon {alpha} of the SNRPN gene reveals methylation specific for the maternal allele. In cases of the usual deletions or UPD, the probe detects absence of an unmethylated allele in PWS and absence of a methylated allele in AS. We have analyzed 21 nondeletion/nonUPD AS patients with this probe and found evidence for an imprinting mutation (absence of a methylated allele) in 3 patients. Southern blotting with methylation-sensitive enzymes using the exon {alpha} probe, like use of the PW71 probe, should detect abnormalities in all known PWS cases and in 3 of the 4 forms of AS: deletion, UPD and imprinting mutations. This analysis provides a valuable diagnostic approach for PWS and AS. In efforts to localize the imprinting mutations in AS, one patient was found with failure to inherit a dinucleotide repeat polymorphism near probe 189-1 (D15S13). Analysis of this locus in AS families and CEPH families demonstrates a polymorphism that impairs amplification and a different polymorphism involving absence of hybridization to the 189-1 probe. The functional significance, if any, of deletion of the 189-1 region is unclear.

  12. Development, validation, and pilot application of a semiquantitative Western blot analysis and an ELISA for bovine adiponectin.

    PubMed

    Mielenz, M; Mielenz, B; Singh, S P; Kopp, C; Heinz, J; Häussler, S; Sauerwein, H

    2013-04-01

    Adiponectin is an adipose tissue-derived glycoprotein circulating as highly abundant multimers. It regulates glucose metabolism and insulin sensitivity. In ruminants, valid data about serum concentrations and tissue-specific protein expression are lacking, and we, therefore, aimed to generate a polyclonal antibody against bovine adiponectin to apply it in immunodetection. The specificity of the purified anti-adiponectin antibody was established by Western blot analysis with the use of reducing and denaturing conditions applied to both the purified protein and the bovine serum samples. Besides bovine serum, the applicability of the antibody for immunodetection of adiponectin was confirmed for the supernatant fluid of in vitro-differentiated bovine adipocytes, for protein extracts from bovine adipose tissue, and also in a multispecies comparison: bands comparable in size with monomeric bovine adiponectin were obtained under denaturing conditions in serum of camel, horse, human, mouse, pig, roe deer, and sheep. In addition, when used in immunohistochemistry on bovine adipose tissue sections, a characteristic adipocyte-specific staining pattern was obtained with this antibody. The antibody was used for establishing a semiquantitative Western blot procedure and the development of an ELISA. Both methods were extensively validated and were first applied to characterize the serum adiponectin concentrations in multiparous dairy cows during the transition from pregnancy to lactation, that is, 3 wk before until 5 wk after calving. With both assays a time effect (P = 0.017, P = 0.026, respectively) with lowest values at the day of parturition was observed. We thus established 2 useful tools to validly assess bovine adiponectin at the protein level. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. MDR-TB Antibody Response (Western Blot) to Fractions of Isoniazid and Rifampicin Resistant Antigens of Mycobacterium tuberculosis.

    PubMed

    Hadizadeh Tasbiti, Alireza; Yari, Shamsi; Ghanei, Mostafa; Shokrgozar, Mohammad Ali; Bahrmand, Ahmadreza

    2015-12-01

    Drug-resistant TB poses a major threat to control of TB worldwide. Despite progress in the detection of Multidrug-resistant TB (MDR-TB) cases, a major diagnostic gap remains: 55% of reported TB patients estimated to have MDR-TB were not detected in 2013. MDR-TB antigens were conjugated to CNBr-activated Sepharose 4B. Specific polyclonal antibodies against MDR-TB Ags were prepared in rabbits using two boosted injections of the MDR-TB antigen. The antibodies were purified and treated with susceptible TB to remove any non-specific and cross-reactive antibodies. In the present study, comparative analysis of electrophoretic pattern of different antigens of INH/RIF-resistant TB were studied for identifying protein profiles. A RIF-resistant TB antigen was shown here to have different protein profiles from INH-resistant TB isolate. The results of Western blotting analysis showed that in the RIF- and INH-resistant antigenic fractions some bands of 14.4 and 45 kDa as immunogenic were common. Moreover, four bands of RIF-resistant TB antigen fractions (16, 19, 21, and 45 KDa) and one band of INH-resistant TB (about 26 KDa) were detected as diagnostic antigens. This study suggests that the Western blot is an accurate test to survey INH- and RIF-resistant TB antigens of M. tuberculosis infection. These findings indicate that MDR-TB diagnosis (based on Ag detection) could be useful in the identification of disease stages that precede symptomatic and microbiologically positive TB, such as subclinical and incipient TB.

  14. Western blot analysis of BK channel β1-subunit expression should be interpreted cautiously when using commercially available antibodies.

    PubMed

    Bhattarai, Yogesh; Fernandes, Roxanne; Kadrofske, Mark M; Lockwood, Lizbeth R; Galligan, James J; Xu, Hui

    2014-10-01

    Large conductance Ca(2+)-activated K(+) (BK) channels consist of pore-forming α- and accessory β-subunits. There are four β-subunit subtypes (β1-β4), BK β1-subunit is specific for smooth muscle cells (SMC). Reduced BK β1-subunit expression is associated with SMC dysfunction in animal models of human disease, because downregulation of BK β1-subunit reduces channel activity and increases SMC contractility. Several anti-BK β1-subunit antibodies are commercially available; however, the specificity of most antibodies has not been tested or confirmed in the tissues from BK β1-subunit knockout (KO) mice. In this study, we tested the specificity and sensitivity of six commercially available antibodies from five manufacturers. We performed western blot analysis on BK β1-subunit enriched tissues (mesenteric arteries and colons) and non-SM tissue (cortex of kidney) from wild-type (WT) and BK β1-KO mice. We found that antibodies either detected protein bands of the appropriate molecular weight in tissues from both WT and BK β1-KO mice or failed to detect protein bands at the appropriate molecular weight in tissues from WT mice, suggesting that these antibodies may lack specificity for the BK β1-subunit. The absence of BK β1-subunit mRNA expression in arteries, colons, and kidneys from BK β1-KO mice was confirmed by RT-PCR analysis. We conclude that these commercially available antibodies might not be reliable tools for studying BK β1-subunit expression in murine tissues under the denaturing conditions that we have used. Data obtained using commercially available antibodies should be interpreted cautiously. Our studies underscore the importance of proper negative controls in western blot analyses. © 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  15. Murine interleukin 1 receptor. Direct identification by ligand blotting and purification to homogeneity of an interleukin 1-binding glycoprotein

    SciTech Connect

    Bird, T.A.; Gearing, A.J.; Saklatvala, J.

    1988-08-25

    Functional receptors (IL1-R) for the proinflammatory cytokine interleukin 1 (IL1) were solubilized from plasma membranes of the NOB-1 subclone of murine EL4 6.1 thymoma cells using the zwitterionic detergent 3((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS). Membrane extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and ligand blotted with /sup 125/I-labeled recombinant human IL1 alpha in order to reveal proteins capable of specifically binding IL1. A single polydisperse polypeptide of Mr approximately equal to 80,000 was identified in this way, which bound IL1 alpha and IL1 beta with the same affinity as the IL1-R on intact NOB-1 cells (approximately equal to 10(-10) M). The IL1-binding polypeptide was only seen in membranes from IL1-R-bearing cells and did not react with interleukin 2, tumor necrosis factor alpha, or interferon. IL1-R was purified to apparent homogeneity from solubilized NOB-1 membranes by affinity chromatography on wheat germ agglutinin-Sepharose and IL1 alpha-Sepharose. Gel electrophoresis and silver staining of purified preparations revealed a single protein of Mr approximately equal to 80,000 which reacted positively in the ligand-blotting procedure and which we identify as the ligand-binding moiety of the murine IL1-R. Purified IL1-R exhibited the same affinity and specificity as the receptor on intact cells. The relationship of this protein to proteins identified by covalent cross-linking studies is discussed.

  16. [Western Blot diagnostic yield for simultaneous antibody-detection in patients with human cysticercosis, hydatidosis, and human fascioliasis].

    PubMed

    Davelois, Kelly; Escalante, Hermes; Jara, César

    2016-01-01

    . To determine the diagnostic yield using western blotting to simultaneously detect antibodies in patients with human cysticercosis, hydatidosis, and human fascioliasis. Materials and methods . Cross-sectional study of diagnostic yield assessment. Excretory/secretory antigens were obtained from Taenia solium larvae, Echinococcus granulosus cysts, and the adult flukes of Fasciola hepática, which were then separated using the polyacrylamide gel electrophoresis technique, transferred, and attached to a nitrocellulose membrane to be probed with sera from the patient infected with the three parasites. The sensitivity of the technique was assessed using 300 individual serum samples, 60 pools of two parasites, and 20 pools of three parasites with 75 sera from patients with other parasites, 10 from patients with other diseases, and 15 from patients without parasites. Results . The technique revealed 13 glycoproteins (GP): GP 35, 31, 24, 23, 18, 17, 14, and 13 kDa for cysticercosis; GP 8, 16, and 21 kDa for hydatidosis; and GP 17 and 23 kDa for fascioliasis. The test detected the presence of antibodies with a sensitivity of 96% (95% confidence interval [CI] = 94.62-98.54%) in the detection of one or the thirteen bands, a specificity of 100% (95% CI = 99.50-100.00%); individually, there was a sensitivity for cysticercosis of 97% (95% CI = 93.16-100.00%), for hydatidosis of 94% (95% CI = 88.85-99.15%) and for fascioliasis of 96% (95% CI = 91.66-100.00%). Conclusions . Western blotting is effective in the simultaneous detection of antibodies in patients with human cysticercosis, hydatidosis, and fascioliasis, and it can be used as a diagnostic test to either rule out or confirm the presence of antibodies in endemic areas.

  17. Saturn, Approaching Northern Summer

    NASA Image and Video Library

    2016-09-15

    Since NASA's Cassini spacecraft arrived at Saturn in mid-2004, the planet's appearance has changed greatly. The shifting angle of sunlight as the seasons march forward has illuminated the giant hexagon-shaped jet stream around the north polar region, and the subtle bluish hues seen earlier in the mission have continued to fade. Earlier views obtained in 2004 and 2009 (see PIA06077 and PIA11667) demonstrate how drastically the illumination has changed. This view shows Saturn's northern hemisphere in 2016, as that part of the planet nears its northern hemisphere summer solstice in May 2017. Saturn's year is nearly 30 Earth years long, and during its long time there, Cassini has observed winter and spring in the north, and summer and fall in the south. The spacecraft will complete its mission just after northern summer solstice, having observed long-term changes in the planet's winds, temperatures, clouds and chemistry. Cassini scanned across the planet and its rings on April 25, 2016, capturing three sets of red, green and blue images to cover this entire scene showing the planet and the main rings. The images were obtained using Cassini's wide-angle camera at a distance of approximately 1.9 million miles (3 million kilometers) from Saturn and at an elevation of about 30 degrees above the ring plane. The view looks toward the sunlit side of the rings from a sun-Saturn-spacecraft angle, or phase angle, of 55 degrees. Image scale on Saturn is about 111 miles (178 kilometers) per pixel. The exposures used to make this mosaic were obtained just prior to the beginning of a 44-hour movie sequence. http://photojournal.jpl.nasa.gov/catalog/PIA21046

  18. Jupiter's Northern Lights

    NASA Image and Video Library

    2017-09-06

    This is a reconstructed view of Jupiter's northern lights through the filters of Juno's Ultraviolet Imaging Spectrometer (UVS) instrument on Dec. 11, 2016, as the Juno spacecraft approached Jupiter, passed over its poles, and plunged towards the equator. Such measurements present a real challenge for the spacecraft's science instruments: Juno flies over Jupiter's poles at 30 miles (50 kilometers) per second -- more than 100,000 miles per hour -- speeding past auroral forms in a matter of seconds. https://photojournal.jpl.nasa.gov/catalog/PIA21938

  19. Northern Summer on Titan

    NASA Image and Video Library

    2017-06-14

    NASA's Cassini spacecraft sees bright methane clouds drifting in the summer skies of Saturn's moon Titan, along with dark hydrocarbon lakes and seas clustered around the north pole. Compared to earlier in Cassini's mission, most of the surface in the moon's northern high latitudes is now illuminated by the sun. The image was taken with the Cassini spacecraft narrow-angle camera on June 9, 2017, using a spectral filter that preferentially admits wavelengths of near-infrared light centered at 938 nanometers. Cassini obtained the view at a distance of about 315,000 miles (507,000 kilometers) from Titan. https://photojournal.jpl.nasa.gov/catalog/PIA21615

  20. Fires in Northern Australia

    NASA Technical Reports Server (NTRS)

    2002-01-01

    Several fires were detected in Northern Australia by MODIS. The fires show up as red dots, superimposed on a surface reflectance product. The image also shows the Clarence Strait, which separates the mainland from Melville Island to the northwest and the smaller Bathurst Island to its west. The Strait connects the more confined, bowl-shaped Van Diemen Gulf to the Beagle Gulf. To the right of the image at the top is the Gulf of Carpentaria, which appears to be full of phytoplankton, as evidenced by the blue-green swirls in the waters

  1. Herpes simplex virus type 2 antibody detection performance in Kisumu, Kenya, using the Herpeselect ELISA, Kalon ELISA, Western blot and inhibition testing.

    PubMed

    Smith, J S; Bailey, R C; Westreich, D J; Maclean, I; Agot, K; Ndinya-Achola, J O; Hogrefe, W; Morrow, R A; Moses, S

    2009-04-01

    In certain parts of Africa, type-specific herpes simplex virus type 2 (HSV-2) ELISAs may have limited specificity. To date, no study has been conducted to validate HerpeSelect and Kalon type-specific HSV-2 ELISAs using both the Western blot and recombinant gG ELISA inhibition testing as reference standards. A total of 120 men who were HIV seronegative (aged 18-24 years) provided blood samples. HSV-2 IgG serum antibodies were detected using four different methods: HerpeSelect HSV-2 ELISA (n = 120), Kalon HSV-2 ELISA (n = 120), University of Washington Western blot (n = 101) and a recombinant inhibition test (n = 93). HSV-2 seroprevalence differed significantly by HSV-2 detection method, ranging from 24.8% with the Western blot to 69.8% with the HerpeSelect ELISA. Using the Western blot as the reference standard, the HerpesSelect had the highest sensitivity for HSV-2 antibody detection (100%) yet lowest specificity (40%). Similar results were obtained using the inhibition test as the reference standard. The sensitivity and specificity of the Kalon test versus the Western blot were 92% and 79%, respectively, and 80% and 82% versus the inhibition test. Using the inhibition test as the reference standard, the sensitivity of the Western blot appeared low (49%). In men in western Kenya who were HIV seronegative, the HerpeSelect and Kalon type-specific ELISAs had high sensitivities yet limited specificities using the Western blot as reference standard. Overall, the Kalon ELISA performed better than the HerpeSelect ELISA in these young men from Kisumu. Further understanding is needed for the interpretation of HSV-2 inhibition or ELISA test positive/ Western blot seronegative results. Before HSV-2 seropositivity may be reliably reported in selected areas of Africa, performance studies of HSV-2 serological assays in individual geographical areas are recommended.

  2. Spectrophotometric quantitation of DNA on blots after ethanol-solubilization of the MTT-formazan from anti-digoxigenin-based detection of nucleic acids.

    PubMed

    Colgan, D J

    1993-01-01

    The tetrazolium salt, 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) has recently been established as a substitute for Nitro-blue tetrazolium (NBT) in stain mixtures using antibody-conjugated alkaline phosphatase for the location of proteins on Western blots (Heegaard, 1990). Experiments reported here show that MTT is as sensitive as NBT in digoxigenin-labeled probe localization (on nucleic acid blots) utilizing alkaline-phosphatase-labelled, anti-digoxigenin antibodies. Moreover, as the formazan from MTT is soluble in ethanol, it is shown that spectrophotometric quantitation can be used to estimate the amount of target DNA on dot and Southern blots. For dot blotting, pBR328 was used as the probe and pBR322 as target. For Southern blots, human rDNA was used as the probe and total genomic calf DNA as the target. Staining response was linear over at least six twofold DNA dilutions in both types of blot.

  3. Expansive Northern Volcanic Plains

    NASA Image and Video Library

    2015-04-16

    Mercury northern region is dominated by expansive smooth plains, created by huge amounts of volcanic material flooding across Mercury surface in the past, as seen by NASA MESSENGER spacecraft. The volcanic lava flows buried craters, leaving only traces of their rims visible. Such craters are called ghost craters, and there are many visible in this image, including a large one near the center. Wrinkle ridges cross this scene and small troughs are visible regionally within ghost craters, formed as a result of the lava cooling. The northern plains are often described as smooth since their surface has fewer impact craters and thus has been less battered by such events. This indicates that these volcanic plains are younger than Mercury's rougher surfaces. Instrument: Mercury Dual Imaging System (MDIS) Center Latitude: 60.31° N Center Longitude: 36.87° E Scale: The large ghost crater at the center of the image is approximately 103 kilometers (64 miles) in diameter http://photojournal.jpl.nasa.gov/catalog/PIA19415

  4. The diagnosis of proventricular dilatation disease: use of a Western blot assay to detect antibodies against avian Borna virus.

    PubMed

    Villanueva, Itamar; Gray, Patricia; Mirhosseini, Negin; Payne, Susan; Hoppes, Sharman; Honkavuori, Kirsi S; Briese, Thomas; Turner, Debra; Tizard, Ian

    2010-07-14

    Avian Borna virus (ABV) has recently been shown to be the causal agent of proventricular dilatation disease (PDD) a lethal neurologic disease of captive psittacines and other birds. An immunoblot assay was used to detect the presence of antibodies against avian Borna virus in the serum of affected birds. A lysate from ABV-infected duck embryo fibroblasts served as a source of antigen. The assay was used to test for the presence of antibodies to ABV in 117 birds. Thirty of these birds had biopsy or necropsy-confirmed proventricular dilatation disease (PDD), while the remaining 87 birds were apparently healthy or were suffering from diseases other than PDD. Sera from 27 of the 30 PDD cases (90%) contained antibodies to ABV. Seventy-three (84%) of the apparently "healthy" birds were seronegative. Additionally, sera from seven macaws and one parrot trapped in the Peruvian Amazon were seronegative. Positive sera recognized the bornaviral nucleoprotein (N-protein). While the presence of antibodies to ABV largely corresponded with the development of clinical PDD, 14 apparently healthy normal birds possessed detectable antibodies to ABV. The existence of a carrier state was confirmed when 13 of 15 apparently healthy cockatiels were shown by PCR to have detectable ABV RNA in their feces. Western blot assays may be of significant assistance in diagnosing proventricular dilatation disease. Many apparently healthy birds may however be seronegative while, at the same time, shedding ABV in their feces.

  5. [Optimization of the immunoelectrophoresis technic (western blot) for the confirmation of human immunodeficiency virus infection (HIV) in Panama].

    PubMed

    Pascale, J M; de Austin, E; de Moreno, N O; Ledezma, C; de Márquez, E; Blanco, R; Quiroz, E; Calzada, J; Vincensini, A R; de Martin, M C

    1995-01-01

    The purpose of this study is to report the results of the authors' investigation to apply the western blot technique (WB UP-LCS) in the diagnosis of human immunodeficiency virus type 1 (HIV-1) infection. To do this, the authors separated the proteins of the HIV-1 virus by electrophoresis, based on their molecular weight, in poliacilamide gel with SDS (SDS-PAGE) during 3 hours at 200 volts. Then they electrotransferred these proteins to nitrocellulose paper during four hours at 200 milliamperes, with the aid of external cooling. The nitrocellulose strips were evaluated considering the incubation time (1 and 16 hours), two conjugates (human anti IgG with Peroxidase and human anti IgG Biotin plus Streptatividine with Peroxidase) and two dilutions of the patients' sera (1/50 and 1/100). Based on their results the Authors conclude that, in the first place, the optimal conditions for the test include a dilution of 1/100 of the patients serum, incubation of the serum for 16 hours and the use of the conjugate of anti human IgG with Biotin and Streptavidine with Peroxidase; secondary, that the immunologic reactivity against proteins p24 and gp 160/120 is the most important diagnostic criterion for the confirmation of infection with HIV-1 and that they obtained a diagnostic correlation of 100% at a cost which was 5 to 7 times less than that of the commercial system.

  6. A Novel Methylation PCR that Offers Standardized Determination of FMR1 Methylation and CGG Repeat Length without Southern Blot Analysis

    PubMed Central

    Grasso, Marina; Boon, Elles M.J.; Filipovic-Sadic, Stela; van Bunderen, Patrick A.; Gennaro, Elena; Cao, Ru; Latham, Gary J.; Hadd, Andrew G.; Coviello, Domenico A.

    2015-01-01

    Fragile X syndrome and associated disorders are characterized by the number of CGG repeats and methylation status of the FMR1 gene for which Southern blot (SB) historically has been required for analysis. This study describes a simple PCR-only workflow (mPCR) to replace SB analysis, that incorporates novel procedural controls, treatment of the DNA in separate control and methylation-sensitive restriction endonuclease reactions, amplification with labeled primers, and two-color amplicon sizing by capillary electrophoresis. mPCR was evaluated in two independent laboratories with 76 residual clinical samples that represented typical and challenging fragile X alleles in both males and females. mPCR enabled superior size resolution and analytical sensitivity for size and methylation mosaicism compared to SB. Full mutation mosaicism was detected down to 1% in a background of 99% normal allele with 50- to 100-fold less DNA than required for SB. A low level of full mutation mosaicism in one sample was detected using mPCR but not observed using SB. Overall, the sensitivity for detection of full mutation alleles was 100% (95% CI: 89%–100%) with an accuracy of 99% (95% CI: 93%–100%). mPCR analysis of DNA from individuals with Klinefelter and Turner syndromes, and DNA from sperm and blood, were consistent with SB. As such, mPCR enables accurate, sensitive, and standardized methods of FMR1 analysis that can harmonize results across different laboratories. PMID:24177047

  7. Exposure to Sarcocystis spp. in horses from Spain determined by Western blot analysis using Sarcocystis neurona merozoites as heterologous antigen.

    PubMed

    Arias, M; Yeargan, M; Francisco, I; Dangoudoubiyam, S; Becerra, P; Francisco, R; Sánchez-Andrade, R; Paz-Silva, A; Howe, D K

    2012-04-30

    Horses serve as an intermediate host for several species of Sarcocystis, all of which utilize canids as the definitive host. Sarcocystis spp. infection and formation of latent sarcocysts in horses often appears to be subclinical, but morbidity can occur, especially when the parasite burden is large. A serological survey was conducted to determine the presence of antibodies against Sarcocystis spp. in seemingly healthy horses from the Galicia region of Spain. Western blot analyses using Sarcocystis neurona merozoites as heterologous antigen suggested greater than 80% seroprevalance of Sarcocystis spp. in a sample set of 138 horses. The serum samples were further tested with enzyme-linked immunosorbent assays (ELISAs) based on recombinant S. neurona-specific surface antigens (rSnSAGs). As expected for horses from the Eastern Hemisphere, less than 4% of the serum samples were positive when analyzed with either the rSnSAG2 or the rSnSAG4/3 ELISAs. An additional 246 horses were tested using the rSnSAG2 ELISA, which revealed that less than 3% of the 384 samples were seropositive. Collectively, the results of this serologic study suggested that a large proportion of horses from this region of Spain are exposed to Sarcocystis spp. Furthermore, the anti-Sarcocystis seroreactivity in these European horses could be clearly distinguished from anti-S. neurona antibodies using the rSnSAG2 and rSnSAG4/3 ELISAs.

  8. A Modified Quantum Dot-Based Dot Blot Assay for Rapid Detection of Fish Pathogen Vibrio anguillarum.

    PubMed

    Zhang, Yang; Xiao, Jingfan; Wang, Qiyao; Zhang, Yuanxing

    2016-08-28

    Vibrio anguillarum, a devastating pathogen causing vibriosis among marine fish, is prevailing in worldwide fishery industries and accounts for grievous economic losses. Therefore, a rapid on-site detection and diagnostic technique for this pathogen is in urgent need. In this study, two mouse monoclonal antibodies (MAbs) against V. anguillarum, 6B3-C5 and 8G3-B5, were generated by using hybridoma technology and their isotypes were characterized. MAb 6B3-C5 was chosen as the detector antibody and conjugated with quantum dots. Based on MAb 6B3- C5 labeled with quantum dots, a modified dot blot assay was developed for the on-site determination of V. anguillarum. It was found that the method had no cross-reactivity with other than V. anguillarum bacteria. The detection limit (LOD) for V. anguillarum was 1 × 10(3) CFU/ml in cultured bacterial suspension samples, which was a 100-fold higher sensitivity than the reported colloidal gold immunochromatographic test strip. When V. anguillarum was mixed with turbot tissue homogenates, the LOD was 1 × 10(3) CFU/ml, suggesting that tissue homogenates did not influence the detection capabilities. Preenrichment with the tissue homogenates for 12 h could raise the LOD up to 1 × 10(2) CFU/ml, confirming the reliability of the method.

  9. Serodiagnosis of grass carp reovirus infection in grass carp Ctenopharyngodon idella by a novel Western blot technique.

    PubMed

    He, Yongxing; Jiang, Yousheng; Lu, Liqun

    2013-12-01

    Frequent outbreaks of grass carp hemorrhagic disease, caused by grass carp reovirus (GCRV) infection, pose as serious threats to the production of grass carp Ctenopharyngodon idella. Although various nucleic acids-based diagnostic methods have been shown effective, lack of commercial monoclonal antibody against grass carp IgM has impeded the development of any reliable immunoassays in detection of GCRV infection. The present study describes the preparation and screening of monoclonal antibodies against the constant region of grass carp IgM protein, and the development of a Western blot (WB) protocol for the specific detection of antibodies against outer capsid VP7 protein of GCRV that serves as antibody-capture antigen in the immunoassay. In comparison to a conventional RT-PCR method, validity of the WB is further demonstrated by testing on clinical fish serum samples collected from a grass carp farm in Jiangxi Province during disease pandemic in 2011. In conclusion, the WB technique established in this study could be employed for specific serodiagnosis of GCRV infection. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Comparison of germ line minisatellite mutation detection at the CEB1 locus by Southern blotting and PCR amplification.

    PubMed

    Taylor, Malcolm; Cieslak, Marcin; Rees, Gwen S; Oojageer, Anthony; Leith, Cheryl; Bristow, Claire; Tawn, E Janet; Winther, Jeanette F; Boice, John D

    2010-07-01

    Identification of de novo minisatellite mutations in the offspring of parents exposed to mutagenic agents offers a potentially sensitive measure of germ line genetic events induced by ionizing radiation and genotoxic chemicals. Germ line minisatellite mutations (GMM) are usually detected by hybridizing Southern blots of unamplified size-fractionated genomic DNA with minisatellite probes. However, this consumes a relatively large amount of DNA, requires several steps and may lack sensitivity. We have developed a polymerase chain reaction (PCR)-based GMM assay, which we applied to the hypermutable minisatellite, CEB1. Here, we compare the sensitivity and specificity of this assay with the conventional Southern hybridization method using DNA from 10 spouse pairs, one parent of each pair being a survivor of cancer in childhood, and their 20 offspring. We report that both methods have similar specificity but that the PCR method uses 250 times less DNA, has fewer steps and is better at detecting GMM with single repeats provided that specific guidelines for allele sizing are followed. The PCR GMM method is easier to apply to families where the amount of offspring DNA sample is limited.

  11. A cost effective non-commercial ECL-solution for Western blot detections yielding strong signals and low background.

    PubMed

    Haan, Claude; Behrmann, Iris

    2007-01-10

    We compared several alternative ECL solutions for Western blot detection of endogenous proteins in whole cell lysates using inexpensive, commercially available reagents. Starting from an existing protocol based on p-coumaric acid (pCA) as enhancer, we found that the ECL solution containing 4-iodophenylboronic acid (4IPBA) generated strong specific signals and low background chemiluminescence. We optimised the luminol, 4IPBA and hydrogenperoxide concentrations of this 4IPBA-ECL solution. The optimised 4IPBA-ECL solution (100 mM Tris/HCl pH 8.8, 1.25 mM luminol, 2 mM 4IPBA, 5.3 mM hydrogenperoxide) shows a greatly increased signal intensity compared to the initial pCA-ECL protocol and to some commercially available ECL solutions. In addition, the optimised 4IPBA-ECL solution also generates much lower background chemiluminescence than other non-commercial ECL solutions using p-coumaric acid or 4-iodophenol as enhancers. The 4IPBA-ECL solution was stable when stored but had the lowest background when prepared freshly from stock solutions. Thus, we present an optimised protocol for a well-performing inexpensive ECL solution which is an alternative to expensive commercial ECL solutions and which achieves a better signal and lower background than the commercial solutions tested.

  12. Optimized solubilization of TRIzol-precipitated protein permits Western blotting analysis to maximize data available from brain tissue

    PubMed Central

    Kopec, Ashley M.; Rivera, Phillip D.; Lacagnina, Michael J.; Hanamsagar, Richa; Bilbo, Staci D.

    2017-01-01

    Background Techniques simultaneously assessing multiple levels of molecular processing are appealing because molecular signaling underlying complex neural phenomena occurs at complementary levels. The TRIzol method isolates RNA and DNA, but protein retrieval is difficult due to inefficient solubilization of precipitated protein pellets. New Method We optimized a protocol for the efficient solubilization of protein from TRIzol-precipitated brain tissue for Western blotting analysis, which was also more effective at directly homogenizing brain tissue than RIPA buffer. Results Protein yield during solubilization, in addition to protein yield via direct homogenization, is increased by optimizing concentrations of chemicals in a standard lysis buffer. Effective incubation parameters for both total protein yield and the analysis of post-translational modifications is remarkably flexible. Importantly, different neural cell types and protein classes are represented in solubilized protein samples. Moreover, we used dissociated mouse brain tissue to isolate microglia from other cell types and successfully resolved cell type-specific proteins from these small and difficult to attain samples. Comparison with Existing Method(s) Solubilization buffers to date have been comprised primarily of SDS or urea; the data herein demonstrate that components common to lysis buffers can also enhance protein solubilization both after direct homogenization and after precipitation. Conclusions This method is suitable for assessing gene and protein expression from a single brain sample, allowing for a more comprehensive evaluation of neural phenomena while minimizing the number of subjects. PMID:28192129

  13. Identification of a Western Blot Pattern for the Specific Diagnosis of Trypanosoma cruzi Infection in Human Sera

    PubMed Central

    Riera, Cristina; Verges, Mireia; Iniesta, Laura; Fisa, Roser; Gállego, Montserrat; Tebar, Silvia; Portús, Montserrat

    2012-01-01

    A Western blot (WB) method using a lysate from Trypanosoma cruzi (Maracay strain) epimastigotes was evaluated. Serum samples from 37 patients with confirmed Chagas disease (cohort I), 27 Spanish patients with visceral leishmaniasis caused by Leishmania infantum (cohort II), and 28 Colombian patients with cutaneous leishmaniasis caused by L. panamensis and negative serology for Chagas disease (cohort III) were tested. The negative controls were 55 healthy seronegative subjects for T. cruzi and Leishmania; 28 of the negative controls were from a region endemic for Chagas disease and Leishmania (cohort IV), and 27 of the negative controls were from a non-endemic area for Leishmania and T. cruzi (cohort V). A homogeneous standard band pattern consisting of six antigenic bands corresponding to 28, 32, 38, 39, 40, and 48 kDa was recognized simultaneously for all Chagasic patients' sera. Sera from Leishmania-infected patients showed a heterogeneous band pattern that was easily differentiated from the pattern of patients with Chagas disease. WB with T. cruzi epimastigote antigen is an efficient method for diagnosis and may be used as an alternative to confirm T. cruzi and detect cross-reactivity with Leishmania. PMID:22403310

  14. Development of rapid, sensitive and non-radioactive tissue-blot diagnostic method for the detection of citrus greening.

    PubMed

    Nageswara-Rao, Madhugiri; Miyata, Shin-Ichi; Ghosh, Dilip; Irey, Mike; Garnsey, Stephen M; Gowda, Siddarame

    2013-01-01

    Citrus huanglongbing (HLB or citrus greening) is one of the most devastating diseases of citrus worldwide. The disease is caused by Gram-negative, phloem-limited α-proteobacterium, 'Candidatus Liberibacter asiaticus', vectored by the psyllid, Diaphorina citri Kuwayama. Citrus plants infected by the HLB bacterium may not show visible symptoms sometimes for years following infection and non-uniform distribution within the tree makes the detection of the pathogen very difficult. Efficient management of HLB disease requires rapid and sensitive detection early in the infection followed by eradication of the source of pathogen and the vector. The polymerase chain reaction (PCR) based method is most commonly employed for screening the infected/suspected HLB plants and psyllids. This is time consuming, cumbersome and not practical for screening large number of samples in the field. To overcome this, we developed a simple, sensitive, non-radioactive, tissue-blot diagnostic method for early detection and screening of HLB disease. Digoxigenin labeled molecular probes specific to 'Ca. L. asiaticus' nucleotide sequences have been developed and used for the detection of the pathogen of the HLB disease. The copy number of the target genes was also assessed using real-time PCR experiments and the optimized real-time PCR protocol allowed positive 'Ca. L. asiaticus' detection in citrus samples infected with 'Ca. L. asiaticus' bacterium. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Detection of antibodies against SARS-CoV in serum from SARS-infected donors with ELISA and Western blot.

    PubMed

    Wang, Yue-Dan; Li, Yan; Xu, Guo-Bin; Dong, Xue-Yuan; Yang, Xiao-Ang; Feng, Zhen-Ru; Tian, Chan; Chen, Wei Feng

    2004-11-01

    Recombinant fragments of S proteins from the Severe Acute Respiratory Syndrome (SARS) coronavirus (SARA-CoV) were generated and used in a Western blot (WB) assay that was compared to a commercial SARS ELISA method. In 85% of confirmed SARS cases (n = 20), the S2 recombinant fragment based WB was positive and this was comparable to the commercial ELISA using heat killed SARS-CoV. WB using the other four recombinant fragments in confirmed SARS cases generated lower rates of detection (S1--75%, S1-N--25%, S1-C--55%). Evaluation of sera from healthy controls (n = 60) resulted in two weakly positive ELISA results with the remainder being negative while the S2 protein WB demonstrated three positive results from the 20 controls with a history of SARS contact and no positive results in 40 noncontact controls. A discrepancy between the ELISA and S2 WB arose when evaluating per-2003 sera from individuals (n = 10) with SARS-like symptoms (ELISA--100% positive, S2 WB--30% positive). These data suggest that the S2 WB assay may be particularly useful in ELISA-negative SARS cases and in some ELISA-positive non-SARS cases.

  16. Far-western blotting as a solution to the non-specificity of the anti-erythropoietin receptor antibody

    PubMed Central

    Fecková, Barbora; Kimáková, Patrícia; Ilkovičová, Lenka; Szentpéteriová, Erika; Debeljak, Nataša; Solárová, Zuzana; Sačková, Veronika; Šemeláková, Martina; Bhide, Mangesh; Solár, Peter

    2016-01-01

    The erythropoietin receptor (EpoR) is a member of the cytokine receptor family. The interaction between erythropoietin (Epo) and EpoR is important for the production and maturation of erythroid cells, resulting in the stimulation of hematopoiesis. The fact that EpoR was also detected in neoplastic cells has opened the question about the relevance of anemia treatment with recombinant Epo in cancer patients. Numerous studies have reported pro-stimulating and anti-apoptotic effects of Epo in cancer cells, thus demonstrating EpoR functionality in these cells. By contrast, a previous study claims the absence of EpoR in tumor cells. This apparent discrepancy is based, according to certain authors, on the use of non-specific anti-EpoR antibodies. With the aim of bypassing the direct detection of EpoR with an anti-EpoR antibody, the present authors propose a far-western blot methodology, which in addition, confirms the interaction of Epo with EpoR. Applying this technique, the presence of EpoR and its interaction with Epo in human ovarian adenocarcinoma A2780 and normal human umbilical vein endothelial cells was confirmed. Furthermore, modified immunoprecipitation of EpoR followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis confirmed a 57 kDa protein as a human Epo-interacting protein in both cell lines. PMID:27446474

  17. Mitigating Radicalism in Northern Nigeria

    DTIC Science & Technology

    2013-08-01

    radicalization in northern Nigeria. u Active engagement of youth and communities in peacebuilding programs that facilitate interactions among individuals...leaders, sustained development investments in marginalized communities , promotion of values of inclusivity to mitigate the spread of extremist ideology...claiming to have repelled Boko Haram, the militants return, regroup, and seek revenge. As a result, social and economic activities in the northern

  18. Report on Ontario's Northern Schools.

    ERIC Educational Resources Information Center

    2003

    Ontario's funding formula fails to recognize the unique needs of northern school boards, which cover immense geographic areas, have many small schools, and enroll a high proportion of Aboriginal students. This report examines school size, enrollment, and staffing in northern Ontario schools, drawing on 2002-03 tracking reports of provincial…

  19. Evaluation of the Bio-Rad Multispot HIV-1/HIV-2 Rapid Test as an alternative to Western blot for confirmation of HIV infection.

    PubMed

    Cárdenas, Ana María; Baughan, Eleonore; Hodinka, Richard L

    2013-12-01

    In the United States, a new HIV diagnostic algorithm has been proposed that uses an HIV-1/HIV-2 antibody differentiation immunoassay instead of Western blot or immunofluoresence for confirmatory testing. To evaluate the Multispot HIV-1/HIV-2 Rapid Test (Multispot) as an alternative to Western blot analysis for confirmation of HIV infection. A series of 205 serum and plasma specimens positive for HIV-1 or HIV-2 were used to compare the performance of Multispot to a standard HIV-1 Western blot. Positive samples included 63 specimens from patients>18 months of age, 33 proficiency survey specimens, and 109 specimens from nine commercial seroconversion and performance panels. In addition, 63 specimens from 51 HIV-exposed, uninfected children≤18 months of age in various stages of seroreversion and 192 HIV-negative samples were tested. Specimens were initially screened using a 4th generation HIV Ag/Ab Combo assay. Multispot readily discriminated between individuals with HIV-1 or HIV-2 infection and those who were uninfected. Of the 205 samples repeatedly reactive by the 4th generation screening assay, infection status was correctly confirmed by Multispot in 83.9% (172/205) compared to 68.8% (141/205) for Western blot. Multispot detected HIV-1 earlier in 27.6% of low-titer antibody specimens called indeterminate by Western blot, and effectively reduced the number of indeterminate results in seroreverting HIV-1 exposed, uninfected infants and for HIV-2 infections misinterpreted as indeterminate or positive by HIV-1 Western blot. Multispot offers speed and simplicity over Western blot and has an excellent performance for differentiation and confirmation of antibodies to HIV-1 and HIV-2. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Northern Sinus Meridiani Stereo

    NASA Technical Reports Server (NTRS)

    2003-01-01

    MGS MOC Release No. MOC2-341, 25 April 2003

    This is a stereo (3-d anaglyph) composite of Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) wide angle images of northern Sinus Meridiani near 2oN, 0oW. The light-toned materials at the south (bottom) end of the picture are considered to be thick (100-200 meters; 300-600 ft) exposures of sedimentary rock. Several ancient meteor impact craters are being exhumed from within these layered materials. To view in stereo, use '3-d' glasses with red over the left eye, and blue over the right. The picture covers an area approximately 113 km (70 mi) wide; north is up.

  1. Glyphosate in northern ecosystems.

    PubMed

    Helander, Marjo; Saloniemi, Irma; Saikkonen, Kari

    2012-10-01

    Glyphosate is the main nonselective, systemic herbicide used against a wide range of weeds. Its worldwide use has expanded because of extensive use of certain agricultural practices such as no-till cropping, and widespread application of glyphosate-resistant genetically modified crops. Glyphosate has a reputation of being nontoxic to animals and rapidly inactivated in soils. However, recent evidence has cast doubts on its safety. Glyphosate may be retained and transported in soils, and there may be cascading effects on nontarget organisms. These processes may be especially detrimental in northern ecosystems because they are characterized by long biologically inactive winters and short growing seasons. In this opinion article, we discuss the potential ecological, environmental and agricultural risks of intensive glyphosate use in boreal regions.

  2. Smoke Blankets Northern California

    NASA Technical Reports Server (NTRS)

    2008-01-01

    Lightning strikes have sparked more than a thousand fires in northern California. This image was captured by the Multi-angle Imaging SpectroRadiometer's nadir (vertical-viewing) camera on 27 June 2008. Cape Mendocino is at the center of the image and Mt. Shasta is near the upper right. Concentrated smoke is visible in several river valleys and the large smoke cloud extends over the Pacific Ocean for hundreds of kilometers.

    MISR was built and is managed by NASA's Jet Propulsion Laboratory, Pasadena, Calif., for NASA's Science Mission Directorate, Washington, D.C. The Terra satellite is managed by NASA's Goddard Space Flight Center, Greenbelt, Md. JPL is a division of the California Institute of Technology.

  3. Saturn Northern Hemisphere

    NASA Image and Video Library

    1998-12-05

    This false color picture of Saturn’s northern hemisphere was assembled from ultraviolet, violet and green images obtained Aug. 19 by Voyager 2 from a range of 7.1 million kilometers (4.4 million miles). The several weather patterns evident include three spots flowing westward at about 15 meters-per-second (33 mph). Although the cloud system associated with the western-most spot is part of this flow, the spot itself moves eastward at about 30 meters-per-second (65 mph). Their joint flow shows the anti-cyclonic rotation of the spot, which is about 3,000 km. (1,900 mi.) in diameter. The ribbon- like feature to the north marks a high-speed jet where wind speeds approach 150 meters-per-second (330 mph). http://photojournal.jpl.nasa.gov/catalog/PIA01365

  4. Northern Sinus Meridiani Stereo

    NASA Technical Reports Server (NTRS)

    2003-01-01

    MGS MOC Release No. MOC2-341, 25 April 2003

    This is a stereo (3-d anaglyph) composite of Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) wide angle images of northern Sinus Meridiani near 2oN, 0oW. The light-toned materials at the south (bottom) end of the picture are considered to be thick (100-200 meters; 300-600 ft) exposures of sedimentary rock. Several ancient meteor impact craters are being exhumed from within these layered materials. To view in stereo, use '3-d' glasses with red over the left eye, and blue over the right. The picture covers an area approximately 113 km (70 mi) wide; north is up.

  5. Evaluation of ELISA coupled with Western blot as a surveillance tool for Trichinella infection in wild boar (Sus scrofa).

    PubMed

    Cuttell, Leigh; Gómez-Morales, Maria Angeles; Cookson, Beth; Adams, Peter J; Reid, Simon A; Vanderlinde, Paul B; Jackson, Louise A; Gray, C; Traub, Rebecca J

    2014-01-31

    Trichinella surveillance in wildlife relies on muscle digestion of large samples which are logistically difficult to store and transport in remote and tropical regions as well as labour-intensive to process. Serological methods such as enzyme-linked immunosorbent assays (ELISAs) offer rapid, cost-effective alternatives for surveillance but should be paired with additional tests because of the high false-positive rates encountered in wildlife. We investigated the utility of ELISAs coupled with Western blot (WB) in providing evidence of Trichinella exposure or infection in wild boar. Serum samples were collected from 673 wild boar from a high- and low-risk region for Trichinella introduction within mainland Australia, which is considered Trichinella-free. Sera were examined using both an 'in-house' and a commercially available indirect-ELISA that used excretory-secretory (E/S) antigens. Cut-off values for positive results were determined using sera from the low-risk population. All wild boar from the high-risk region (352) and 139/321 (43.3%) of the wild boar from the low-risk region were tested by artificial digestion. Testing by Western blot using E/S antigens, and a Trichinella-specific real-time PCR was also carried out on all ELISA-positive samples. The two ELISAs correctly classified all positive controls as well as one naturally infected wild boar from Gabba Island in the Torres Strait. In both the high- and low-risk populations, the ELISA results showed substantial agreement (k-value=0.66) that increased to very good (k-value=0.82) when WB-positive only samples were compared. The results of testing sera collected from the Australian mainland showed the Trichinella seroprevalence was 3.5% (95% C.I. 0.0-8.0) and 2.3% (95% C.I. 0.0-5.6) using the in-house and commercial ELISA coupled with WB respectively. These estimates were significantly higher (P<0.05) than the artificial digestion estimate of 0.0% (95% C.I. 0.0-1.1). Real-time PCR testing of muscle from

  6. Deep proteomic profiling of vasopressin-sensitive collecting duct cells. I. Virtual Western blots and molecular weight distributions.

    PubMed

    Yang, Chin-Rang; Tongyoo, Pumipat; Emamian, Milad; Sandoval, Pablo C; Raghuram, Viswanathan; Knepper, Mark A

    2015-12-15

    The mouse mpkCCD cell line is a continuous cultured epithelial cell line with characteristics of renal collecting duct principal cells. This line is widely used to study epithelial transport and its regulation. To provide a data resource useful for experimental design and interpretation in studies using mpkCCD cells, we have carried out "deep" proteomic profiling of these cells using three levels of fractionation (differential centrifugation, SDS-PAGE, and HPLC) followed by tandem mass spectrometry to identify and quantify proteins. The analysis of all resulting samples generated 34.6 gigabytes of spectral data. As a result, we identified 6,766 proteins in mpkCCD cells at a high level of stringency. These proteins are expressed over eight orders of magnitude of protein abundance. The data are provided to users as a public data base (https://helixweb.nih.gov/ESBL/Database/mpkFractions/). The mass spectrometry data were mapped back to their gel slices to generate "virtual Western blots" for each protein. For most of the 6,766 proteins, the apparent molecular weight from SDS-PAGE agreed closely with the calculated molecular weight. However, a substantial fraction (>15%) of proteins was found to run aberrantly, with much higher or much lower mobilities than predicted. These proteins were analyzed to identify mechanisms responsible for altered mobility on SDS-PAGE, including high or low isoelectric point, high or low hydrophobicity, physiological cleavage, residence in the lysosome, posttranslational modifications, and expression of alternative isoforms due to alternative exon usage. Additionally, this analysis identified a previously unrecognized isoform of aquaporin-2 with apparent molecular mass <20 kDa.

  7. Detection of Potentially Diagnostic Leishmania Antigens with Western Blot Analysis of Sera from Patients with Cutaneous and Visceral Leishmaniases.

    PubMed

    Seyyedtabaei, Seyyed Javad; Rostami, Ali; Haghighi, Ali; Mohebali, Mehdi; Kazemi, Bahram; Fallahi, Shirzad; Spotin, Adel

    2017-01-01

    Visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) are important public health problems in Iran. We aimed to evaluate the diagnostic potential of Western blot (WB) compared with indirect immunofluorescence test (IFAT) to serodiagnosis of leishmaniasis. This study was performed from 2010-2014 and participants were different parts of Iran. Serum samples were obtained from 43 patients with proven CL, 33 patients with proven VL, 39 patients with other parasitic diseases and 23 healthy individuals. WB sensitivity for CL and VL was 100% and 91%, compared to IFA 4.6% and 87.8%, respectively. Sera from patients with CL and VL recognized numerous antigens with molecular weights ranging from 14 to 68 kDa and 12 to 94 kDa, respectively. The most sensitive antigens were 14 and 16 kDa for CL recognized by 100% of the sera from patients with proven CL and 12, 14 and 16 kDa for VL, recognized by 63.6%, 100% and 63.6% of the sera from patients with proven VL respectively. WB analysis is more sensitive than IFAT for the diagnosis of leishmaniasis particularly in cases of cutaneous leishmaniasis. The 12, 14 and 16 kDa can be valuable diagnostic molecules for serodiagnosis of leishmaniasis because at least two immunogenic molecules were simultaneously detected by all patient sera, as well as produced antibodies against these antigens have no cross-reactivity with other control groups. WB could be useful for screening and serodiagnosis of CL and VL in epidemiologic studies in endemic areas.

  8. A study of the technique of western blot for diagnosis of lyme disease caused by Borrelia afzelii in China.

    PubMed

    Liu, Zhi Yun; Hao, Qin; Hou, Xue Xia; Jiang, Yi; Geng, Zhen; Wu, Yi Mou; Wan, Kang Lin

    2013-03-01

    To study the technique of Western blot for the diagnosis of Lyme disease caused by Borrelia afzelii in China and to establish the standard criteria by operational procedure. FP1, which is the representative strain of B. afzelii in China, was analyzed by SDS-PAGE, electro transfer and immunoblotting assays. The molecular weights of the protein bands of FP1 were analyzed by Gel-Pro analysis software. In a study using 451 serum samples (159 patients with Lyme disease and 292 controls), all observed bands were recorded. The accuracy of the WB as a diagnostic test was established by using the ROC curve and Youden index. Criteria for a positive diagnosis of Lyme disease were established as at least one band of P83/100, P58, P39, OspB, OspA, P30, P28, OspC, P17, and P14 in the IgG test and at least one band of P83/100, P58, P39, OspA, P30, P28, OspC, P17, and P41 in the IgM test. For IgG criteria, the sensitivity, specificity and Youden index were 69.8%, 98.3%, and 0.681, respectively; for IgM criteria, the sensitivity, specificity and Youden index were 47%, 94.2%, and 0.412, respectively. Establishment of WB criteria for B. afzelii is important in validating the diagnostic assays for Lyme disease in China. Copyright © 2013 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  9. Proviral Features of Human T Cell Leukemia Virus Type 1 in Carriers with Indeterminate Western Blot Analysis Results.

    PubMed

    Kuramitsu, Madoka; Sekizuka, Tsuyoshi; Yamochi, Tadanori; Firouzi, Sanaz; Sato, Tomoo; Umeki, Kazumi; Sasaki, Daisuke; Hasegawa, Hiroo; Kubota, Ryuji; Sobata, Rieko; Matsumoto, Chieko; Kaneko, Noriaki; Momose, Haruka; Araki, Kumiko; Saito, Masumichi; Nosaka, Kisato; Utsunomiya, Atae; Koh, Ki-Ryang; Ogata, Masao; Uchimaru, Kaoru; Iwanaga, Masako; Sagara, Yasuko; Yamano, Yoshihisa; Okayama, Akihiko; Miura, Kiyonori; Satake, Masahiro; Saito, Shigeru; Itabashi, Kazuo; Yamaguchi, Kazunari; Kuroda, Makoto; Watanabe, Toshiki; Okuma, Kazu; Hamaguchi, Isao

    2017-09-01

    Western blotting (WB) for human T cell leukemia virus type 1 (HTLV-1) is performed to confirm anti-HTLV-1 antibodies detected at the initial screening of blood donors and in pregnant women. However, the frequent occurrence of indeterminate results is a problem with this test. We therefore assessed the cause of indeterminate WB results by analyzing HTLV-1 provirus genomic sequences. A quantitative PCR assay measuring HTLV-1 provirus in WB-indeterminate samples revealed that the median proviral load was approximately 100-fold lower than that of WB-positive samples (0.01 versus 0.71 copy/100 cells). Phylogenic analysis of the complete HTLV-1 genomes of WB-indeterminate samples did not identify any specific phylogenetic groups. When we analyzed the nucleotide changes in 19 HTLV-1 isolates from WB-indeterminate samples, we identified 135 single nucleotide substitutions, composed of four types, G to A (29%), C to T (19%), T to C (19%), and A to G (16%). In the most frequent G-to-A substitution, 64% occurred at GG dinucleotides, indicating that APOBEC3G is responsible for mutagenesis in WB-indeterminate samples. Moreover, interestingly, five WB-indeterminate isolates had nonsense mutations in Pol and/or Tax, Env, p12, and p30. These findings suggest that WB-indeterminate carriers have low production of viral antigens because of a combination of a low proviral load and mutations in the provirus, which may interfere with host recognition of HTLV-1 antigens. Copyright © 2017 American Society for Microbiology.

  10. Application of Western blot analysis for the diagnosis of Encephalitozoon cuniculi infection in rabbits: example of a quantitative approach.

    PubMed

    Desoubeaux, Guillaume; Pantin, Ana; Peschke, Roman; Joachim, Anja; Cray, Carolyn

    2017-02-01

    Diagnosis of Encephalitozoon cuniculi infection in rabbits remains a major veterinary issue. ELISA or immunofluorescence assays are the current reference standards of serological tests. However, these conventional techniques suffer from a lack of accuracy for distinguishing active from past infections, as a positive serostatus is common in clinically normal rabbits. In this study, we assessed the diagnostic performance of Western blot (WB) to detect both anti-E. cuniculi immunoglobulin G (IgG) and immunoglobulin M (IgM) in comparison with ELISA and to address the intensity of the immune response through a quantitative approach. Positive WB results were highly correlated with the E. cuniculi-related diseased status (P < 0.0001). Although it was more labor intensive and less standardized, quantitative WB provided detailed comparable analysis regarding the humoral response and diagnostic performance similar to ELISA testing with statistically higher sensitivity (88.4 vs. 76.1% for IgG detection and 84.3 vs. 70.4% for IgM, P < 0.01). Several specific WB bands were shown to be significantly associated with concomitant clinical signs, like the one located at 50 kDa (OR = 8.2, [2.4-27.7], P = 0.0008) for IgG and (OR = 27.9, [4.2-187.9], P = 0.0006) for IgM. Therefore, the quantitative WB may have application in veterinary diagnostic laboratories to increase the accuracy of the clinical diagnosis of E. cuniculi infection. In addition, this tool may help to further understand the development and function of the humoral immune response to this infectious agent.

  11. Molecular Detection and Genotyping of Male-Specific Coliphages by Reverse Transcription-PCR and Reverse Line Blot Hybridization

    PubMed Central

    Vinjé, Jan; Oudejans, Sjon J. G.; Stewart, Jill R.; Sobsey, Mark D.; Long, Sharon C.

    2004-01-01

    In recent years, there has been increased interest in the use of male-specific or F+ coliphages as indicators of microbial inputs to source waters. Sero- or genotyping of these coliphages can also be used for microbial source tracking (MST). Among the male-specific coliphages, the F+ RNA (FRNA) viruses are well studied, while little is known about the F+ DNA (FDNA) viruses. We have developed a reverse line blot hybridization (RLB) assay which allows for the simultaneous detection and genotyping of both FRNA as well as FDNA coliphages. These assays included a novel generic duplex reverse transcription-PCR (RT-PCR) assay for FRNA viruses as well as a generic PCR for FDNA viruses. The RT-PCR assays were validated by using 190 field and prototype strains. Subsequent DNA sequencing and phylogenetic analyses of RT-PCR products revealed the classification of six different FRNA clusters, including the well-established subgroups I through IV, and three different FDNA clusters, including one (CH) not previously described. Within the leviviruses, a potentially new subgroup (called JS) including strains having more than 40% nucleotide sequence diversity with the known levivirus subgroups (MS2 and GA) was identified. We designed subgroup-specific oligonucleotides that were able to genotype all nine (six FRNA, three FDNA) different clusters. Application of the method to a panel of 351 enriched phage samples from animal feces and wastewater, including known prototype strains (MS2, GA, Qβ, M11, FI, and SP for FRNA and M13, f1, and fd for FDNA), resulted in successful genotyping of 348 (99%) of the samples. In summary, we developed a novel method for standardized genotyping of F+ coliphages as a useful tool for large-scale MST studies. PMID:15466543

  12. Development of enzyme immunoassays (ELISA and Western blot) for the serological diagnosis of dermatophytosis in symptomatic and asymptomatic cats.

    PubMed

    Santana, Aline Elisa; Taborda, Carlos Pelleschi; Severo, Julia So; Rittner, Glauce Mary Gomes; Muñoz, Julian Esteban; Larsson, Carlos Eduardo; Larsson, Carlos Eduardo

    2017-03-11

    Dermatophytosis is the most common fungal infection in cats worldwide and plays an important role in both animal and human health due to their high zoonotic potential. Effective screening is a strong preventive measure and the fungal culture is quite useful but requires full laboratorial experience and it takes a long time to obtain the result. A rapid and accurate screening test for dermatophytosis in cats is crucial for the effective control of disease outbreaks. The aim of this study was to develop and evaluate the diagnostic efficacy of enzyme immunoassays (ELISA and Western blot [WB]) for the rapid and precise diagnosis of dermatophytosis in cats. Seventy cats of various ages were divided into three groups: S (symptomatic, n = 20), AS (asymptomatic, n = 30), and N (negative, n = 20). All animals were submitted to fungal culture and blood samples for carrying out the serological tests. A significant difference (P < 0.05) was found between IgG-specific levels of sera of Microsporum canis positive and negative animals. There was no statistic difference between groups symptomatic and asymptomatic. The ELISA test showed sensitivity of 94% and specificity of 75%. Receiver operating characteristic (ROC) analysis also showed higher diagnostic accuracy (AUC 0.925). The WB technique detected 13 bands, and the 50 kDa protein was considered the most immunogenic protein, observing reactivity in 83.3% in the symptomatic group and 66.6% in the asymptomatic group. The study concluded that ELISA and WB were useful tools to reliably detect cats that have been exposed to M. canis. © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. Molecular mass dependence of hyaluronan detection by sandwich ELISA-like assay and membrane blotting using biotinylated hyaluronan binding protein

    PubMed Central

    Yuan, Han; Tank, Mihir; Alsofyani, Abeer; Shah, Naman; Talati, Nishant; LoBello, Jaclyn C; Kim, Jin Ryoun; Oonuki, Yoji; de la Motte, Carol A; Cowman, Mary K

    2013-01-01

    Hyaluronan (HA) is widely detected in biological samples and its concentration is most commonly determined by the use of a labeled specific HA binding protein (aggrecan G1-IGD-G2, HABP), employing membrane blotting and sandwich enzyme-linked immunosorbent assay (ELISA)-like methods. However, the detected signal intensity or the quantified value obtained by using these surface-based methods is related to the molecular mass (M) of HA, especially for HA in the low M range below ∼150 kDa. At the same mass or mass concentration, higher M HA gives a higher signal than lower M HA. We have experimentally determined the quantitative relationship between the M of HA (in the range 20–150 kDa) and the relative signal intensity in comparison with a standard HA, in a sandwich ELISA-like assay. An M-dependent signal correction factor (SCF) was calculated and used to correct the signal intensity, so that the corrected concentration value would more accurately reflect the true HA concentration in solution. The SCF for polydisperse low M HA was also calculated and compared with experimental results. When the molecular mass distribution of an HA sample is determined by a method such as gel electrophoresis, then its appropriately averaged SCF can be calculated and used to correct the signal in sandwich ELISA to obtain a more accurate concentration estimation. The correction method works for HA with M between ∼150 and 20 kDa, but lower M HA is too poorly detected for useful analysis. The physical basis of the M-dependent detection is proposed to be the increase in detector-accessible fraction of each surface-bound molecule as M increases. PMID:23964097

  14. Immunohistochemical and Western blot analyses of collar enamel in the jaw teeth of gars, Lepisosteus oculatus, an actinopterygian fish.

    PubMed

    Sasagawa, Ichiro; Ishiyama, Mikio; Yokosuka, Hiroyuki; Mikami, Masato; Shimokawa, Hitoyata; Uchida, Takashi

    2014-06-01

    Although most fish have no enamel layer in their teeth, those belonging to Lepisosteus (gars), an extant actinopterygian fish genus, do and so can be used to study amelogenesis. In order to examine the collar enamel matrix in gar teeth, we subjected gar teeth to light and electron microscopic immunohistochemical examinations using an antibody against bovine amelogenin (27 kDa) and antiserum against porcine amelogenin (25 kDa), as well as region-specific antibodies and antiserum against the C-terminus and middle region, and N-terminus of porcine amelogenin, respectively. The enamel matrix exhibited intense immunoreactivity to the anti-bovine amelogenin antibody and the anti-porcine amelogenin antiserum in addition to the C-terminal and middle region-specific antibodies, but not to the N-terminal-specific antiserum. These results suggest that the collar enamel matrix of gar teeth contains amelogenin-like proteins and that these proteins possess domains that closely resemble the C-terminal and middle regions of porcine amelogenin. Western blot analyses of the tooth germs of Lepisosteus were also performed. As a result, protein bands with molecular weights of 78 kDa and 65 kDa were clearly stained by the anti-bovine amelogenin antibody as well as the antiserum against porcine amelogenin and the middle-region-specific antibody. It is likely that the amelogenin-like proteins present in Lepisosteus do not correspond to the amelogenins found in mammals, although they do possess domains that are shared with mammalian amelogenins.

  15. TESA-blot for the diagnosis of Chagas disease in dogs from co-endemic regions for Trypanosoma cruzi, Trypanosoma evansi and Leishmania chagasi.

    PubMed

    Umezawa, E S; Souza, A I; Pinedo-Cancino, V; Marcondes, M; Marcili, A; Camargo, L M A; Camacho, A A; Stolf, A M S; Teixeira, M M G

    2009-07-01

    We standardized serodiagnosis of dogs infected with Trypanosoma cruzi using TESA (trypomastigote excreted-secreted antigen)-blot developed for human Chagas disease. TESA-blot showed 100% sensitivity and specificity. In contrast, ELISA using TESA (TESA-ELISA) or epimastigotes (epi-ELISA) as antigen yielded 100% sensitivity but specificity of 94.1% and 49.4%, respectively. When used in field studies in an endemic region for Chagas disease, visceral leishmaniasis and Trypanosoma evansi (Mato Grosso do Sul state, Central Brazil), positivities were 9.3% for TESA-blot, 10.7% for TESA-ELISA and 32% for epi-ELISA. Dogs from a non-endemic region for these infections (Rondonia state, western Amazonia) where T. cruzi is enzootic showed positivity of 4.5% for TESA-blot and epi-ELISA and 6.8% for TESA-ELISA. Sera from urban dogs from Santos, São Paulo, where these diseases are absent, yielded negative results. TESA-blot was the only method that distinguished dogs infected with T. cruzi from those infected with Leishmania chagasi and/or Trypanosoma evansi.

  16. Discrepancies between a new highly sensitive Toxoplasma gondii ELISA assay and other reagents: interest of Toxo IgG Western blot.

    PubMed

    Leslé, F; Touafek, F; Fekkar, A; Mazier, D; Paris, L

    2011-10-01

    Immunodiagnostic assays are commonly used to screen for maternal toxoplasmic seroconversion during pregnancy. The introduction to the market of a new highly sensitive IgG assay, the Elecsys Toxo IgG test, has resulted in discrepancy issues with other immunoassays because of a lack of standardisation. Western blot appears to be a good alternative gold standard to the dye test, as the latter is not routinely available. For the present prospective study, we compared the analytical performances of two immunoassays, Elecsys Toxo IgG (Roche Diagnostics) and Platelia Toxo IgG (Bio-Rad, Marnes la Coquette, France), to Toxo II IgG Western blot (LDBio, Lyon, France) using 231 consecutive sera with low or equivocal IgG titres. Of these 231 sera, 213 presented discrepancies, which showed the importance of a confirmation test. Of the Elecsys Toxo IgG-positive results, 100% were confirmed by the Western blot with a positive threshold of 30 IU/ml for Elecsys; in the equivocal area (1-30 IU/ml), Western blot is negative in 54% of cases. Our results suggest that the lower diagnostic cut-off of Platelia Toxo IgG should be further reduced. Our study indirectly confirms that monitoring, especially for pregnant women, must be done in the same laboratory using the same technique. The ability to diagnose very early seroconversion using Western blot merits further study.

  17. Human T lymphotropic virus types I and II proviral sequences in Argentinian blood donors with indeterminate Western blot patterns.

    PubMed

    Mangano, A M; Remesar, M; del Pozo, A; Sen, Luisa

    2004-10-01

    Human T-cell lymphotropic virus (HTLV) seroindeterminate blood donors have been reported worldwide including Argentina. To investigate the significance of HTLV-I/II seroindeterminate Western blot (WB) patterns, we conducted an 8-year cross-sectional study. Of 86,238 Argentinian blood donors, 146 sera were reactive by screening tests. The WB results indicated that 20% were HTLV-I reactive, 8% HTLV-II reactive, 61% indeterminate, and 11% negative. The overall seroprevalence was 0.034% for HTLV-I, 0.014% for HTLV-II, and 0.103% for indeterminate. In 57 reactive specimens, HTLV-I/II provirus could be examined by type specific PCR for tax, pol, and env regions. When at least two gene fragments were amplified HTLV-I/II infection was considered confirmed. PCR results confirmed all WB seropositive samples for HTLV-I (n = 15), and HTLV-II (n = 7), and the only WB negative case was also PCR negative, showing a complete concordance between PCR and WB. However, of 34 WB seroindeterminate sera studied by PCR, in 5 was proviral DNA amplified. According to our criteria PCR confirmed one to be HTLV-I, and one HTLV-II, 3 remained indeterminate since only tax sequences were amplified. Among WB indeterminate samples tested by PCR, most of their serological profile showed reactivity to gag codified proteins but lacked env reactivities (70%). One sample with a WB gag pattern showed proviral tax sequences, but of the four samples with reactivity to env proteins GD21 (n = 3) or rgp46II (n = 1) PCR results indicated that one was HTLV-I, one was HTLV-II, and two were indeterminate (only tax sequences). In conclusion, the majority of HTLV-seroindeterminate WB donors exhibited a gag indeterminate profile lacking HTLV provirus, and were thus considered uninfected. However, seroreactivity to env proteins, in particular to GD21, may indicate infection and a follow-up study of each seroreactive blood donor should be considered.

  18. Enzyme-linked immunoelectrotransfer blot analysis of a cryptosporidiosis outbreak on a United States Coast Guard cutter.

    PubMed

    Moss, D M; Bennett, S N; Arrowood, M J; Wahlquist, S P; Lammie, P J

    1998-01-01

    Symptoms consistent with an outbreak of cryptosporidiosis (diarrhea, vomiting, nausea, and abdominal cramps) occurred on a U.S. Coast Guard cutter within 0-18 days after the cutter filled its tanks with Milwaukee, Wisconsin city water in March 1993. At three-weeks postdocking (PD), the suspected water was removed, and serum samples and stool specimens were collected from 47 of the 58 crew members, as well as questionnaire data on their water consumption and symptoms aboard the cutter. At 10-weeks PD and/or at 28-weeks PD, additional serum specimens were collected. Intensitometric data from enzyme-linked immunoelectrotransfer blot (EITB) were obtained on IgA responses to a 17-kD antigen group, IgM responses to a 27-kD antigen group, and IgG responses to 27-, 17-, and 15-kD antigen groups extracted from oocysts. In addition, IgG responses to crude oocyst antigens were obtained by ELISA. Based on reported symptoms, EITB results, and stool examination, the crew members were classified as confirmed (10), probable (10), suspected (22), and noncases (16). Of the 10 confirmed cases (all symptomatic) and the 10 probable cases (eight symptomatic) whose stools were positive and negative, respectively, for Cryptosporidium oocysts by microscopy, all showed changes in EITB intensities to the antigen groups and were considered EITB positive. The remaining 38 crew members, 22 suspected cases (all symptomatic), and 16 noncases (all asymptomatic), if tested, had negative stool examinations and were considered EITB negative. Of the 10 confirmed cases, only four showed a significant change in IgG responses (P < 0.05) between three-weeks PD and follow-up serum specimens by ELISA. Crew members considered confirmed cases consumed significantly more water (P < or = 0.005) aboard the cutter than noncases. Crew members considered EITB positive consumed more water (P < or = 0.04) than crew members considered EITB negative while there was no significant difference in water consumption (P > or

  19. A mass screening survey of cystic echinococcosis by ultrasonography, Western blotting, and ELISA among university students in Manisa, Turkey.

    PubMed

    Kilimcioğlu, Ali Ahmet; Girginkardeşler, Nogay; Korkmaz, Metin; Özkol, Mine; Düzgün, Fatih; Östan, Ipek; Pabuşcu, Yüksel; Dinç, Gönül; Ok, Ulgen Zeki

    2013-12-01

    Cystic echinococcosis (CE) is one of the most important zoonotic diseases in a wide geographic area, including Turkey. In the present project, a total of 4275 students from Celal Bayar University, Manisa, Turkey, were screened by ultrasonography (US) and specific antibodies for CE were examined by Western blotting (WB) and ELISA in finger prick blood samples of 2034 of 4275 volunteered students. We aimed to report the apparent prevalence of CE based on different diagnostic procedures and to compare WB and ELISA with US in diagnosis of CE in a mass screening setting. Six new cases were diagnosed as CE by US during the survey. In addition to these cases, three students were also detected to have been previously operated and pathologically confirmed for hepatic CE. US revealed parenchymal changes in these cases in concordance with their operation history; so, the prevalence of CE by US was calculated as 0.21% (9/4275) (95%CI, 0.11-0.39%) among university students in Manisa. Bands were detected at 8, 28, 32, 38, 42, 47, 70 and 90kDa by WB and the cases were considered to be positive for CE when at least three of the bands were seen together. Apparent prevalence of CE by ELISA and WB were found to be 2.11% (43/2034) (95%CI, 1.57-2.83%) and 0.25% (5/2034) (95%CI, 0.10-0.57%), respectively. Of the six US positive cases, WB was positive in only one case with two cysts in the liver. All of four cases with liver involvement were positive by ELISA. The high prevalence of CE among university students in Manisa indicated that CE is a major health problem in this area of Turkey. Our results supported that WB is rather difficult and not feasible as a mass screening test and may not be effective for confirmation especially in asymptomatic cases. As a result, we recommend US to be used initially in mass screening surveys for CE followed by confirmation by ELISA for suspected cases. Further examination primarily by chest X-ray followed by computed tomography and/or magnetic

  20. Relative performance of Organon kit in comparison to Du Pont for confirmatory serological testing of HIV infection by western blot test in sera from blood donors.

    PubMed

    Aggarwal, R K; Chatterjee, R; Chattopadhya, D; Kumari, S

    1992-06-01

    A total of 32 specimens with different categories of reactivity by Du Pont Western Blot kit comprising of specimens showing full spectrum of HIV-I antigen specific bands, 19 specimens showing total absence of bands and four specimens showing non-specific bands (without any interpretative importance) were subjected to Western Blot testing by Organon test. Of the nine specimens showing full spectrum of bands by Du Pont the correlation with Organon kit was 100 per cent based on WHO criteria. Four specimens with non-specific indeterminate band pattern by Du Pont failed to show any band in Organon kit, indicating that latter to be more specific.

  1. Identification of recombinant human EPO variants in greyhound plasma and urine by ELISA, LC-MS/MS and western blotting: a comparative study.

    PubMed

    Timms, Mark; Steel, Rohan; Vine, John

    2016-02-01

    The recombinant human erythropoietins epoetin alfa (Eprex®), darbepoetin (Aranesp®) and methoxy polyethylene glycol-epoetin beta (Mircera®) were administered to greyhounds for 7, 10 and 14 days respectively. Blood and urine samples were collected and analysed for erythropoietin by ELISA, LC-MS/MS and western blotting. Limits of confirmation in plasma for western blotting and LC-MS/MS methods ranged from a low of 2.5mIU/mL, and closely matched the sensitivity of ELISA screening.

  2. Snow in northern Alaska

    NASA Image and Video Library

    2017-09-28

    As autumn colors moved across much of the lower forty-eight states in mid-October 2015, winter weather had already arrived in Alaska. The Moderate Resolution Imaging Spectroradiometer (MODIS) aboard NASA’s Terra satellite captured this true-color image of the icy scene on October 16 as it passed over the region. Point Barrow, the northern-most location in the United States sits between the Chukchi Sea (west) and the Beaufort Sea on the east. The rugged peaks of the Brooks Range can be seen along the southern section of the image. North of the Brooks Range the land is almost entirely covered with snow; to the south the tan and browns visible between snow marks uncovered land. Sea ice lies over the waters near the coasts of much of Alaska’s North Slope, especially east of Point Barrow. White cloud banks are notable in the northeast and southeast sections of the image. Credit: NASA/GSFC/Jeff Schmaltz/MODIS Land Rapid Response Team NASA image use policy. NASA Goddard Space Flight Center enables NASA’s mission through four scientific endeavors: Earth Science, Heliophysics, Solar System Exploration, and Astrophysics. Goddard plays a leading role in NASA’s accomplishments by contributing compelling scientific knowledge to advance the Agency’s mission. Follow us on Twitter Like us on Facebook Find us on Instagram

  3. Estimating cull in northern hardwoods

    Treesearch

    W.M. Zillgitt; S.R. Gevorkiantz

    1946-01-01

    Cull in northern hardwood stands is often very heavy and is difficult to estimate. To help clarify this situation and aid the average cruiser to become more accurate in his estimates, the study reported here should prove very helpful.

  4. MISR Views Northern Australia

    NASA Technical Reports Server (NTRS)

    2000-01-01

    MISR images of tropical northern Australia acquired on June 1, 2000 (Terra orbit 2413) during the long dry season. Left: color composite of vertical (nadir) camera blue, green, and red band data. Right: multi-angle composite of red band data only from the cameras viewing 60 degrees aft, 60 degrees forward, and nadir. Color and contrast have been enhanced to accentuate subtle details. In the left image, color variations indicate how different parts of the scene reflect light differently at blue, green, and red wavelengths; in the right image color variations show how these same scene elements reflect light differently at different angles of view. Water appears in blue shades in the right image, for example, because glitter makes the water look brighter at the aft camera's view angle. The prominent inland water body is Lake Argyle, the largest human-made lake in Australia, which supplies water for the Ord River Irrigation Area and the town of Kununurra (pop. 6500) just to the north. At the top is the southern edge of Joseph Bonaparte Gulf; the major inlet at the left is Cambridge Gulf, the location of the town of Wyndham (pop. 850), the port for this region. This area is sparsely populated, and is known for its remote, spectacular mountains and gorges. Visible along much of the coastline are intertidal mudflats of mangroves and low shrubs; to the south the terrain is covered by open woodland merging into open grassland in the lower half of the pictures.

    MISR was built and is managed by NASA's Jet Propulsion Laboratory, Pasadena, CA, for NASA's Office of Earth Science, Washington, DC. The Terra satellite is managed by NASA's Goddard Space Flight Center, Greenbelt, MD. JPL is a division of the California Institute of Technology.

  5. Isolated Northern Dunes

    NASA Technical Reports Server (NTRS)

    2005-01-01

    [figure removed for brevity, see original site]

    Our topic for the weeks of April 4 and April 11 is dunes on Mars. We will look at the north polar sand sea and at isolated dune fields at lower latitudes. Sand seas on Earth are often called 'ergs,' an Arabic name for dune field. A sand sea differs from a dune field in two ways: 1) a sand sea has a large regional extent, and 2) the individual dunes are large in size and complex in form.

    This VIS image was taken at 81 degrees North latitude during Northern spring. In this region, the dunes are isolated from each other. The dunes are just starting to emerge from the winter frost covering appearing dark with bright crests. These dunes are located on top of ice.

    Image information: VIS instrument. Latitude 82.1, Longitude 191.3 East (168.7 West). 19 meter/pixel resolution.

    Note: this THEMIS visual image has not been radiometrically nor geometrically calibrated for this preliminary release. An empirical correction has been performed to remove instrumental effects. A linear shift has been applied in the cross-track and down-track direction to approximate spacecraft and planetary motion. Fully calibrated and geometrically projected images will be released through the Planetary Data System in accordance with Project policies at a later time.

    NASA's Jet Propulsion Laboratory manages the 2001 Mars Odyssey mission for NASA's Office of Space Science, Washington, D.C. The Thermal Emission Imaging System (THEMIS) was developed by Arizona State University, Tempe, in collaboration with Raytheon Santa Barbara Remote Sensing. The THEMIS investigation is led by Dr. Philip Christensen at Arizona State University. Lockheed Martin Astronautics, Denver, is the prime contractor for the Odyssey project, and developed and built the orbiter. Mission operations are conducted jointly from Lockheed Martin and from JPL, a division of the California Institute of Technology in Pasadena.

  6. Northern Sand Sea

    NASA Technical Reports Server (NTRS)

    2005-01-01

    [figure removed for brevity, see original site]

    Our topic for the weeks of April 4 and April 11 is dunes on Mars. We will look at the north polar sand sea and at isolated dune fields at lower latitudes. Sand seas on Earth are often called 'ergs,' an Arabic name for dune field. A sand sea differs from a dune field in two ways: 1) a sand sea has a large regional extent, and 2) the individual dunes are large in size and complex in form.

    This VIS image was taken at 82 degrees North latitude during Northern spring. The image is completely dominated by dunes. In sand seas, it is very common for a single type of dune to occur, and for a single predominate wind to control the alignment of the dunes.

    Image information: VIS instrument. Latitude 82.2, Longitude 152.5 East (207.5 West). 19 meter/pixel resolution.

    Note: this THEMIS visual image has not been radiometrically nor geometrically calibrated for this preliminary release. An empirical correction has been performed to remove instrumental effects. A linear shift has been applied in the cross-track and down-track direction to approximate spacecraft and planetary motion. Fully calibrated and geometrically projected images will be released through the Planetary Data System in accordance with Project policies at a later time.

    NASA's Jet Propulsion Laboratory manages the 2001 Mars Odyssey mission for NASA's Office of Space Science, Washington, D.C. The Thermal Emission Imaging System (THEMIS) was developed by Arizona State University, Tempe, in collaboration with Raytheon Santa Barbara Remote Sensing. The THEMIS investigation is led by Dr. Philip Christensen at Arizona State University. Lockheed Martin Astronautics, Denver, is the prime contractor for the Odyssey project, and developed and built the orbiter. Mission operations are conducted jointly from Lockheed Martin and from JPL, a division of the California Institute of Technology in Pasadena.

  7. MISR Views Northern Australia

    NASA Technical Reports Server (NTRS)

    2000-01-01

    MISR images of tropical northern Australia acquired on June 1, 2000 (Terra orbit 2413) during the long dry season. Left: color composite of vertical (nadir) camera blue, green, and red band data. Right: multi-angle composite of red band data only from the cameras viewing 60 degrees aft, 60 degrees forward, and nadir. Color and contrast have been enhanced to accentuate subtle details. In the left image, color variations indicate how different parts of the scene reflect light differently at blue, green, and red wavelengths; in the right image color variations show how these same scene elements reflect light differently at different angles of view. Water appears in blue shades in the right image, for example, because glitter makes the water look brighter at the aft camera's view angle. The prominent inland water body is Lake Argyle, the largest human-made lake in Australia, which supplies water for the Ord River Irrigation Area and the town of Kununurra (pop. 6500) just to the north. At the top is the southern edge of Joseph Bonaparte Gulf; the major inlet at the left is Cambridge Gulf, the location of the town of Wyndham (pop. 850), the port for this region. This area is sparsely populated, and is known for its remote, spectacular mountains and gorges. Visible along much of the coastline are intertidal mudflats of mangroves and low shrubs; to the south the terrain is covered by open woodland merging into open grassland in the lower half of the pictures.

    MISR was built and is managed by NASA's Jet Propulsion Laboratory, Pasadena, CA, for NASA's Office of Earth Science, Washington, DC. The Terra satellite is managed by NASA's Goddard Space Flight Center, Greenbelt, MD. JPL is a division of the California Institute of Technology.

  8. Tornadoes Strike Northern Wisconsin

    NASA Technical Reports Server (NTRS)

    2007-01-01

    A series of tornadoes ripped through the Upper Midwest region of the United States in the evening of June 7, 2007. At least five different tornadoes touched down in Wisconsin, according to the Associated Press, one of which tore through the Bear Paw Resort in northern Wisconsin. Despite dropping as much as fifteen centimeters (six inches) of rain in some places and baseball-size hail in others, authorities were reporting no deaths attributable to the storm system, and only a smattering of injuries, but considerable property damage in some areas. When the MODIS instrument on NASA's Terra satellite observed the area on June 9, 2007, the track torn through the woods by one of the tornadoes stands out quite clearly. This photo-like image uses data collected by MODIS in the normal human vision range to give a familiar natural-looking appearance. The landscape is largely a checkerboard of farms, towns, roads, and cities. The pale land is predominantly farmland where crops have not fully grown in yet. Dark blue shows the winding path of rivers and lakes dotting the landscape. The large blue lake on the east (right) side of the image is Lake Michigan. Towns and cities, including the city of Green Bay, are gray. To the north side, farmland gives way to dark green as land use shifts from agriculture to the Menominee Indian Reservation and Nicolet National Forest. The diagonal slash through the dark green forested land shows the tornado track. Bare land was revealed where the tornado tore down trees or stripped vegetation off the branches. The high-resolution image provided above is at MODIS' full spatial resolution (level of detail) of 250 meters per pixel. The MODIS Rapid Response System provides this image at additional resolutions.

  9. Tornadoes Strike Northern Wisconsin

    NASA Technical Reports Server (NTRS)

    2007-01-01

    A series of tornadoes ripped through the Upper Midwest region of the United States in the evening of June 7, 2007. At least five different tornadoes touched down in Wisconsin, according to the Associated Press, one of which tore through the Bear Paw Resort in northern Wisconsin. Despite dropping as much as fifteen centimeters (six inches) of rain in some places and baseball-size hail in others, authorities were reporting no deaths attributable to the storm system, and only a smattering of injuries, but considerable property damage in some areas. When the MODIS instrument on NASA's Terra satellite observed the area on June 9, 2007, the track torn through the woods by one of the tornadoes stands out quite clearly. This photo-like image uses data collected by MODIS in the normal human vision range to give a familiar natural-looking appearance. The landscape is largely a checkerboard of farms, towns, roads, and cities. The pale land is predominantly farmland where crops have not fully grown in yet. Dark blue shows the winding path of rivers and lakes dotting the landscape. The large blue lake on the east (right) side of the image is Lake Michigan. Towns and cities, including the city of Green Bay, are gray. To the north side, farmland gives way to dark green as land use shifts from agriculture to the Menominee Indian Reservation and Nicolet National Forest. The diagonal slash through the dark green forested land shows the tornado track. Bare land was revealed where the tornado tore down trees or stripped vegetation off the branches. The high-resolution image provided above is at MODIS' full spatial resolution (level of detail) of 250 meters per pixel. The MODIS Rapid Response System provides this image at additional resolutions.

  10. Evaluation of Western blot, ELISA and latex agglutination tests to detect Toxoplasma gondii serum antibodies in farmed red deer.

    PubMed

    Patel, Kandarp Khodidas; Howe, Laryssa; Heuer, Cord; Asher, Geoffery William; Wilson, Peter Raymond

    2017-09-15

    Abortion due to Toxoplasma gondii has been suspected in New Zealand farmed red deer. However, knowledge around the epidemiology and prevalence of T. gondii in farmed red deer is limited. The aim of this study was to firstly, assess the sensitivity and specificity of two commercially available assays, ELISA and latex agglutination test (LAT), for use in deer and secondly, to estimate the sero-prevalence of T. gondii in red deer. A total of 252 sera from rising 2-year-old and adult hinds from 17 New Zealand red deer herds at early and late pregnancy scanning and from known aborted and/or non-aborted hinds were tested for the presence of T. gondii antibodies. Each assays' sensitivity and specificity was evaluated by both the Western Blot (WB) as a gold standard method and Bayesian latent class (BLC) analysis in the absence of a gold standard. The sensitivity and specificity for WB were 95.8% (95% credible interval: 89.5-99.2%) and 95.1% (95% credible interval: 90.6-98.1%), respectively. For the LAT at the manufacturer's recommended ≥1:32 cut-off titre, the sensitivity (88.7%, 95% credible interval: 80.8-94.7%) and specificity (74.3%, 95% credible interval: 67.5-80.5%) were lower and higher than the sensitivity (76.2%, 95% credible interval: 66.7-84.5%) and specificity (89.7%, 95% credible interval: 84.5-93.9%) at a ≥1:64 cut-off, using (BLC) analysis. Sensitivity and specificity of the LAT at cut-off titre of 1:32 were estimated to be 84.4% (95% CI: 74.9-90.9%) and 73.5% (95% CI: 65.8-79.9%) against WB. The LAT had better agreement with WB at cut-off titre of ≥1:64 than ≥1:32 (Kappa=0.63 vs 0.54). At optimised cut-off S/P of 15.5%, the sensitivity (98.8%, 95% credible interval 96.1-99.8%) and specificity (92.8%, 95% credible interval 88.9-95.7%) of the ELISA were higher and lower, respectively, than the sensitivity (85.1%, 95% credible interval 76.2-91.9%) and specificity (98.5%, 95% credible interval 96.9-99.4%) at manufacturer's cut-off S/P of 30%, from BLC

  11. Development and evaluation of a dot blot assay for rapid determination of invasion-associated gene ibeA directly in fresh bacteria cultures of E. coli.

    PubMed

    Niu, Chunling; Wang, Shaohui; Lu, Chengping

    2012-11-01

    The ibeA gene, one of the important invasion-associated genes in neonatal meningitis Escherichia coli (NMEC), has been recently detected in avian pathogenic E. coli (APEC). Thus, it is necessary to close monitor the possible contamination of the poultry farms and its products to people. Here, a dot blot method for detecting the ibeA gene in E. coli was developed and validated. For the present study, probe sequence was designed and optimized for the specificity of dot blot. A 342-bp conserved fragment of ibeA gene was selected and labeled with digoxigenin (DIG)-dUTP according to the manufacturer's guidelines, which indicated that this probe hybridizes with ibeA. In our established method, the bacteria culture samples were directly spotted on the membrane, following simple lyses on the membrane. Hence, the extraction of genomic DNA is not required, which reduces the workload and shortens the time. Furthermore, this assay was very sensitive, which could detect as few as 2.5 × 10(3) CFU bacteria. The diagnostic reliability of this dot blot was evaluated on 467 APEC bacteria samples by using PCR analysis. Both methods showed that the result was in complete concordance. The dot blot assay was proved to be a simple, rapid, highly accurate, and cost-effective method to identify invasion-associated genes ibeA, which could be applied for initial screening of a large number of clinical samples or direct detection of bacteria culture.

  12. Profile of the MP Diagnostics HTLV Blot 2.4 test: a supplemental assay for the confirmation and differentiation of antibodies to HTLV-1 and HTLV-2.

    PubMed

    Miller, Liane

    2016-01-01

    As the first US FDA-approved assay for supplemental HTLV testing, the MP Diagnostics HTLV Blot 2.4 is an effective and efficient method for confirming and differentiating HTLV type infection in repeatedly reactive samples. Novel and patented antigens added increased sensitivity in identifying specimens from infected individuals while differentiating those from uninfected individuals with false reactivity.

  13. Carbonic anhydrase IX as a specific biomarker for clear cell renal cell carcinoma: comparative study of Western blot and immunohistochemistry and implications for diagnosis.

    PubMed

    Giménez-Bachs, José M; Salinas-Sánchez, Antonio S; Serrano-Oviedo, Leticia; Nam-Cha, Syong H; Rubio-Del Campo, Antonio; Sánchez-Prieto, Ricardo

    2012-10-01

    This study aimed to evaluate the usefulness of carbonic anhydrase IX (CA-IX) expression in clear cell renal cell carcinoma (CCRCC) using two different techniques to detect protein expression. An experimental, cross-sectional, analytical study was conducted to analyse proteins in renal tumour and healthy tissue specimens from 38 consecutive patients who underwent nephrectomy for renal cancer. CA-IX protein expression was measured by immunohistochemistry and Western blot analysis and quantified. Statistical analysis was performed with the positive and negative specific agreements and kappa coefficient. The sensitivity and specificity of both techniques were assessed. Statistical tests were conducted to analyse the association between CA-IX expression quantitation and normal prognosis factors (TNM stage and Fuhrman nuclear grade), only in CCRCC. The mean patient age was 65 years, 78.9% of patients were men and 57.9% of tumours were CCRCC. CA-IX protein expression was positive in 63.2% of tumours by immunohistochemistry and in 60.5% by Western blot. Both techniques detected CA-IX expression only in CCRCC and unclassifiable tumours. High concordance indices were observed for CCRCC diagnosis. Western blot and immunohistochemistry had a sensitivity of 95.5% and 100%, respectively; the specificity was 100% in both techniques. CA-IX expression quantitation did not correlate with tumour stage or Fuhrman nuclear grade. Immunochemistry and Western blot techniques can be used to detect abnormal CA-IX protein expression in CCRCC and to support morphology-based diagnostic techniques.

  14. Resolution and identification of major peanut allergens using a combination of fluorescence two-dimensional differential gel electrophoresis, western blotting and Q-TOF mass spectrometry.

    USDA-ARS?s Scientific Manuscript database

    Peanut allergy is triggered by several proteins known as allergens. The matching resolution and identification of major peanut allergens in 2D protein maps, was accomplished by the use of fluorescence two-dimensional differential gel electrophoresis (2D DIGE), Western blotting and quadrupole time-of...

  15. Staging of recent HIV-1 infection using Geenius rapid confirmatory assay compared to INNO-LIA, New Lav and Blot 2.2 assays.

    PubMed

    Tuaillon, E; Sanosyan, A; Pisoni, A; Liscouët, J; Makinson, A; Perre, P Van de

    2017-10-01

    Besides confirmation of HIV seropositivity, Western Blot (WB) assays play an important role for identification of recent infection based on incomplete antibody reactivity and lack of p31 band. We evaluated the capacities of the Geenius™ HIV1/2 Confirmatory Assay (Bio-Rad), a new generation rapid confirmatory assay based on immune-chromatography and automated reading, for staging of HIV-1 infection. Sixteen samples collected during early HIV-1 infections (Fiebig stage III-VI) were tested using the Geenius assay, and compared to HIV Blot 2.2 WB assay (MP Diagnostics), New Lav Blot I WB assay (Bio-Rad) and INNO-LIA™ HIV I/II Score Dot Blot assay (Fujirebio). Results obtained with Geenius and INNO LIA in 47 newly diagnosed chronic HIV-1 infections were also compared. The p24 band was less frequently detected in early HIV-1 infections using the Geenius (3/16) compared to the New Lav (15/16, p<0.0001), INNO-LIA (13/16, p=0.0011), and Blot 2.2 (13/16, p=0.0011). Testing samples collected during chronic infection allowed to confirm that p31 band and complete Gag, Pol, Env profiles were less frequently observed using the Geenius assay compared to the INNO LIA assay (p=0.027 for p31, and p=0.0015 for complete profile). The Geenius assay is a simple and rapid test showing a high sensitivity to detect Env bands and to confirm HIV-1 seropositivity during the early phases of infection. However, this test is less suitable for distinguishing between later stages of acute and chronic infections because of a reduced sensitivity to detect the p31 and p24 bands compared to INNO LIA and New Lav assays. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Western blot analysis of stray cat sera against Toxoplasma gondii and the diagnostic availability of monoclonal antibodies in sandwich-ELISA

    PubMed Central

    Sohn, Woon-Mok

    1999-01-01

    A total of 198 sera from stray cats was assayed against Toxoplasma gondii antigen by western blot. Out of 198 sera assayed, 26 sera (13.1%) showed typical blot patterns against T. gondii. When spotted by ELISA absorbance and indirect latex agglutination test (ILAT) titer, all 26 cases were distributed over the cut-off value of ELISA whereas 24 cases (92.3%) were in the positive range of 1:32 or higher and 2 cases in negative range by ILAT. Among western blot negative 172 sera, 162 cases were negative in both ILAT and ELISA while 10 cases were reactive falsely such that three cases were ILAT positive with 1:32 titer and 9 cases were ELISA positive (2 cases overlapped). These 10 cases reacted peculiarly without typical binding pattern in Western blot. Sandwich-ELISA was performed with monoclonal antibodies (mAbs) of Tg563 (30 kDa, SAG1), Tg505 (22 kDa, SAG2), Tg605 (43 kDa, SAG3), Tg556 (28 kDa, GRA2), Tg737 (32 kDa, GRA6), Tg695 (66 kDa, ROP2), Tg786 (42 kDa, ROP6), and Tg621 (32 kDa, anonymous but cytosolic) clone, respectively. All western blot-positive cases were in the positive range and negative cases in the negative range clearly. Among the 10 false reactive cases, 3 cases were in the positive range with one or more mAbs. All mAbs used in this study were confirmed to be specific to T. gondii infection as a standardized sandwich-ELISA to differentiate it from other pathogens. PMID:10634041

  17. Northern Arizona Volcanoes

    NASA Technical Reports Server (NTRS)

    2006-01-01

    Northern Arizona is best known for the Grand Canyon. Less widely known are the hundreds of geologically young volcanoes, at least one of which buried the homes of local residents. San Francisco Mtn., a truncated stratovolcano at 3887 meters, was once a much taller structure (about 4900 meters) before it exploded some 400,000 years ago a la Mt. St. Helens. The young cinder cone field to its east includes Sunset Crater, that erupted in 1064 and buried Native American homes. This ASTER perspective was created by draping ASTER image data over topographic data from the U.S. Geological Survey National Elevation Data.

    With its 14 spectral bands from the visible to the thermal infrared wavelength region, and its high spatial resolution of 15 to 90 meters (about 50 to 300 feet), ASTER images Earth to map and monitor the changing surface of our planet.

    ASTER is one of five Earth-observing instruments launched December 18, 1999, on NASA's Terra satellite. The instrument was built by Japan's Ministry of Economy, Trade and Industry. A joint U.S./Japan science team is responsible for validation and calibration of the instrument and the data products.

    The broad spectral coverage and high spectral resolution of ASTER provides scientists in numerous disciplines with critical information for surface mapping, and monitoring of dynamic conditions and temporal change. Example applications are: monitoring glacial advances and retreats; monitoring potentially active volcanoes; identifying crop stress; determining cloud morphology and physical properties; wetlands evaluation; thermal pollution monitoring; coral reef degradation; surface temperature mapping of soils and geology; and measuring surface heat balance.

    The U.S. science team is located at NASA's Jet Propulsion Laboratory, Pasadena, Calif. The Terra mission is part of NASA's Science Mission Directorate.

    Size: 20.4 by 24.6 kilometers (12.6 by 15.2 miles) Location: 35.3 degrees North latitude, 111

  18. Dual infection of chickens with pox and infectious laryngotracheitis (ILT) confirmed with specific pox and ILT DNA dot-blot hybridization assays.

    PubMed

    Fatunmbi, O O; Reed, W M; Schwartz, D L; Tripathy, D N

    1995-01-01

    Dual infection with fowl pox (FP) and infectious laryngotracheitis (ILT) was diagnosed as the cause of acute mortality in a flock of three age groups of Hy-Line leghorn layers. The affected chickens had not been previously vaccinated against either FP or ILT. The diagnosis was confirmed by virus isolation, histopathology, and the use of specific pox and ILT genomic DNA probes in a dot-blot hybridization assay. FP and ILT vaccinations were recommended to control mortality. The use of FP- and ILT-specific DNA dot-blot hybridization may be used as a routine diagnostic tool to differentiate between the two diseases, especially in atypical cases of either infection or to confirm the existence of the two diseases as a mixed infection in a flock of chickens.

  19. Titan's Stratospheric Condensibles at High Northern Latitudes During Northern Winter

    NASA Technical Reports Server (NTRS)

    Anderson, Carrie; Samuelson, R.; Achterberg, R.

    2012-01-01

    The Infrared Interferometer Spectrometer (IRIS) instrument on board Voyager 1 caught the first glimpse of an unidentified particulate feature in Titan's stratosphere that spectrally peaks at 221 per centimeter. Until recently, this feature that we have termed 'the haystack,' has been seen persistently at high northern latitudes with the Composite Infrared Spectrometer (CIRS) instrument onboard Cassini, The strength of the haystack emission feature diminishes rapidly with season, becoming drastically reduced at high northern latitudes, as Titan transitions from northern winter into spring, In contrast to IRIS whose shortest wavenumber was 200 per centimeter, CIRS extends down to 10 per centimeter, thus revealing an entirely unexplored spectral region in which nitrile ices have numerous broad lattice vibration features, Unlike the haystack, which is only found at high northern latitudes during northern winter/early northern spring, this geometrically thin nitrile cloud pervades Titan's lower stratosphere, spectrally peaking at 160 per centimeter, and is almost global in extent spanning latitudes 85 N to 600 S, The inference of nitrile ices are consistent with the highly restricted altitude ranges over which these features are observed, and appear to be dominated by a mixture of HCN and HC3N, The narrow range in altitude over which the nitrile ices extend is unlike the haystack, whose vertical distribution is significantly broader, spanning roughly 70 kilometers in altitude in Titan's lower stratosphere, The nitrile clouds that CIRS observes are located in a dynamically stable region of Titan's atmosphere, whereas CH4 clouds, which ordinarily form in the troposphere, form in a more dynamically unstable region, where convective cloud systems tend to occur. In the unusual situation where Titan's tropopause cools significantly from the HASI 70.5K temperature minimum, CH4 should condense in Titan's lower stratosphere, just like the aforementioned nitrile clouds, although

  20. Titan's stratospheric condensibles at high northern latitudes during northern winter

    NASA Astrophysics Data System (ADS)

    Anderson, C.; Samuelson, R.; Achterberg, R.

    2012-04-01

    The Infrared Interferometer Spectrometer (IRIS) instrument on board Voyager 1 caught the first glimpse of an unidentified particulate feature in Titan’s stratosphere that spectrally peaks at 221 cm-1. Until recently, this feature that we have termed ‘the haystack,’ has been seen persistently at high northern latitudes with the Composite Infrared Spectrometer (CIRS) instrument onboard Cassini. The strength of the haystack emission feature diminishes rapidly with season, becoming drastically reduced at high northern latitudes, as Titan transitions from northern winter into spring. In contrast to IRIS whose shortest wavenumber was 200 cm-1, CIRS extends down to 10 cm-1, thus revealing an entirely unexplored spectral region in which nitrile ices have numerous broad lattice vibration features. Unlike the haystack, which is only found at high northern latitudes during northern winter/early northern spring, this geometrically thin nitrile cloud pervades Titan’s lower stratosphere, spectrally peaking at 160 cm-1, and is almost global in extent spanning latitudes 85°N to 60°S. The inference of nitrile ices are consistent with the highly restricted altitude ranges over which these features are observed, and appear to be dominated by a mixture of HCN and HC3N. The narrow range in altitude over which the nitrile ices extend is unlike the haystack, whose vertical distribution is significantly broader, spanning roughly 70 km in altitude in Titan’s lower stratosphere. The nitrile clouds that CIRS observes are located in a dynamically stable region of Titan’s atmosphere, whereas CH4 clouds, which ordinarily form in the troposphere, form in a more dynamically unstable region, where convective cloud systems tend to occur. In the unusual situation where Titan’s tropopause cools significantly from the HASI 70.5K temperature minimum, CH4 should condense in Titan’s lower stratosphere, just like the aforementioned nitrile clouds, although in significantly larger

  1. Avoiding pitfalls of internal controls: validation of reference genes for analysis by qRT-PCR and Western blot throughout rat retinal development.

    PubMed

    Rocha-Martins, Maurício; Njaine, Brian; Silveira, Mariana S

    2012-01-01

    Housekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development. We applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (β-actin, cyclophilin b, α-tubulin and lamin A/C), while MAPK1 was stably expressed. Putative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot.

  2. Mycoplasma agassizii strain variation and distinct host antibody responses explain differences between enzyme-linked immunosorbent assays and Western blot assays.

    PubMed

    Wendland, Lori D; Klein, Paul A; Jacobson, Elliott R; Brown, Mary B

    2010-11-01

    The precarious status of desert (Gopherus agassizii) and gopher (G. polyphemus) tortoises has resulted in conservation efforts that now include health assessment as an important component of management decision-making. Mycoplasmal upper respiratory tract disease (URTD) is one of very few diseases in chelonians for which comprehensive and rigorously validated diagnostic tests exist. In this study, serum samples obtained from eight Gopherus tortoises documented at necropsy to (i) be enzyme-linked immunosorbent assay (ELISA) seropositive using the PS6 antigen, (ii) be infected with Mycoplasma agassizii as indicated by direct isolation of the pathogen from the respiratory surfaces, and (iii) have histological lesions of mycoplasmal URTD were used to evaluate four distinct clinical isolates of M. agassizii as antigens for ELISA and Western blot analyses. Each animal sample reacted in the Western blot with its homologous M. agassizii strain, but recognition of heterologous M. agassizii strains was variable. Further, individual animals varied significantly with respect to the specific proteins recognized by the humoral immune response. An additional 114 Gopherus serum samples were evaluated using ELISA antigens prepared from the four distinct M. agassizii strains; A₄₀₅ values were significantly correlated (r² goodness of fit range, 0.708 to 0.771; P < 0.0001) for all antigens tested. The results confirm that strain variation is responsible for the observed differences between Western blot binding patterns. Thus, reliance on a single M. agassizii strain as an antigen in Western blot assays may provide false-negative results. This could have adverse consequences for the well-being of these environmentally sensitive hosts if false-negative animals were relocated to sites consisting of true-negative populations.

  3. Expansion of HIV screening to non-clinical venues is aided by the use of dried blood spots for Western blot confirmation.

    PubMed

    Sullivan, Timothy J; Antonio-Gaddy, Mara San; Richardson-Moore, April; Styer, Linda M; Bigelow-Saulsbery, Deborah; Parker, Monica M

    2013-12-01

    HIV rapid testing programs in New York State (NYS) are required to collect a specimen for confirmation of a preliminary positive result; however, some venues have limited capacity to collect venous blood, and confirmation using oral fluid is restricted by cost and availability. To evaluate the feasibility of using dried blood spots (DBS) at non-clinical HIV rapid testing sites for Western blot testing. The New York State Department of Health facilitated registration of 48 non-clinical HIV test sites and provided training on DBS procedures. Following a reactive rapid test, DBS were collected by fingerstick onto filter paper cards, dried and mailed to the NYS public health laboratory for Western blot testing. From October 2010 to December 2012, 280 DBS specimens were submitted for confirmation. Four (1.4%) were unsatisfactory for testing and 276 (98.6%) DBS were tested. Of these, 235 (85.1%) were positive, 37 (13.4%) were negative and 4 (1.4%) were indeterminate. During this period, the laboratory also received 1033 venous blood specimens for rapid test confirmation, and 35 (3.4%) were unsatisfactory. Of the 998 tested by Western blot, 784 (78.6%) were positive, 197 (19.7%) were negative and 17 (1.7%) were indeterminate. Compared to venous blood, the percentage of rapid test referral specimens with a positive Western blot was significantly greater for DBS specimens and the frequency of unsatisfactory specimens did not differ significantly. These results indicate that DBS are a suitable alternative to venous blood for confirmation of HIV rapid tests conducted at non-clinical sites. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Comparison of dot blot hybridization, polymerase chain reaction, and virus isolation for detection of bovine herpesvirus-1 (BHV-1) in artificially infected bovine semen.

    PubMed Central

    Xia, J Q; Yason, C V; Kibenge, F S

    1995-01-01

    Bovine semen samples spiked with bovine herpesvirus 1 (BHV-1) were used to compare dot blot hybridization, polymerase chain reaction (PCR), and virus isolation for detection of BHV-1 in bovine semen. The PCR amplification used primers targeting the BHV-1 thymidine kinase gene and a nucleic acid releasing cocktail (GeneReleaser); the PCR product was used as the DNA probe in dot blot hybridization; virus isolation was done in primary bovine fetal testis (BFT) cell cultures. Semen diluted 1:20 in tissue culture medium had the least cytotoxicity and inhibition of viral cytopathic effects in BFT cells, allowing detection of 1 TCID50/100 microL of BHV-1 suspension by virus isolation. The presence of foreign DNA such as bovine sperm DNA or salmon sperm DNA increased the sensitivity of dot blot hybridization in detecting BHV-1, allowing detection of 20,000 TCID50/100 microL of neat semen. The inhibition of PCR amplification of BHV-1 DNA in bovine semen was eliminated by diluting the samples 1:20 in tissue culture medium. The best PCR amplification was obtained when semen was diluted 1:20 and when a reaction buffer of pH 9.0, with 1.0 mM MgCl2 was used. Under these conditions, the PCR followed by ethidium bromide staining of agarose gels could detect 1 TCID20/100 microL of sample, whereas PCR followed by Southern blot hybridization could detect 0.01 TCID50/100 microL of sample.(ABSTRACT TRUNCATED AT 250 WORDS) Images Fig. 1. Fig. 2. Fig. 3. PMID:7648521

  5. Clinical Application of a Dot Blot Test for Diagnosis of Enteric Fever Due to Salmonella enterica Serovar Typhi in Patients with Typhoid Fever from Colombia and Peru

    PubMed Central

    Cardona-Castro, Nora; Gotuzzo, Eduardo; Rodriguez, Monica; Guerra, Humberto

    2000-01-01

    Clinical application of a dot blot test to detect immunoglobulin G (IgG) (88% sensitivity and specificity) and IgM (12.1% sensitivity and 97% specificity) against flagellar antigen from Salmonella enterica serovar Typhi was performed in Peruvian and Colombian patients with typhoid fever. This test can be used as a good predictor of serovar Typhi infection in regions lacking laboratory facilities and in field studies. PMID:10702512

  6. Avoiding Pitfalls of Internal Controls: Validation of Reference Genes for Analysis by qRT-PCR and Western Blot throughout Rat Retinal Development

    PubMed Central

    Rocha-Martins, Maurício; Njaine, Brian; Silveira, Mariana S.

    2012-01-01

    Background Housekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development. Methodology/Principal Findings We applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (β-actin, cyclophilin b, α-tubulin and lamin A/C), while MAPK1 was stably expressed. Conclusion Putative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot. PMID:22916200

  7. High-resolution methylation polymerase chain reaction for fragile X analysis: evidence for novel FMR1 methylation patterns undetected in Southern blot analyses.

    PubMed

    Chen, Liangjing; Hadd, Andrew; Sah, Sachin; Houghton, Jeffrey F; Filipovic-Sadic, Stela; Zhang, Wenting; Hagerman, Paul J; Tassone, Flora; Latham, Gary J

    2011-06-01

    Fragile X syndrome is associated with the expansion of CGG trinucleotide repeats and subsequent methylation of the FMR1 gene. Molecular diagnosis of fragile X currently requires Southern blot analysis to assess methylation. This study describes the evaluation of a polymerase chain reaction-only workflow for the determination of methylation status across a broad range of FMR1 genotypes in male and female specimens. We evaluated a novel method that combines allele-specific methylation polymerase chain reaction and capillary electrophoresis with eight cell line and 80 clinical samples, including 39 full mutations. Methylation status was determined using a three-step workflow: (1) differential treatment of genomic DNA using a methylation-sensitive restriction enzyme; (2) polymerase chain reaction with two sets of dye-tagged primers; and (3) amplicon sizing by capillary electrophoresis. All samples were analyzed by both methylation polymerase chain reaction and Southern blot analysis. FMR1 methylation status and CGG repeat sizing were accurately and reproducibly determined in a set of methylation controls and genomic DNA samples representing a spectrum of CGG repeat lengths and methylation states. Moreover, methylation polymerase chain reaction revealed allele-specific methylation patterns in premutation alleles that were unobtainable using Southern blot analysis. Methylation polymerase chain reaction enabled high throughput, high resolution, and semiquantitative methylation assessments of FMR1 alleles, as well as determinations of CGG repeat length. Results for all samples were concordant with corresponding Southern blot analyses. As a result, this study presents a polymerase chain reaction-based method for comprehensive FMR1 analysis. In addition, the identification of novel methylation mosaic patterns revealed after polymerase chain reaction and capillary electrophoresis may be relevant to several FMR1 disorders.

  8. Development of EMab-51, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Flow Cytometry, Western Blot, and Immunohistochemistry.

    PubMed

    Itai, Shunsuke; Kaneko, Mika K; Fujii, Yuki; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Chang, Yao-Wen; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

    2017-09-11

    The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG1, kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.

  9. Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis.

    PubMed

    Schüchner, Stefan; Andorfer, Peter; Mudrak, Ingrid; Ogris, Egon

    2016-08-17

    Western blotting is one of the most widely used techniques in molecular biology and biochemistry. Prestained proteins are used as molecular weight standards in protein electrophoresis. In the chemiluminescent Western blot analysis, however, these colored protein markers are invisible leaving researchers with the unsatisfying situation that the signal for the protein of interest and the signal for the markers are not captured simultaneously and have to be merged in an error-prone step. To allow the simultaneous detection of marker proteins we generated monoclonal antibodies specific for the protein dyes. To elicit a dye rather than protein specific immune response we immunized mice sequentially with dye-carrier protein complexes, in which a new carrier protein was used for each subsequent immunization. Moreover, by sequentially immunizing with dye-carrier protein complexes, in which different but structurally related dyes were used, we could also generate an antibody, termed anti-RAINBOW, that cross-reacted even with structurally related dyes not used in the immunizations. Our novel antibodies represent convenient tools for the simultaneous Western blot detection of commercially available prestained marker proteins in combination with the detection of any specific protein of interest. These antibodies will render obsolete the anachronistic tradition of manually charting marker bands on film.

  10. Western blot analysis of a limited number of cells: a valuable adjunct to proteome analysis of paraffin wax-embedded, alcohol-fixed tissue after laser capture microdissection.

    PubMed

    Martinet, Wim; Abbeloos, Vanessa; Van Acker, Nathalie; De Meyer, Guido R Y; Herman, Arnold G; Kockx, Mark M

    2004-03-01

    In recent years, laser capture microdissection (LCM) has been used successfully to obtain distinct populations of cells for subsequent molecular analysis. Because of the limited sample availability and the absence of in vitro amplification steps for proteins, the use of LCM for proteome analysis largely depends on highly sensitive protein detection methods. In this study, a western blot protocol was developed and validated for the detection of beta-actin and the moderately expressed cell death protein caspase-3 in small numbers of cells. Initially, cultured human U937 monocytes and whole sections of paraffin wax-embedded, alcohol-fixed human tonsils were used to optimize protein electrophoresis and western blotting conditions. High-performance NuPAGE Bis-Tris gels in combination with high-quality transfer membranes, optimized antibody concentrations, and a sensitive chemiluminescent substrate provided a strong signal for beta-actin with approximately 500 U937 cells. In the same way, procaspase-3 could be identified with approximately 1000 cells. Similar results were obtained with germinal centre cells that were procured from paraffin wax-embedded, alcohol-fixed human tonsils by LCM. Treatment of U937 cells with etoposide rapidly induced cell death and allowed the detection of active caspase-3 with approximately 2500 cells (0.8 pg of protein). The findings of this study suggest that western blotting is a valuable adjunct to proteome analysis of LCM procured cells. Copyright 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  11. Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis

    PubMed Central

    Schüchner, Stefan; Andorfer, Peter; Mudrak, Ingrid; Ogris, Egon

    2016-01-01

    Western blotting is one of the most widely used techniques in molecular biology and biochemistry. Prestained proteins are used as molecular weight standards in protein electrophoresis. In the chemiluminescent Western blot analysis, however, these colored protein markers are invisible leaving researchers with the unsatisfying situation that the signal for the protein of interest and the signal for the markers are not captured simultaneously and have to be merged in an error-prone step. To allow the simultaneous detection of marker proteins we generated monoclonal antibodies specific for the protein dyes. To elicit a dye rather than protein specific immune response we immunized mice sequentially with dye-carrier protein complexes, in which a new carrier protein was used for each subsequent immunization. Moreover, by sequentially immunizing with dye-carrier protein complexes, in which different but structurally related dyes were used, we could also generate an antibody, termed anti-RAINBOW, that cross-reacted even with structurally related dyes not used in the immunizations. Our novel antibodies represent convenient tools for the simultaneous Western blot detection of commercially available prestained marker proteins in combination with the detection of any specific protein of interest. These antibodies will render obsolete the anachronistic tradition of manually charting marker bands on film. PMID:27531616

  12. "Subtractive" Bilingualism in Northern Belize.

    ERIC Educational Resources Information Center

    Rubinstein, Robert A.

    "Subtractive" bilingualism in Northern Belize is analyzed based on an extension of a model by Wallace Lambert. The impact of English language instruction on Spanish speaking children in Corozal Town, the northernmost urban center in the British colony of Belize, Central America, is described. This description extends an earlier account…

  13. The outlook for northern forests

    Treesearch

    Stephen R. Shifley; W. Keith Moser; Sherri Wormstead; Francisco X. Aguilar

    2016-01-01

    The forests of the Northern United States have been remarkably resilient to nearly four centuries of immigration and settlement with associated anthropogenic disturbances caused by farming, logging, burning, urbanization, trade, and many other factors. Although resilient, forests conditions have been significantly impacted by humans as is illustrated by the extent of...

  14. Primary Science in Northern Ireland

    ERIC Educational Resources Information Center

    McAllister, Peter

    2005-01-01

    Since 1990 the science curriculum in Northern Ireland has gone through three major changes. In the beginning, fifteen attainment targets were introduced to an unsuspecting and largely unprepared teaching population: these were eventually reduced to five in 1993 and then to the present two in 1996. Unlike in England, technology has never stood as…

  15. BARRIERS TO NORTHERN SCHOOL DESEGREGATION.

    ERIC Educational Resources Information Center

    DENTLER, ROBERT A.

    UNLESS THE RATE OF INTEGRATION IN NORTHERN SCHOOLS IN LARGE CITIES ACCELERATES, THERE WILL BE EXTENSIVE URBAN SEGREGATION UNTIL AT LEAST THE MID-21ST CENTURY. HOWEVER, DATA FROM SMALLER CITIES SHOW THAT THERE SEEM TO BE "UNIFORM" CONDITIONS WHICH FAVOR DESEGREGATION--NEGRO PROTEST ACTION, STIMULUS FROM EXTRA-LOCAL AUTHORITY, AND A LESS HIGHLY…

  16. NUMA: A Northern Paiute History.

    ERIC Educational Resources Information Center

    Inter-Tribal Council of Nevada, Reno.

    One in a series of four histories of native Nevadans, this volume presents the story of the Northern Paiute people, or Numa, who lived, hunted, and travelled in the Great Basin area which occupies one-third of present day Nevada and parts of Oregon, Idaho, and California. Based on interviews with tribal elders and research conducted at numerous…

  17. Vesta Partly Shadowed Northern Regions

    NASA Image and Video Library

    2012-06-01

    This image from NASA Dawn spacecraft shows a part of asteroid Vesta surface reasonably far north into the northern hemisphere which appears washed out because it has been stretched to make features visible that would otherwise be too dark to see.

  18. Northern Storm in Full Force

    NASA Image and Video Library

    2013-01-31

    This mosaic of images from NASA Cassini spacecraft shows the trail of a great northern storm on Saturn raging in full force. The contrast in the images has been enhanced to make the turbulent parts of the storm in white stand out.

  19. Motivating Learners in Northern Communities.

    ERIC Educational Resources Information Center

    Swanson, Sharon

    2003-01-01

    A teacher at a northern Ontario adult Native literacy program describes how she cultivates student motivation. The teacher-student relationship is the most influential factor for motivating students. By focusing on cultural awareness, cultural teaching practices, and a sense of community, teachers can help students be successful. Program…

  20. The combination of quantitative PCR and western blot detecting CP4-EPSPS component in Roundup Ready soy plant tissues and commercial soy-related foodstuffs.

    PubMed

    Xiao, Xiao; Wu, Honghong; Zhou, Xinghu; Xu, Sheng; He, Jian; Shen, Wenbiao; Zhou, Guanghong; Huang, Ming

    2012-06-01

    With the widespread use of Roundup Ready soy (event 40-3-2) (RRS), the comprehensive detection of genetically modified component in foodstuffs is of significant interest, but few protein-based approaches have been found useful in processed foods. In this report, the combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different RRS plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing both meat and plant protein concentrates. The validity of the 2 methods was confirmed first. We also showed that the CP4-EPSPS protein existed in different RRS plant tissues. In certain cases, the results from the western blot and the qPCR were not consistent. To be specific, at least 2 degraded fragments of CP4-EPSPS protein (35.5 and 24.6 kDa) were observed. For dried bean curd crust and deep-fried bean curd, a degraded protein fragment with the size of 24.6 kDa appeared, while cp4-epsps gene could not be traced by qPCR. In contrast, we found a signal of cp4-epsps DNA in 3 foodstuffs, including soy-containing ham cutlet product, meat ball, and sausage by qPCR, while CP4-EPSPS protein could not be detected by western blot in such samples. Our study therefore concluded that the combination of DNA- and protein-based methods would compensate each other, thus resulting in a more comprehensive detection from nucleic acid and protein levels. The combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different Roundup Ready soy (event 40-3-2) plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing a combination of both meat and plant protein concentrates. This study indicated that the combination of DNA- and protein-based methods

  1. Northern Border Pipeline Company NPDES Permit

    EPA Pesticide Factsheets

    Under NPDES permit MT-0030791, the Northern Border Pipeline Company is authorized to discharge from locations along the Northern Border Gas Transmission Pipeline located within the exterior boundaries of the Fort Peck Indian Reservation, Montana.

  2. The epidemiology of tick-borne haemoparasites as determined by the reverse line blot hybridization assay in an intensively studied cohort of calves in western Kenya

    PubMed Central

    Njiiri, Nyawira E.; Bronsvoort, B. Mark deC.; Collins, Nicola E.; Steyn, Helena C.; Troskie, Milana; Vorster, Ilse; Thumbi, S.M.; Sibeko, Kgomotso P.; Jennings, Amy; van Wyk, Ilana Conradie; Mbole-Kariuki, Mary; Kiara, Henry; Poole, E. Jane; Hanotte, Olivier; Coetzer, Koos; Oosthuizen, Marinda C.; Woolhouse, Mark; Toye, Philip

    2015-01-01

    The development of sensitive surveillance technologies using PCR-based detection of microbial DNA, such as the reverse line blot assay, can facilitate the gathering of epidemiological information on tick-borne diseases, which continue to hamper the productivity of livestock in many parts of Africa and elsewhere. We have employed a reverse line blot assay to detect the prevalence of tick-borne parasites in an intensively studied cohort of indigenous calves in western Kenya. The calves were recruited close to birth and monitored for the presence of infectious disease for up to 51 weeks. The final visit samples from 453 calves which survived for the study period were analyzed by RLB. The results indicated high prevalences of Theileria mutans (71.6%), T. velifera (62.8%), Anaplasma sp. Omatjenne (42.7%), A. bovis (39.9%), Theileria sp. (sable) (32.7%), T. parva (12.9%) and T. taurotragi (8.5%), with minor occurrences of eight other haemoparasites. The unexpectedly low prevalence of the pathogenic species Ehrlichia ruminantium was confirmed by a species-specific PCR targeting the pCS20 gene region. Coinfection analyses of the seven most prevalent haemoparasites indicated that they were present as coinfections in over 90% of the cases. The analyses revealed significant associations between several of the Theileria parasites, in particular T. velifera with Theileria sp. sable and T. mutans, and T. parva with T. taurotragi. There was very little coinfection of the two most common Anaplasma species, although they were commonly detected as coinfections with the Theileria parasites. The comparison of reverse line blot and serological results for four haemoparasites (T. parva, T. mutans, A. marginale and B. bigemina) indicated that, except for the mostly benign T. mutans, indigenous cattle seem capable of clearing infections of the three other, pathogenic parasites to below detectable levels. Although the study site was located across four agroecological zones, there was

  3. Reverse Cross Blot Hybridization Assay for Rapid Detection of PCR-Amplified DNA from Candida Species, Cryptococcus neoformans, and Saccharomyces cerevisiae in Clinical Samples

    PubMed Central

    Posteraro, Brunella; Sanguinetti, Maurizio; Masucci, Luca; Romano, Lucio; Morace, Giulia; Fadda, Giovanni

    2000-01-01

    A PCR-based assay was developed to detect and identify medically important yeasts in clinical samples. Using a previously described set of primers (G. Morace et al., J. Clin. Microbiol. 35:667–672, 1997), we amplified a fragment of the ERG11 gene for cytochrome P-450 lanosterol 14α-demethylase, a crucial enzyme in the biosynthesis of ergosterol. The PCR product was analyzed in a reverse cross blot hybridization assay with species-specific probes directed to a target region of the ERG11 gene of Candida albicans (pCal), C. guilliermondii (pGui), C. (Torulopsis) glabrata (pGla), C. kefyr (pKef), C. krusei (pKru), C. parapsilosis (pPar), C. tropicalis (pTro), the newly described species C. dubliniensis (pDub), Saccharomyces cerevisiae (pSce), and Cryptococcus neoformans (pCry). The PCR-reverse cross blot hybridization assay correctly identified multiple isolates of each species tested. No cross-hybridization was detected with any other fungal, bacteria, or human DNAs tested. The method was tested against conventional identification on 140 different clinical samples, including blood and cerebrospinal fluid, from patients with suspected fungal infections. The results agreed with those of culture and phenotyping for all but six specimens (two of which grew yeasts not included in the PCR panel of probes and four in which PCR positivity-culture negativity was justified by clinical findings). Species identification time was reduced from a mean of 4 days with conventional identification to 7 h with the molecular method. The PCR-reverse cross blot hybridization assay is a rapid method for the direct detection and identification of yeasts in clinical samples. PMID:10747151

  4. Discovery of novel hematopoietic cell adhesion molecules from human bone marrow stromal cell membrane protein extracts by a new cell-blotting technique.

    PubMed

    Seshi, B

    1994-05-01

    In an attempt to define the role of cell adhesion molecules (CAMs) within the bone marrow (BM) microenvironment in normal hematopoiesis and in leukemia development, a novel cell-blotting technique that involved cell adhesion to protein bands after separation by lithium dodecyl sulfate-polyacrylamide gel electrophoresis (LDS-PAGE) and blotting onto polyvinylidene difluoride (PVDF) membrane has been developed. Human BM stromal cell membrane fractions have been prepared from Dexter-type cultures after cell lysis by sonification and differential centrifugations of the sonification contents. The 20,000 g pellets representing membrane fractions have been solubilized by 2% Triton X-100, 0.575% LDS, and 8 mol/L urea in sequential order. The protein extracts are fractionated by LDS-PAGE and screened for CAMs by the new cell-blotting technique. This led to identification of nine protein bands in lanes containing LDS extracts showing adhesion of KG1a (CD34+ progenitor myeloid) cells. Evidence that the BM proteins exhibiting KG1a cell adhesion are novel CAMs is based on the observations that these proteins, in comparison with known CAMs, specifically VCAM-1, CD54, and CD44, show (1) contrasting detergent-solubility properties, (2) different temperature requirement for mediating cell adhesion function, and (3) markedly distinct electrophoretic mobilities. The various cell types tested, notably KG1a, NALM-6, WIL-2, Ramos, HS-Sultan, K562, JY B lymphoblastoid cells, and T lymphoblasts, showed distinctive patterns of binding to different subsets of BM CAMs. These results demonstrate a new approach to studies of molecular mechanisms that may determine specificity of hematopoietic cellular localization within BM microenvironment and may play an important role in controlling hematopoiesis.

  5. Rapid Preparation of a Plasma Membrane Fraction: Western Blot Detection of Translocated Glucose Transporter 4 from Plasma Membrane of Muscle and Adipose Cells and Tissues.

    PubMed

    Yamamoto, Norio; Yamashita, Yoko; Yoshioka, Yasukiyo; Nishiumi, Shin; Ashida, Hitoshi

    2016-08-01

    Membrane proteins account for 70% to 80% of all pharmaceutical targets, indicating their clinical relevance and underscoring the importance of identifying differentially expressed membrane proteins that reflect distinct disease properties. The translocation of proteins from the bulk of the cytosol to the plasma membrane is a critical step in the transfer of information from membrane-embedded receptors or transporters to the cell interior. To understand how membrane proteins work, it is important to separate the membrane fraction of cells. This unit provides a protocol for rapidly obtaining plasma membrane fractions for western blot analysis. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  6. Hammersley Range, northern Western Australia

    NASA Technical Reports Server (NTRS)

    1990-01-01

    The oval shaped basin of the sedimentary rocks of the Hammersley Range, northern Western Australia (23.0S, 119.0E) dominates the center of this near nadir view. The Fortescue River is the remarkably straight, fault controlled feature bordering the Hammersley on the north. Sand dunes are the main surface features in the northeast and southwest. Many dry lakebeds can be seen to the east as light grey colored patches along the watercourses.

  7. Carrion use by northern goshawks

    Treesearch

    John R. Squires

    1995-01-01

    Northern goshawks (Accipiter gentilis) feed on a wide variety of birds and mammals (R. T. Reynolds, et al. 1992, USDA For. Ser. Gen. Tech. Rep. RM-217. Fort Collins, CO U.S.A.), but few accounts describe goshawks feeding on carrion. J.H. Schnell (1958, Condor 60:377-403) stated that, "It seems highly unlikely that the goshawk would forage for carrion under normal...

  8. Ceres Northern Hemisphere in Survey

    NASA Image and Video Library

    2015-06-10

    Craters in the northern hemisphere of dwarf planet Ceres are seen in this image taken by NASA's Dawn spacecraft on June 6, 2015. This is among the first snapshots from Dawn's second mapping orbit, which is 2,700 miles (4,400 kilometers) in altitude. The resolution is 1,400 feet (410 meters) per pixel. http://photojournal.jpl.nasa.gov/catalog/PIA19570

  9. Hammersley Range, northern Western Australia

    NASA Technical Reports Server (NTRS)

    1990-01-01

    The oval shaped basin of the sedimentary rocks of the Hammersley Range, northern Western Australia (23.0S, 119.0E) dominates the center of this near nadir view. The Fortescue River is the remarkably straight, fault controlled feature bordering the Hammersley on the north. Sand dunes are the main surface features in the northeast and southwest. Many dry lakebeds can be seen to the east as light grey colored patches along the watercourses.

  10. Hammersley Range, northern Western Australia

    NASA Image and Video Library

    1990-04-29

    The oval shaped basin of the sedimentary rocks of the Hammersley Range, northern Western Australia (23.0S, 119.0E) dominates the center of this near nadir view. The Fortescue River is the remarkably straight, fault controlled feature bordering the Hammersley on the north. Sand dunes are the main surface features in the northeast and southwest. Many dry lakebeds can be seen to the east as light grey colored patches along the watercourses.

  11. The northern Egyptian continental margin

    NASA Astrophysics Data System (ADS)

    Badawy, Ahmed; Mohamed, Gad; Omar, Khaled; Farid, Walid

    2015-01-01

    Africa displays a variety of continental margin structures, tectonics and sedimentary records. The northern Egyptian continental margin represents the NE portion of the North African passive continental margin. Economically, this region is of great importance as a very rich and productive hydrocarbon zone in Egypt. Moreover, it is characterized by remarkable tectonic setting accompanied by active tectonic processes from the old Tethys to recent Mediterranean. In this article, seismicity of the northern Egyptian continental margin has been re-evaluated for more than 100-years and the source parameters of three recent earthquakes (October 2012, January 2013 and July 2013) have been estimated. Moment tensor inversions of 19th October 2012 and 17th January 2013 earthquakes reveal normal faulting mechanism with strike-slip component having seismic moment of 3.5E16 N m and 4.3E15 N m respectively. The operation of the Egyptian National Seismic Network (ENSN) since the end of 1997 has significantly enhanced the old picture of earthquake activity across northern Egyptian continental margin whereas; the record-ability (annual rate) has changed from 2-events/year to 54-event/year before and after ENSN respectively. The spatial distribution of earthquakes foci indicated that the activity tends to cluster at three zones: Mediterranean Ridge (MR), Nile Cone (NC) and Eratosthenes Seamount (ERS). However, two seismic gaps are reported along Levant Basin (LEV) and Herodotus Basin (HER).

  12. Human T-cell lymphotropic virus type I (HTLV-I) confirmed by PCR-Southern blot and sequencing analysis in a woman with tropical spastic paraparesis.

    PubMed

    Perandin, F; Cariani, A; Bonfanti, C; Trainini, L; Magoni, M; Manca, N

    2006-09-01

    Human T-cell lymphotropic virus type I (HTLV-I) is a human retrovirus and the aetiological agent of a progressive neurological disease called tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM), as confirmed by evidence accumulated in HTLV-I seroprevalence studies. TSP/HAM is rarely diagnosed in Italy, given the low prevalence of HTLV-I in the population. TSP/HAM begins insidiously in the fourth decade, mainly with spastic paraparesis of the lower extremities and positive Babinski reflex, as well as interfering with bowel and bladder functions. In this study we report the clinical, virological and haemato chemical data of a 54-year-old woman, born in the Ivory Cost, with symptoms suggestive of TSP. The presence of HTLV-I infection was demonstrated by the detection of antibodies in serum and in cerebrospinal fluid by immunoenzymatic assay and Western blot analysis. In addition, viral isolation was carried out in peripheral blood cells, and the presence of HTLV-I proviral DNA was confirmed by polymerase chain reaction/Southern blot and sequencing analysis. According to our results, HTLV-I testing might be useful when TSP/HAM is suspected.

  13. Analysis of the insulin receptor gene in noninsulin-dependent diabetes mellitus by denaturing gradient gel blots: A clinical research center study

    SciTech Connect

    Magre, J.; Goldfine, A.B.; Warram, J.H.

    1995-06-01

    We have used a new technique of denaturing gradient gel blotting to determine the prevalence of alterations in the intracellular domain of the insulin receptor in normal individuals and subjects with non-insulin-dependent diabetes mellitus (NIDDM). This method detects DNA sequence differences as restriction fragment melting polymorphisms (RFMP) and is sensitive to changes in sequence at both restriction sites and within the fragments themselves. Using restriction digests with AluI, HaeIII, HinfI, RsaI, Sau3A, and Sau96, 12 RFMPs were found to localize to the region of the {beta}-subunit of the insulin receptor gene. Using exon-specific probes, these RFMPs could be localized to specific regions surrounding individual exons, including exons, 14, 15, 16, 18, 20, and 22. In general, linkage disequilibrium between polymorphisms was inversely related to their distance in the gene structure, although there was a {open_quotes}hot spot{close_quotes} for recombination between exons 19 and 20. No difference in melting temperatures or allele frequency was observed between NIDDM patients and controls. These data indicate that the region of the insulin receptor gene coding for the intracellular portion of the {beta}-subunit is highly polymorphic and that polymorphisms surrounding specific exons can be identified by denaturing gradient gel blotting, but there is no evidence that variation at this locus contributes to NIDDM susceptibility in most individuals. 36 refs., 3 figs., 3 tabs.

  14. A research design for the quantification of the neuropeptides substance p and calcitonin gene-related Peptide in rat skin using Western blot analysis.

    PubMed

    Lapin, Guilherme Abbud Franco; Hochman, Bernardo; Nishioka, Michele Akemi; Maximino, Jessica Ruivo; Chadi, Gerson; Ferreira, Lydia Masako

    2015-06-01

    To describe and standardize a protocol that overcomes the technical limitations of Western blot (WB) analysis in the quantification of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) following nociceptive stimuli in rat skin. Male Wistar rats (Rattus norvegicus albinus) weighing 250 to 350 g were used in this study. Elements of WB analysis were adapted by using specific manipulation of samples, repeated cycles of freezing and thawing, more thorough maceration, and a more potent homogenizer; increasing lytic reagents; promoting greater inhibition of protease activity; and using polyvinylidene fluoride membranes as transfer means for skin-specific protein. Other changes were also made to adapt the WB analysis to a rat model. University research center. Western blot analysis adapted to a rat model. This research design has proven effective in collecting and preparing skin samples to quantify SP and CGRP using WB analysis in rat skin. This study described a research design that uses WB analysis as a reproducible, technically accessible, and cost-effective method for the quantification of SP and CGRP in rat skin that overcomes technical biases.

  15. IgG avidity Western blot using Toxoplasma gondii rGRA-7 cloned from nucleotides 39-711 for serodiagnosis of acute toxoplasmosis.

    PubMed

    Deshpande, Poonam S; Kotresha, Dupadahalli; Noordin, Rahmah; Yunus, Muhammad Hafiznur; Saadatnia, Geita; Golkar, Majid; Osman, Sabariah; Karim, Izzati Zahidah Abdul; Ghaffarifar, Fatemeh

    2013-01-01

    Toxoplasmosis is an important cause of congenital infection. The present study was performed to evaluate the usefulness of recombinant (r) GRA-7 cloned from nucleotides (n) 39-711 in discriminating between acute and chronic toxoplasmosis. First, commercial IgM, IgG and IgG avidity ELISAs were used to determine the serological profile of the sera. Serum samples were from 20 symptomatic patients with acute infection (low IgG avidity, IgM positive), 10 with chronic infection (high IgG avidity, IgM negative) and 10 with indeterminate IgG avidity (IgM positive) which were tested for IgG avidity status with an in-house developed IgG avidity Western blot using the rGRA-7 recombinant antigen. All 20 sera from cases of probable acute infection showed bands which either faded out completely or reduced significantly in intensity after treatment with 8 M urea, whereas the band intensities of the 10 serum samples from chronic cases remained the same. Of the 10 sera with indeterminate IgG avidity status, after treatment with 8 M urea the band intensities with six sera remained the same, two sera had completely faded bands and another two sera had significantly reduced band intensities. Discrimination between acute and chronic toxoplasmosis was successfully performed by the in-house IgG avidity Western blot.

  16. Characterization of 65 epitope-specific dystrophin monoclonal antibodies in canine and murine models of duchenne muscular dystrophy by immunostaining and western blot.

    PubMed

    Kodippili, Kasun; Vince, Lauren; Shin, Jin-Hong; Yue, Yongping; Morris, Glenn E; McIntosh, Mark A; Duan, Dongsheng

    2014-01-01

    Epitope-specific monoclonal antibodies can provide unique insights for studying cellular proteins. Dystrophin is one of the largest cytoskeleton proteins encoded by 79 exons. The absence of dystrophin results in Duchenne muscular dystrophy (DMD). Over the last two decades, dozens of exon-specific human dystrophin monoclonal antibodies have been developed and successfully used for DMD diagnosis. Unfortunately, the majority of these antibodies have not been thoroughly characterized in dystrophin-deficient dogs, an outstanding large animal model for translational research. To fill the gap, we performed a comprehensive study on 65 dystrophin monoclonal antibodies in normal and dystrophic dogs (heart and skeletal muscle) by immunofluorescence staining and western blot. For comparison, we also included striated muscles from normal BL10 and dystrophin-null mdx mice. Our analysis revealed distinctive species, tissue and assay-dependent recognition patterns of different antibodies. Importantly, we identified 15 antibodies that can consistently detect full-length canine dystrophin in both immunostaining and western blot. Our results will serve as an important reference for studying DMD in the canine model.

  17. Determining the cleavage site for the mature antimicrobial peptide of Nile tilapia β-defensin using 2D electrophoresis, western blot, and mass spectrometry analysis.

    PubMed

    Chang, Chin-I; Chen, Li-Hao; Hu, Yeh-Fang; Wu, Chia-Che; Tsai, Jyh-Ming

    2017-03-01

    Several proteomic techniques were used to determine the cleavage site of the mature antimicrobial peptide of Nile tilapia β-defensin. The computer-predicted Nile tilapia β-defensin ((25)ASFPWSCLSLSGVCRKVCLPTELFFGPLGCGKGSLCCVSHFL(66)) composed of 42 amino acids was chemically synthesized and prepared to produce an antibody for Western blotting. Total proteins from the skin of the Nile tilapia were separated on two-dimensional electrophoresis, and the spot of Nile tilapia β-defensin was recognized using Western blot analysis. It was then excised and extracted from the gel. The precise molecular mass of this spot was determined by LC-MS/MS spectrometry. Four major peptides were discovered, with molecular weights of 4293.2 Da, 4306.5 Da, 4678.9 Da, and 4715.0 Da. The calculated mass of the 40-amino-acid sequence ((27)FPWSCLSLSGVCRKVCLPTELFFGPLGCGKGSLCCVSHFL(66)) of Nile tilapia β-defensin starting from Phe27 and ending with Leu66 was 4293.18 Da, which completely matched the 4293.2 Da peptide that was obtained from the mass spectrometry analysis. This result confirmed that the cleavage site for the mature C-terminal Nile tilapia β-defensin is at residue Ser26-Phe27, not at Ala24-25 as predicted by computer analysis. This study provides a simple but reliable model to determine the cleavage site for a mature antimicrobial peptide. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Positively charged polymer brush-functionalized filter paper for DNA sequence determination following Dot blot hybridization employing a pyrrolidinyl peptide nucleic acid probe.

    PubMed

    Laopa, Praethong S; Vilaivan, Tirayut; Hoven, Voravee P

    2013-01-07

    As inspired by the Dot blot analysis, a well known technique in molecular biology and genetics for detecting biomolecules, a new paper-based platform for colorimetric detection of specific DNA sequences employing peptide nucleic acid (PNA) as a probe has been developed. In this particular study, a pyrrolidinyl PNA bearing a conformationally rigid d-prolyl-2-aminocyclopentanecarboxylic acid backbone (acpcPNA) was used as a probe. The filter paper was modified to be positively charged with grafted polymer brushes of quaternized poly(dimethylamino)ethyl methacrylate (QPDMAEMA) prepared by surface-initiated polymerization of 2-(dimethylamino)ethyl methacrylate from the filter paper via ARGET ATRP followed by quaternization with methyl iodide. Following the Dot blot format, a DNA target was first immobilized via electrostatic interactions between the positive charges of the QPDMAEMA brushes and negative charges of the phosphate backbone of DNA. Upon hybridization with the biotinylated pyrrolidinyl peptide nucleic acid (b-PNA) probe, the immobilized DNA can be detected by naked eye observation of the yellow product generated by the enzymatic reaction employing HRP-labeled streptavidin. It has been demonstrated that this newly developed assay was capable of discriminating between complementary and single base mismatch targets at a detection limit of at least 10 fmol. In addition, the QPDMAEMA-grafted filter paper exhibited a superior performance to the commercial membranes, namely Nylon 66 and nitrocellulose.

  19. A Comparison of Antibacterial Activity of Selected Thyme (Thymus) Species by Means of the Dot Blot Test with Direct Bioautographic Detection.

    PubMed

    Orłowska, Marta; Kowalska, Teresa; Sajewicz, Mieczysław; Jesionek, Wioleta; Choma, Irena M; Majer-Dziedzic, Barbara; Szymczak, Grażyna; Waksmundzka-Hajnos, Monika

    2015-01-01

    Bioautography carried out with the aid of thin-layer chromatographic adsorbents can be used to assess antibacterial activity in samples of different origin. It can either be used as a simple and cost-effective detection method applied to a developed chromatogram, or to the dot blot test performed on a chromatographic plate, where total antibacterial activity of a sample is scrutinized. It was an aim of this study to compare antibacterial activity of 18 thyme (Thymus) specimens and species (originating from the same gardening plot and harvested in the same period of time) by means of a dot blot test with direct bioautography. A two-step extraction of herbal material was applied, and at step two the polar fraction of secondary metabolites was obtained under the earlier optimized extraction conditions [methanol-water (27+73, v/v), 130°C]. This fraction was then tested for its antibacterial activity against Bacillus subtilis bacteria. It was established that all investigated extracts exhibited antibacterial activity, yet distinct differences were perceived in the size of the bacterial growth inhibition zones among the compared thyme species. Based on the results obtained, T. citriodorus "golden dwarf" (sample No. 5) and T. marschallianus (sample No. 6) were selected as promising targets for further investigations and possible inclusion in a herbal pharmacopeia, which is an essential scientific novelty of this study.

  20. Polymerase chain reaction and Southern blot-based analysis of the C9orf72 hexanucleotide repeat in different motor neuron diseases.

    PubMed

    Hübers, Annemarie; Marroquin, Nicolai; Schmoll, Birgit; Vielhaber, Stefan; Just, Marlies; Mayer, Benjamin; Högel, Josef; Dorst, Johannes; Mertens, Thomas; Just, Walter; Aulitzky, Anna; Wais, Verena; Ludolph, Albert C; Kubisch, Christian; Weishaupt, Jochen H; Volk, Alexander E

    2014-05-01

    The GGGGCC-hexanucleotide repeat expansion in C9orf72 is the most common genetic cause of familial amyotrophic lateral sclerosis and frontotemporal dementia. This study determined the frequency of C9orf72 repeat expansions in different motor neuron diseases (amyotrophic lateral sclerosis (ALS), motor neuron diseases affecting primarily the first or the second motor neuron and hereditary spastic paraplegia). Whereas most studies on C9orf72 repeat expansions published so far rely on a polymerase chain reaction-based screening, we applied both polymerase chain reaction-based techniques and Southern blotting. Furthermore, we determined the sensitivity and specificity of Southern blotting of the C9orf72 hexanucleotide repeat in DNA derived from lymphoblastoid cell lines. C9orf72 repeat expansions were found in 27.1% out of 166 familial ALS patients, only once in 68 sporadic ALS patients, and not in 61 hereditary spastic paraplegia patients or 52 patients with motor neuron diseases affecting clinically primarily either the first or the second motor neuron. We found hints for a correlation between C9orf72 repeat length and the age of onset. Somatic instability of the C9orf72 repeat was observed in lymphoblastoid cell lines compared with DNA derived from whole blood from the same patient and therefore caution is warranted for repeat length determination in immortalized cell lines.

  1. Application of a reverse dot blot DNA-DNA hydridization method to quantify host-feeding tendencies of two sibling species in the Anopheles gambiae complex.

    PubMed

    Fritz, M L; Miller, J R; Bayoh, M N; Vulule, J M; Landgraf, J R; Walker, E D

    2013-12-01

    A DNA-DNA hybridization method, reverse dot blot analysis (RDBA), was used to identify Anopheles gambiae s.s. and Anopheles arabiensis (Diptera: Culicidae) hosts. Of 299 blood-fed and semi-gravid An. gambiae s.l. collected from Kisian, Kenya, 244 individuals were identifiable to species; of these, 69.5% were An. arabiensis and 29.5% were An. gambiae s.s. Host identifications with RDBA were comparable with those of conventional polymerase chain reaction (PCR) followed by direct sequencing of amplicons of the vertebrate mitochondrial cytochrome b gene. Of the 174 amplicon-producing samples used to compare these two methods, 147 were identifiable by direct sequencing and 139 of these were identifiable by RDBA. Anopheles arabiensis bloodmeals were mostly (94.6%) bovine in origin, whereas An. gambiae s.s. fed upon humans more than 91.8% of the time. Tests by RDBA detected that two of 112 An. arabiensis contained blood from more than one host species, whereas PCR and direct sequencing did not. Recent use of insecticide-treated bednets in Kisian is likely to have caused the shift in the dominant vector species from An. gambiae s.s. to An. arabiensis. Reverse dot blot analysis provides an opportunity to study changes in host-feeding by members of the An. gambiae complex in response to the broadening distribution of vector control measures targeting host-selection behaviours. © 2013 The Royal Entomological Society.

  2. Assessment and modification of a Western blot assay for detection of central nervous system tissue in meat products in the United States.

    PubMed

    Salman, M D; Jemmi, T; Triantis, J; Dewell, R D

    2005-08-01

    Health hazards associated with meat contaminated by the bovine spongiform encephalopathy agent have led to the development of tests for the presence of this agent. The objective of this study was to optimize a neuron-specific enolase Western blot assay for use in the United States. We compared the original test with a modified protocol to evaluate the detection limit for the presence of central nervous system (CNS) tissue in experimentally inoculated samples and compared and evaluated the utility of these tests for detecting CNS tissue in retail sausages. Sensitivity and specificity of the original and modified protocols were evaluated using the kappa statistic to assess agreement between the results of the two protocols. The original protocol resulted in 100% specificity and 92% sensitivity for raw samples and 92% specificity and 72% sensitivity for cooked samples. The modified protocol resulted in 92% specificity and 89% sensitivity for raw samples and 83% specificity and 75% sensitivity for cooked samples. The kappa statistic for protocol comparison was 0.94 for raw samples and 0.74 for cooked samples. Both protocols correctly identified CNS tissue in positive controls for each replicate. Although the Western blot technique should be considered for screening for the presence of bovine CNS tissue in meat samples, the techniques should be further optimized to address problems of low sensitivity. A test with higher sensitivity is needed to protect consumers from food safety threats associated with bovine CNS tissue.

  3. Multiplexed quantification of plant thylakoid proteins on Western blots using lanthanide-labeled antibodies and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS).

    PubMed

    de Bang, Thomas Christian; Pedas, Pai; Schjoerring, Jan Kofod; Jensen, Poul Erik; Husted, Søren

    2013-05-21

    We have developed a novel calibration method that allows concurrent quantification of multiple proteins by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) after Western blotting. Calibrants were made of nitrocellulose membranes doped with lanthanide standards. Excellent linearity was obtained in the interval from 0 to 24 ng lanthanide cm(-2). Cerium-labeled lysozyme was introduced as an internal reference protein, enabling correction for up to 50% difference in transfer efficiency during the blotting of membranes. The sensitivity of the LA-ICP-MS method was comparable to state-of-the-art chemiluminescence detection and was further improved by a factor of 20, using a polymer tag. Our method allowed reproducible and multiplexed quantification of five thylakoid proteins extracted from chloroplasts of the plant species Arabidopsis thaliana (relative standard deviation (RSD) of ≤ 5% in three independent analytical series). The method was capable of measuring the L subunit in photosystem I of an Arabidopsis mutant containing <5% of this particular protein, relative to the wild type. We conclude that the developed calibration method is highly suited for multiplexed and comparative protein studies, allowing for intermembrane comparisons with high sensitivity and reproducibility.

  4. A combined RT-PCR and dot-blot hybridization method reveals the coexistence of SJNNV and RGNNV betanodavirus genotypes in wild meagre (Argyrosomus regius).

    PubMed

    Lopez-Jimena, B; Cherif, N; Garcia-Rosado, E; Infante, C; Cano, I; Castro, D; Hammami, S; Borrego, J J; Alonso, M C

    2010-10-01

    To detect the possible coexistence of striped jack nervous necrosis virus (SJNNV) and red-spotted grouper nervous necrosis virus (RGNNV) genotypes in a single fish, a methodology based on the combination of PCR amplification and blot hybridization has been developed and applied in this study. The degenerate primers designed for the PCR procedure target the T4 region within the capsid gene, resulting in the amplification of both genotypes. The subsequent hybridization of these amplification products with two different specific digoxigenin-labelled probes resulted in the identification of both genotypes separately. The application of the RT-PCR protocol to analyse blood samples from asymptomatic wild meagre (Argyrosomus regius) specimens has shown a 46.87% of viral nervous necrosis virus carriers. The combination of RT-PCR and blot hybridization increases the detection rate up to 90.62%, and, in addition, it has shown the coexistence of both genotypes in 18 out of the 32 specimens analysed (56.25%). This study reports the coexistence of betanodaviruses belonging to two different genotypes (SJNNV and RGNNV) in wild fish specimens. This is the first report demonstrating the presence of SJNNV and RGNNV genotypes in the same specimen. This study also demonstrates a carrier state in this fish species for the first time. © 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.

  5. Analysis of differentially expressed genes in two immunologically distinct strains of Eimeria maxima using suppression subtractive hybridization and dot-blot hybridization

    PubMed Central

    2014-01-01

    Background It is well known that different Eimeria maxima strains exhibit significant antigenic variation. However, the genetic basis of these phenotypes remains unclear. Methods Total RNA and mRNA were isolated from unsporulated oocysts of E. maxima strains SH and NT, which were found to have significant differences in immunogenicity in our previous research. Two subtractive cDNA libraries were constructed using suppression subtractive hybridization (SSH) and specific genes were further analyzed by dot-blot hybridization and qRT-PCR analysis. Results A total of 561 clones were selected from both cDNA libraries and the length of the inserted fragments was 0.25–1.0 kb. Dot-blot hybridization revealed a total of 86 differentially expressed clones (63 from strain SH and 23 from strain NT). Nucleotide sequencing analysis of these clones revealed ten specific contigs (six from strain SH and four from strain NT). Further analysis found that six contigs from strain SH and three from strain NT shared significant identities with previously reported proteins, and one contig was presumed to be novel. The specific differentially expressed genes were finally verified by RT-PCR and qRT-PCR analyses. Conclusions The data presented here suggest that specific genes identified between the two strains may be important molecules in the immunogenicity of E. maxima that may present potential new drug targets or vaccine candidates for coccidiosis. PMID:24894832

  6. Characterization of antigenic property of Toxocara canis and Toxascaris leonina adults and larvae through immunodiagnostic electrophoresis (SDS-PAGE) and western blot technique.

    PubMed

    el-Massry, A A

    1999-08-01

    Differential molecular studies were performed by sodium dodecyle sulphate polyacrylamide gel electrophoresis--SDS-PAGE--between somatic antigen of Toxocara canis and Toxascaris leonina, adults and larvae, recovered from dogs. SDS-PAGE of both adults somatic antigen showed two closely similar bands (90.00, 91.95 KDa and 69.25-70.56 KDa). Each parasite had characteristic bands clustered in different molecular weights. While for their larval antigen, T. canis showed a very different antigenic profile when analysed in comparison to T. leonina antigen except at one band (66.85-66.89 KDa). The Western blot analysis showed four prominent bands represented immunoreaction between the separated somatic antigen of T. canis adults and experimentally immunized rabbit with the corresponding parasite (125.37, 117.73, 90.00 and 69.25 KDa). While separated antigen of T. leonina adults immune reacted with the corresponding hyperimmune rabbit sera at 119.04, 91.95 and 70.56 KDa. The Western blot showed cross reactive immune bands between T. canis and T. leonina adults somatic antigen at two bands (90.00, 91.95 KDa and 69.25-70.56 KDa). The polypeptide bands reacted at 125.37 KDa and 117.73 KDa can be used as specific finger print for T. canis adults while that at 119.04 KDa was specific for T. leonina adult worm.

  7. Beta-thalassaemia mutations in northern India (Delhi).

    PubMed

    Madan, N; Sharma, S; Rusia, U; Sen, S; Sood, S K

    1998-03-01

    The present study was undertaken to define beta-thalassaemia mutations prevalent in northern India (Delhi). Forty six children of beta-thalassaemia major and their families were investigated. DNA was extracted from leucocytes and screened for mutations prevalent in the Indian population. These mutations included 619bp deletion, IVS 1-1 (G-T), IVS 1-5 (G-C), frameshift mutations FS 8/9 (+G), FS 41/42 (-CTTT), Codon 16(-C), Codon 15 (G-A), codon 30 (G-C), IVS 1-110 (G-A) and -88 (C-T). 619 bp deletion mutation was detected directly by amplification of DNA by PCR followed by agarose gel electrophoresis. Other mutations were studied by DNA amplification and dot blot hybridization using synthetic normal and mutant oligonucleotide probes labelled at 5' end with gamma-32 P-ATP. Five mutations accounted for all the chromosomes in 46 patients. 619 bp deletion mutation was found to be the commonest mutation (34.8%) followed by IVS 1-5 (G-C) in 22.8 per cent, IVS 1-1 (G-T) in 19.6 per cent, FS 8/9 (+G) in 13 per cent and FS 41/42 (-CTTT) in 9.8 per cent. Nineteen (41.3%) patients were homozygous and 27 (58.7%) double heterozygous for different beta-thalassaemia mutations. This observation of limited number of mutations is significant and will be useful in planning strategies for prenatal diagnosis of beta-thalassaemia in northern India.

  8. Geothermal systems of northern Nevada

    USGS Publications Warehouse

    Hose, Richard Kenneth; Taylor, Bruce Edward

    1974-01-01

    Hot springs are numerous and nearly uniformly distributed in northern Nevada. Most occur on the flanks of basins, along Basin and Range (late Miocene to Holocene) faults, while some occur in the inner parts of the basins. Surface temperatures of the springs range from slightly above ambient to, boiling; some springs are superheated. Maximum subsurface water temperatures calculated on the basis of quartz solubility range as high as 252?C, although most are below 190?C. Flows range from a trickle to several hundred liters per minute. The Nevada geothermal systems differ markedly from the power-producing system at The Geysers, Calif., and from those areas with a high potential, for power production (e.g., Yellowstone Park, Wyo.; Jemez Mountains, N. Mex.). These other systems are associated with Quaternary felsic volcanic rocks and probably derive their heat from cooling magma rather high in the crust. In northern Nevada, however, felsic volcanic rocks are virtually all older than 10 million years, and. analogous magmatic heat sources are, therefore, probably lacking. Nevada is part of an area of much higher average heat flow than the rest of the United States. In north-central Nevada, geothermal gradients are as great as 64?C per kilometer in bedrock and even higher in basin fill. The high gradients probably result from a combination of thin crust and high temperature upper mantle. We suggest that the geothermal systems of northern Nevada result from circulation of meteoric waters along Basin and Range faults and that their temperature chiefly depends upon (1) depth of circulation and (2) the geothermal gradient near the faults.

  9. The Northern Manitoba Mining Academy

    NASA Astrophysics Data System (ADS)

    Alexandre, Paul

    2017-04-01

    The Northern Manitoba Mining Academy (NMMA, miningacademy.ca) is a new educational institution located in Flin Flon, Manitoba. It is associated with the University College of the North and is specifically intended to serve the needs of the Northern Manitoban communities with regards to job creation by providing training in a variety of mining, construction, and exploration related areas. NMMA's mission is to provide innovative and responsible solutions for the creation of a knowledgeable, skilled, and sustainable workforce within a vibrant, mineral-rich resource industry. It facilitates strategic training initiatives and research activities in order to strengthen the social, economic, and environmental benefits of a robust mining and resources sector. In terms of education, NMMA offers its own programs, mostly short courses in health and safety, courses organized by the University College of the North (wilderness safety, prospecting, and exploration), and courses organized in association with provincial Industries-Based Safety Programs and Associations (a variety of construction-related trades). However, the programming is not limited to those courses already on the syllabus: the Academy operates on open-doors policy and welcomes people with their unique and diverse needs; it prides itself in its ability to tailor or create specific on-demand courses and deliver them locally in the North. The Northern Manitoba Mining Academy also provides access to its world-class facilities for field-based undergraduate courses, as well as graduate students and researchers doing field work. Full sample preparation facilities are offered to students and scientists in all natural and environmental sciences.

  10. Detection of immunoglobulin M antibodies to glycoprotein G-2 by western blot (immunoblot) for diagnosis of initial herpes simplex virus type 2 genital infections.

    PubMed Central

    Ho, D W; Field, P R; Irving, W L; Packham, D R; Cunningham, A L

    1993-01-01

    Western blots (immunoblots) for the detection of immunoglobulin M (IgM) antibodies specific for herpes simplex virus type 1 (HSV-1) and HSV-2 in patients' sera were developed. The locations of the type-specific glycoprotein G (gpG-2) of HSV-2 (92- and 140-kDa forms) and glycoprotein C of HSV-1 (gpC-1), which carries mostly type-specific antigenic epitopes, were checked with specific monoclonal antibodies. Western blot assays for IgM antibody to gpC-1 or gpG-2 were performed after depletion of IgG by precipitation with anti-human IgG. In patients with primary HSV-2 genital infections, seroconversion of IgM and IgG antibodies to both the 92- and 140-kDa forms of gpG-2 was observed, although both antibodies appeared in convalescent-phase serum after the first week. IgM and IgG antibodies to low-molecular-size polypeptides (40 to 65 kDa) were the first antibodies observed in patients with primary infection, but these antibodies were cross-reactive with HSV-1 and HSV-2. However, in patients with recurrent HSV-2 infections, IgG antibodies to both forms of gpG-2 and the low-molecular-size polypeptides were found no matter how early after onset the patient was bled, and IgM to gpG-2 did not appear. In patients with nonprimary initial genital HSV-2 infections, IgG antibody to HSV-1 was demonstrated in the first serum specimen, and HSV-2-specific IgM was found in 39% of the serum specimens. Hence, the Western blot assay can be used to test for IgM antibody to gpG-2, allowing for the retrospective diagnosis of inital HSV-2 infections and its use as a supplementary test to the gpG-2 IgG enzyme-linked immunosorbent assays developed elsewhere. In contrast, IgM antibody to gpG-2 is not usually detected in patients with recurrent HSV-2 infections. Images PMID:7508453

  11. A theoretical timeline for myocardial infarction: immunohistochemical evaluation and western blot quantification for Interleukin-15 and Monocyte chemotactic protein-1 as very early markers

    PubMed Central

    2014-01-01

    Background Experimental and human studies have demonstrated that innate immune mechanisms and consequent inflammatory reaction play a critical role in cardiac response to ischemic injury. Thus, the detection of immuno-inflammatory and cellular phenomena accompanying cardiac alterations during the early inflammatory phase of myocardial infarction (MI) may be an excellent diagnostic tool. Current knowledge of the chronology of the responses of myocardial tissue following the occurrence of ischemic insult, as well as the existence of numerous studies aiming to identify reliable markers in dating MI, induced us to investigate the myocardial specimens of MI fatal cases in order to better define the age of MI. Methods We performed an immunohistochemical study and a Western blot analysis to evaluate detectable morphological changes in myocardial specimens of fatal MI cases and to quantify the effects of cardiac expression of inflammatory mediators (CD15, IL-1β, IL-6, TNF-α, IL-15, IL-8, MCP-1, ICAM-1, CD18, tryptase) and structural and functional cardiac proteins. Results We observed a biphasic course of MCP-1: it was strongly expressed in the very early phase (0-4 hrs), to diminish in the early period (after 6-8 hrs). Again, our choice of IL-15 is explained by the synergism with neutrophilic granulocytes (CD15) and our study shows the potential for striking cytokine synergy in promoting fast, local neutrophil response in damaged tissues. A progressively stronger immunoreaction for the CD15 antibody was visible in the areas where the margination of circulating inflammatory cells was detectable, up to very strong expression in the oldest ones (>12 hours). Further, the induction of CD15, IL-15, MCP-1 expression levels was quantified by Western blot analysis. The results were as follows: IL-15/β-actin 0.80, CD15/β-actin 0.30, and MCP-1/β-actin 0.60, matching perfectly with the results of immunohistochemistry. Control hearts from traumatic death cases did not show any

  12. The epidemiology of tick-borne haemoparasites as determined by the reverse line blot hybridization assay in an intensively studied cohort of calves in western Kenya.

    PubMed

    Njiiri, Nyawira E; Bronsvoort, B Mark deC; Collins, Nicola E; Steyn, Helena C; Troskie, Milana; Vorster, Ilse; Thumbi, S M; Sibeko, Kgomotso P; Jennings, Amy; van Wyk, Ilana Conradie; Mbole-Kariuki, Mary; Kiara, Henry; Poole, E Jane; Hanotte, Olivier; Coetzer, Koos; Oosthuizen, Marinda C; Woolhouse, Mark; Toye, Philip

    2015-05-30

    The development of sensitive surveillance technologies using PCR-based detection of microbial DNA, such as the reverse line blot assay, can facilitate the gathering of epidemiological information on tick-borne diseases, which continue to hamper the productivity of livestock in many parts of Africa and elsewhere. We have employed a reverse line blot assay to detect the prevalence of tick-borne parasites in an intensively studied cohort of indigenous calves in western Kenya. The calves were recruited close to birth and monitored for the presence of infectious disease for up to 51 weeks. The final visit samples from 453 calves which survived for the study period were analyzed by RLB. The results indicated high prevalences of Theileria mutans (71.6%), T. velifera (62.8%), Anaplasma sp. Omatjenne (42.7%), A. bovis (39.9%), Theileria sp. (sable) (32.7%), T. parva (12.9%) and T. taurotragi (8.5%), with minor occurrences of eight other haemoparasites. The unexpectedly low prevalence of the pathogenic species Ehrlichia ruminantium was confirmed by a species-specific PCR targeting the pCS20 gene region. Coinfection analyses of the seven most prevalent haemoparasites indicated that they were present as coinfections in over 90% of the cases. The analyses revealed significant associations between several of the Theileria parasites, in particular T. velifera with Theileria sp. sable and T. mutans, and T. parva with T. taurotragi. There was very little coinfection of the two most common Anaplasma species, although they were commonly detected as coinfections with the Theileria parasites. The comparison of reverse line blot and serological results for four haemoparasites (T. parva, T. mutans, A. marginale and B. bigemina) indicated that, except for the mostly benign T. mutans, indigenous cattle seem capable of clearing infections of the three other, pathogenic parasites to below detectable levels. Although the study site was located across four agroecological zones, there was

  13. A theoretical timeline for myocardial infarction: immunohistochemical evaluation and western blot quantification for Interleukin-15 and Monocyte chemotactic protein-1 as very early markers.

    PubMed

    Turillazzi, Emanuela; Di Paolo, Marco; Neri, Margherita; Riezzo, Irene; Fineschi, Vittorio

    2014-07-02

    Experimental and human studies have demonstrated that innate immune mechanisms and consequent inflammatory reaction play a critical role in cardiac response to ischemic injury. Thus, the detection of immuno-inflammatory and cellular phenomena accompanying cardiac alterations during the early inflammatory phase of myocardial infarction (MI) may be an excellent diagnostic tool. Current knowledge of the chronology of the responses of myocardial tissue following the occurrence of ischemic insult, as well as the existence of numerous studies aiming to identify reliable markers in dating MI, induced us to investigate the myocardial specimens of MI fatal cases in order to better define the age of MI. We performed an immunohistochemical study and a Western blot analysis to evaluate detectable morphological changes in myocardial specimens of fatal MI cases and to quantify the effects of cardiac expression of inflammatory mediators (CD15, IL-1β, IL-6, TNF-α, IL-15, IL-8, MCP-1, ICAM-1, CD18, tryptase) and structural and functional cardiac proteins. We observed a biphasic course of MCP-1: it was strongly expressed in the very early phase (0-4 hrs), to diminish in the early period (after 6-8 hrs). Again, our choice of IL-15 is explained by the synergism with neutrophilic granulocytes (CD15) and our study shows the potential for striking cytokine synergy in promoting fast, local neutrophil response in damaged tissues. A progressively stronger immunoreaction for the CD15 antibody was visible in the areas where the margination of circulating inflammatory cells was detectable, up to very strong expression in the oldest ones (>12 hours). Further, the induction of CD15, IL-15, MCP-1 expression levels was quantified by Western blot analysis. The results were as follows: IL-15/β-actin 0.80, CD15/β-actin 0.30, and MCP-1/β-actin 0.60, matching perfectly with the results of immunohistochemistry. Control hearts from traumatic death cases did not show any immunoreactivity to the

  14. A simple, inexpensive, robust and sensitive dot-blot assay for equal detection of the nonstructural-1 glycoprotein of all dengue virus serotypes.

    PubMed

    Falconar, Andrew K I; Romero-Vivas, Claudia M E

    2013-04-22

    Detection of dengue virus (DENV) soluble/excreted (s/e) form of the nonstructural-1 (NS1) glycoprotein in patient acute-phase sera is ideal for diagnosis. The commercially-available detection assays are, however, too expensive for routine use and have low specificity, particularly for the s/e NS1 glycoprotein of DENV-2 and DENV-4, which are important causes of lethal human disease worldwide. Mouse monoclonal antibodies (MAbs) were generated and screened against s/e NS1 glycoprotein purified from each DENV serotype to obtain those that reacted equally with each serotype, but not with yellow fever virus (YFV) s/e NS1 glycoprotein or human serum proteins. One MAb, MAb 2C4.6, was further tested against these DENV glycoproteins in human sera using simple, peroxidase-labelled secondary antibody/substrate-developed dot-blot assays. Optimal quenching of endogenous human serum peroxidases was attained using 3% H(2)O(2) in H(2)0 for 5 min. MAb 2C4.6 showed an acceptable detection sensitivity of < 32 ng/ml for the s/e NS1 glycoprotein of each DENV serotype but did not cross-react with the YFV s/e NS1 glycoprotein or human serum proteins. By contrast, the LX1 epitope-specific MAb, 3D1.4, showed similar detection sensitivity against only the DENV-1 NS1 glycoprotein, consistent with results from commercial DENV s/e NS1 glycoprotein detection assays.DENV s/e NS1 glycoproteins were stable in human sera after drying on the nitrocellulose membranes and storage for one month at ambient temperature (28°C) before being processed. The total assay time was reduced to 3 h without any loss of detection sensitivity. This dot-blot format was ideal for the circulating immune complex disruption step, which is required for increased DENV s/e NS1 glycoprotein detection. This is the first study to determine the detection sensitivity of MAbs against known concentrations of s/e NS1 glycoprotein from each DENV serotype. The preparation of patient serum samples for dot-blot assays can be performed

  15. A simple, inexpensive, robust and sensitive dot-blot assay for equal detection of the nonstructural-1 glycoprotein of all dengue virus serotypes

    PubMed Central

    2013-01-01

    Background Detection of dengue virus (DENV) soluble/excreted (s/e) form of the nonstructural-1 (NS1) glycoprotein in patient acute-phase sera is ideal for diagnosis. The commercially-available detection assays are, however, too expensive for routine use and have low specificity, particularly for the s/e NS1 glycoprotein of DENV-2 and DENV-4, which are important causes of lethal human disease worldwide. Methods Mouse monoclonal antibodies (MAbs) were generated and screened against s/e NS1 glycoprotein purified from each DENV serotype to obtain those that reacted equally with each serotype, but not with yellow fever virus (YFV) s/e NS1 glycoprotein or human serum proteins. One MAb, MAb 2C4.6, was further tested against these DENV glycoproteins in human sera using simple, peroxidase-labelled secondary antibody/substrate-developed dot-blot assays. Results Optimal quenching of endogenous human serum peroxidases was attained using 3% H2O2 in H20 for 5 min. MAb 2C4.6 showed an acceptable detection sensitivity of < 32 ng/ml for the s/e NS1 glycoprotein of each DENV serotype but did not cross-react with the YFV s/e NS1 glycoprotein or human serum proteins. By contrast, the LX1 epitope-specific MAb, 3D1.4, showed similar detection sensitivity against only the DENV-1 NS1 glycoprotein, consistent with results from commercial DENV s/e NS1 glycoprotein detection assays. DENV s/e NS1 glycoproteins were stable in human sera after drying on the nitrocellulose membranes and storage for one month at ambient temperature (28°C) before being processed. The total assay time was reduced to 3 h without any loss of detection sensitivity. This dot-blot format was ideal for the circulating immune complex disruption step, which is required for increased DENV s/e NS1 glycoprotein detection. Conclusions This is the first study to determine the detection sensitivity of MAbs against known concentrations of s/e NS1 glycoprotein from each DENV serotype. The preparation of patient serum samples for

  16. Geothermal development plan: northern Arizona

    SciTech Connect

    White, D.H.; Goldstone, L.A.

    1981-01-01

    Much of the northern counties (Apache, Coconino, Gila, Mohave, Navajo and Yavapai) is located in the Colorado Plateau province, a region of low geothermal potential. Two areas that do show some potential are the Flagstaff - San Francisco Peaks area and the Springerville area. Flagstaff is rapidly becoming the manufacturing center of Arizona and will have many opportunities to use geothermal energy to satisfy part of its increasing need for energy. Using a computer simulation model, projections of geothermal energy on line as a function of time are made for both private and city-owned utility development of a resource.

  17. Rattlesnake Bites in Northern California

    PubMed Central

    Butner, Alfred N.

    1983-01-01

    In a series of 59 cases of rattlesnake bites at two major northern California hospitals, no deaths occurred, no amputations or fasciotomies were required and only one patient had tissue necrosis requiring a graft. Because patients are being seen in major medical facilities earlier, envenomation is encountered in earlier stages. Less specific national standards of treatment, therefore, should receive less emphasis than treatment based on the virulence of the snakes in the particular geographic region. Initial doses of antivenin given intravenously should be based on the degree of envenomation, with additional titration done for worsening symptoms. PMID:6636730

  18. Studies of ancient crania from northern Africa.

    PubMed

    Keita, S O

    1990-09-01

    Historical sources and archaeological data predict significant population variability in mid-Holocene northern Africa. Multivariate analyses of crania demonstrate wide variation but also suggest an indigenous craniometric pattern common to both late dynastic northern Egypt and the coastal Maghreb region. Both tropical African and European metric phenotypes, as well intermediate patterns, are found in mid-Holocene Maghreb sites. Early southern predynastic Egyptian crania show tropical African affinities, displaying craniometric trends that differ notably from the coastal northern African pattern. The various craniofacial patterns discernible in northern Africa are attributable to the agents of microevolution and migration.

  19. Application of glyco-blotting for identification of structures of polysaccharides causing membrane fouling in a pilot-scale membrane bioreactor treating municipal wastewater.

    PubMed

    Kimura, Katsuki; Nishimura, Shin-Ichiro; Miyoshi, Risho; Hoque, Asiful; Miyoshi, Taro; Watanabe, Yoshimasa

    2015-03-01

    A new approach for the analysis of polysaccharides in membrane bioreactor (MBR) is proposed in this study. Enrichment of polysaccharides by glyco-blotting, in which polysaccharides are specifically collected via interactions between the aldehydes in the polysaccharides and aminooxy groups on glycoblotting beads, enabled MALDI-TOF/MS analysis at a high resolution. Structures of polysaccharides extracted from fouled membranes used in a pilot-scale MBR treating municipal wastewater and those in the supernatant of the mixed liquor suspension in the MBR were investigated. It was found that the overlap between polysaccharides found in the supernatants and those extracted from the fouled membrane was rather limited, suggesting that polysaccharides that dominate in supernatants may not be important in membrane fouling in MBRs. Analysis using a bacterial carbohydrate database suggested that capsular polysaccharides (CPS) and/or lipo-polysaccharides (LPS) produced by gram-negative bacteria are key players in the evolution of membrane fouling in MBRs.

  20. [Analysis of antigens recognized by anti-gamma-seminoprotein antibody and anti-prostate-specific antigen antibody by means of Ouchterlony method and western blotting method].

    PubMed

    Deguchi, T; Matsui, H; Ehara, H; Kobayashi, K; Saito, I; Shinoda, I; Takahashi, Y; Kuriyama, M; Ban, Y; Kawada, Y

    1989-03-01

    Antigens recognized by anti-gamma-seminoprotein (gamma-Sm) antibody and anti-prostate-specific antigen (PA) antibody were analyzed immunologically by Ouchterlony method and Western blotting method. The double immunodiffusion test demonstrated that anti-gamma-Sm antibody and anti-PA antibody reacted against an identical antigen in seminal fluid or prostate homogenate. In the immunoblotting examination, anti-gamma-Sm antibody and anti-PA antibody formed a single and identical band, which gave a molecular weight of 33,000, in seminal fluid and prostate homogenate. This study demonstrated that anti-gamma-Sm antibody and anti-PA antibody recognized a single and identical antigen in seminal fluid and prostate homogenate and suggested that an identical material could be measured in sera of patients with prostate cancer by immunoassay systems using anti-gamma-Sm antibody and anti-PA antibody.

  1. Identification of Helicobacter pylori by immunological dot blot method based on reaction of a species-specific monoclonal antibody with a surface-exposed protein.

    PubMed Central

    Bölin, I; Lönroth, H; Svennerholm, A M

    1995-01-01

    Monoclonal antibodies (MAbs) against membrane preparations of Helicobacter pylori were produced. One MAb was found to be specific for H. pylori, because it did not react with a number of other bacterial species, including Helicobacter felis and Campylobacter jejuni. This MAb reacted with a 30-kDa protein found in outer membrane preparations of H. pylori. The protein was also detected on the cell surface on intact bacteria when analyzed by immunoelectron microscopy. To facilitate the identification of H. pylori isolates after culturing of biopsies, an immunodot blot assay based on the reaction of this MAb was developed. This assay was found to be highly specific for H. pylori. Sixty-six clinical isolates typed as H. pylori by conventional biochemical tests were found to be positive, whereas no other bacterial species tested gave a positive result. By this method, reliable and rapid identification of H. pylori could be accomplished. PMID:7714196

  2. Serum detection of IgG antibodies against Demodex canis by western blot in healthy dogs and dogs with juvenile generalized demodicosis.

    PubMed

    Ravera, Ivan; Ferreira, Diana; Gallego, Laia Solano; Bardagí, Mar; Ferrer, Lluís

    2015-08-01

    The aim of this study was to investigate the presence of canine immunoglobulins (Ig) G against Demodex proteins in the sera of healthy dogs and of dogs with juvenile generalized demodicosis (CanJGD) with or without secondary pyoderma. Demodex mites were collected from dogs with CanJGD. Protein concentration was measured and a western blot technique was performed. Pooled sera from healthy dogs reacted mainly with antigen bands ranging from 55 to 72 kDa. Pooled sera from dogs with CanJGD without secondary pyoderma reacted either with 10 kDa antigen band or 55 to 72 kDa bands. Pooled sera from dogs with CanJGD with secondary pyoderma reacted only with a 10 kDa antigen band. The results of this study suggest that both healthy dogs and dogs with CanJGD develop a humoral response against different proteins of Demodex canis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Investigation of cross-reactions against Trichinella spiralis antigens by enzyme-linked immunosorbent assay and enzyme-linked immunoelectrotransfer blot assay in patients with various diseases.

    PubMed Central

    De-la-Rosa, J L; Alcantara, P; Correa, D

    1995-01-01

    Data regarding cross-reactions against Trichinella spiralis in humans are scarce and controversial. For this reason, we tested serum samples from patients with typhoid fever, brucellosis, toxoplasmosis, amoebiasis, cysticercosis, trichocephaliasis, ascariasis, and onchocerciasis against an antigenic extract of T. spiralis infective larvae in an enzyme-linked immunosorbent assay (ELISA) and an enzyme-linked immunoelectrotransfer blot (EITB) assay. All except one serum sample from the group of patients with onchocerciasis were negative in the ELISA; in the EITB assay, only faint bands were observed with the samples from patients with onchocerciasis and ascariasis and negative results were obtained with the samples from patients with other diseases. In conclusion, cross-reactions were found only in the groups of patients with other nematode infections and were of very low magnitude, most of them virtually negative. PMID:7719905

  4. Detection and chromosomal assignment of SV40-DNA integration in Chinese hamster cell lines by chromosome sorting and dot blot hybridization.

    PubMed

    Hutter, K J; Klefenz, H; Goerttler, K

    1990-01-01

    A combination of cytometric (chromosome sorting), molecular (dot blot hybridization using radio-active and/or biotinylated DNA probes) and cytogenetic (G-banding) evaluation is described which allows the rapid identification of single copy and repetitive viral integrates and their assignment to chromosome groups or even individual chromosomes. In the case of Chinese hamster cell line CO 631 it could be demonstrated that SV40 DNA was solely integrated into a submetacentric marker chromosome. Such a cytometric/molecular/cytogenetic "identogram" may prove to be a useful tool in many areas of cell and tumor biology. Furthermore, amounts of chromosomes sufficient for analysis as well as subsequent cloning experiments can be accumulated.

  5. Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts.

    PubMed

    Arnold, Stefan A; Albiez, Stefan; Bieri, Andrej; Syntychaki, Anastasia; Adaixo, Ricardo; McLeod, Robert A; Goldie, Kenneth N; Stahlberg, Henning; Braun, Thomas

    2017-03-01

    We present a sample preparation method for cryo-electron microscopy (cryo-EM) that requires only 3-20nL of sample to prepare a cryo-EM grid, depending on the protocol used. The sample is applied and spread on the grid by a microcapillary. The procedure does not involve any blotting steps, and real-time monitoring allows the water film thickness to be assessed and decreased to an optimum value prior to vitrification. We demonstrate that the method is suitable for high-resolution cryo-EM and will enable alternative electron microscopy approaches, such as single-cell visual proteomics. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. Detection of Ca(2+)-binding proteins by electrophoretic migration in the presence of Ca2+ combined with 45Ca2+ overlay of protein blots

    SciTech Connect

    Garrigos, M.; Deschamps, S.; Viel, A.; Lund, S.; Champeil, P.; Moller, J.V.; le Maire, M. , Gif-sur-Yvette )

    1991-04-01

    When high affinity Ca(2+)-binding proteins like calmodulin, or proteins with a high Ca(2+)-binding capacity like calsequestrin, underwent sodium dodecyl sulfate-gel electrophoresis in Laemmli systems, their electrophoretic migration rates were much higher in gels containing 1 mM Ca2+ than in gels containing ethylene glycol bis(beta-aminoethyl ether) N,N{prime}-tetraacetic acid (EGTA). Replacement of EGTA by Ca2+ in the gel, combined with the blotting of electrophoretically separated proteins on polyvinylidene difluoride membranes and subsequent 45Ca2+ overlay, proved a very effective means of detecting Ca(2+)-binding proteins. This combined approach is important since artifacts occur in both techniques when used separately. We found that the usual procedure of adding Ca2+ to the sample before electrophoresis without including it in the gel itself permitted the detection of only very high affinity Ca(2+)-binding proteins.

  7. Detection of Cryptosporidium parvum oocysts by dot-blotting using monoclonal antibodies to Cryptosporidium parvum virus 40-kDa capsid protein.

    PubMed

    Jenkins, Mark C; O'Brien, Celia N; Trout, James M

    2008-02-01

    Monoclonal antibodies (MAb) were prepared against the 40-kDa capsid protein of Cryptosporidium parvum virus (CPV) by immunizing mice with purified recombinant CPV40 protein. In immunoblotting analysis, MAbCPV40-1 bound to a 40-kDa protein in extracts of C. parvum oocysts. This 40-kDa protein was localized in the sporozoite cytoplasm by immunofluorescence (IFA) staining with MAbCPV40-1. In a dot-blot assay, MAbCPV40-1 was capable of detecting 10(2) non-bleach-treated and 10(2)-10(3) bleach-treated C. parvum oocysts. MAbCPV40-1 was capable of detecting CPV40 antigen in both soluble and total C. parvum oocyst protein extracts, indicating a potential use for detecting this parasite in environmental samples.

  8. Twenty-two year follow-up of an Indian family with dysferlinopathy-clinical, immunocytochemical, western blotting and genetic features.

    PubMed

    Khadilkar, Satish V; Singh, Rakesh K; Agarwal, Pankaj; Krahn, Martin; Levy, Nicolas

    2008-01-01

    Long-term observations over a period of 22 years in an Indian family with primary dysferlinopathy are recorded, defining phenotypic variability. In the propositus, the dystrophy began distally in the tibialis anterior muscles, before involving the gastrocnemius. Transient painful calf hypertrophy, followed by calf wasting was observed. The proximal lower and upper limbs weakened after three to four years. The younger sibling presented with the proximo-distal phenotype. Both patients showed very high creatine kinase values early into the illness. Disease progression was slow. The younger sibling lost ambulation 14 years after onset, while the elder one remains ambulatory 22 years into the illness. Muscle biopsy showed dystrophic features and absence of dysferlin. Monocyte western blotting confirmed absence of dysferlin. Genetic analysis detected a heterozygous mutation in Exon 54 [c.6124C>T] in the DYSF gene. This is the first family with a diagnosis of dysferlinopathy supported by genetic data, reported from India.

  9. A new and frequent human T-cell leukemia virus indeterminate Western blot pattern: epidemiological determinants and PCR results in central African inhabitants.

    PubMed

    Filippone, Claudia; Bassot, Sylviane; Betsem, Edouard; Tortevoye, Patricia; Guillotte, Micheline; Mercereau-Puijalon, Odile; Plancoulaine, Sabine; Calattini, Sara; Gessain, Antoine

    2012-05-01

    Human T-cell leukemia virus (HTLV) indeterminate Western blot (WB) serological patterns are frequently observed in plasma/serum from persons living in intertropical areas. In the framework of ongoing projects on HTLV-1/2 and related viruses in Central Africa, we systematically analyzed plasma from villagers living in South Cameroon by WB. The group included 1,968 individuals (mean age, 44 years; age range, 5 to 90 years; 978 women/990 men), both Bantus (1,165) and Pygmies (803). Plasma samples were tested by WB analysis (MPD HTLV Blot 2.4) and interpreted according to the manufacturer's instructions. Only clear bands were considered in the analysis. Among the 1,968 plasma samples, 38 (1.93%) were HTLV-1, 13 (0.66%) were HTLV-2, and 6 (0.3%) were HTLV WB seropositive. Furthermore, 1,292 (65.65%) samples were WB sero-indeterminate, including 104 (5.28%) with an HTLV-1 Gag-indeterminate pattern (HGIP) and 68 (3.45%) with a peculiar yet unreported pattern exhibiting mostly a strong shifted GD21 and a p28. The other 619 (31.45%) samples were either WB negative or exhibited other patterns, mostly with unique p19 or p24 bands. DNA, extracted from peripheral blood buffy coat, was subjected to PCR using several primer pairs known to detect HTLV-1/2/3/4. Most DNAs from HTLV-1- and HTLV-seropositive individuals were PCR positive. In contrast, all the others, from persons with HTLV-2, HGIP, new WB, and other indeterminate patterns, were PCR negative. Epidemiological determinant analysis of the persons with this new peculiar WB pattern revealed that seroprevalence was independent from age, sex, or ethnicity, thus resembling the indeterminate profile HGIP rather than HTLV-1. Moreover, this new pattern persists over time.

  10. Development and validation of a novel protein extraction methodology for quantitation of protein expression in formalin-fixed paraffin-embedded tissues using western blotting.

    PubMed

    Nirmalan, Niroshini J; Harnden, Patricia; Selby, Peter J; Banks, Rosamonde E

    2009-03-01

    The development of efficient formaldehyde cross-link reversal strategies will make the vast diagnostic tissue archives of pathology departments amenable to prospective and retrospective translational research, particularly in biomarker-driven proteomic investigations. Heat-induced antigen retrieval strategies (HIARs) have achieved varying degrees of cross-link reversal, potentially enabling archival tissue usage for proteomic applications outside its current remit of immunohistochemistry (IHC). While most successes achieved so far have been based on retrieving tryptic peptide fragments using shot-gun proteomic approaches, attempts at extracting full-length, non-degraded, immunoreactive proteins from archival tissue have proved challenging. We have developed a novel heat-induced antigen retrieval strategy using SDS-containing Laemmli buffer for efficient intact protein recovery from formalin-fixed tissues for subsequent analysis by western blotting. Protocol optimization and comparison of extraction efficacies with frozen tissues and current leader methodology is presented. Quantitative validation of methodology was carried out in a cohort of matched tumour/normal, frozen/FFPE renal tissue samples from 10 patients, probed by western blotting for a selected panel of seven proteins known to be differentially expressed in renal cancer. Our data show that the protocol enables efficient extraction of non-degraded, full-length, immunoreactive protein, with tumour versus normal differential expression profiles for a majority of the panel of proteins tested being comparable to matched frozen tissue controls (rank correlation, r = 0.7292, p < 1.825e-09). However, the variability observed in extraction efficacies for some membrane proteins emphasizes the need for cautious interpretation of quantitative data from this subset of proteins. The method provides a viable, cost-effective quantitative option for the validation of potential biomarker panels through a range of clinical

  11. Use of rapid HIV assays as supplemental tests in specimens with repeatedly reactive screening immunoassay results not confirmed by HIV-1 Western blot.

    PubMed

    Wesolowski, Laura G; Delaney, Kevin P; Meyer, William A; Blatt, Amy J; Bennett, Berry; Chavez, Pollyanna; Granade, Timothy C; Owen, Michele

    2013-09-01

    An alternate HIV testing algorithm has been proposed which includes a fourth-generation immunoassay followed by an HIV-1/HIV-2 antibody differentiation supplemental test for reactive specimens and a nucleic acid test (NAT) for specimens with discordant results. To evaluate the performance of five rapid tests (Alere Clearview, Bio-Rad Multispot, OraSure OraQuick, MedMira Reveal, and Trinity Biotech Unigold) as the supplemental antibody assay in the algorithm. A total of 3273 serum and plasma specimens that were third-generation immunoassay repeatedly reactive and Western blot (WB) negative or indeterminate were tested with rapid tests and NAT. Specimens were classified by NAT: (1) HIV-1 infected (NAT-reactive; n=184, 5.6%), (2) HIV-status unknown (NAT nonreactive; n=3078, 94.2%) or by Multispot, (3) HIV-2 positive (n=5), and (4) HIV-1 and HIV-2 positive (n=6). Excluding HIV-2 positive specimens, we calculated the proportion of reactive rapid tests among specimens with reactive and nonreactive NAT. The proportion of infected specimens with reactive rapid test results and negative or indeterminate WB ranged from 30.4% (56) to 47.8% (88) depending on the rapid test. From 1% to 2% of NAT-negative specimens had reactive rapid test results. In these diagnostically challenging specimens, all rapid tests identified infections that were missed by the Western blot, but only Multispot could differentiate HIV-1 from HIV-2. Regardless of which rapid test is used as a supplemental test in the alternative algorithm, false-positive algorithm results (i.e., reactive screening and rapid test in uninfected person) may occur, which will need to be resolved during the baseline medical evaluation. Published by Elsevier B.V.

  12. A Secondary Antibody-Detecting Molecular Weight Marker with Mouse and Rabbit IgG Fc Linear Epitopes for Western Blot Analysis.

    PubMed

    Lin, Wen-Wei; Chen, I-Ju; Cheng, Ta-Chun; Tung, Yi-Ching; Chu, Pei-Yu; Chuang, Chih-Hung; Hsieh, Yuan-Chin; Huang, Chien-Chiao; Wang, Yeng-Tseng; Kao, Chien-Han; Roffler, Steve R; Cheng, Tian-Lu

    2016-01-01

    Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. Here, we describe M&R LE protein markers which contain linear epitopes derived from the heavy chain constant regions of mouse and rabbit immunoglobulin G (IgG Fc LE). These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. We selected three mouse (M1, M2 and M3) linear IgG1 and three rabbit (R1, R2 and R3) linear IgG heavy chain epitope candidates based on their respective crystal structures. Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. We fused the M2 and R2 epitopes (M&R LE) and incorporated the polypeptide in a range of 15-120 kDa auto-detecting markers (M&R LE protein marker). The M&R LE protein marker can be auto-detected by anti-mouse and anti-rabbit IgG secondary antibodies in standard immunoblots. Linear regression analysis of the M&R LE protein marker plotted as gel mobility versus the log of the marker molecular weights revealed good linearity with a correlation coefficient R2 value of 0.9965, indicating that the M&R LE protein marker displays high accuracy for determining protein molecular weights. This accurate, regular and auto-detected M&R LE protein marker may provide a simple, efficient and economical tool for protein analysis.

  13. GAPDH and β-actin protein decreases with aging, making Stain-Free technology a superior loading control in Western blotting of human skeletal muscle.

    PubMed

    Vigelsø, Andreas; Dybboe, Rie; Hansen, Christina Neigaard; Dela, Flemming; Helge, Jørn W; Guadalupe Grau, Amelia

    2015-02-01

    Reference proteins (RP) or the total protein (TP) loaded is used to correct for uneven loading and/or transfer in Western blotting. However, the signal sensitivity and the influence of physiological conditions may question the normalization methods. Therefore, three widely used reference proteins [β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and α-tubulin], as well as TP loaded measured by Stain-Free technology (SF) as normalization tool were tested. This was done using skeletal muscle samples from men subjected to physiological conditions often investigated in applied physiology where the intervention has been suggested to impede normalization (ageing, muscle atrophy, and different muscle fiber type composition). The linearity of signal and the methodological variation coefficient was obtained. Furthermore, the inter- and intraindividual variation in signals obtained from SF and RP was measured in relation to ageing, muscle atrophy, and different muscle fiber type composition, respectively. A stronger linearity of SF and β-actin compared with GAPDH and α-tubulin was observed. The methodological variation was relatively low in all four methods (4-11%). Protein level of β-actin and GAPDH was lower in older men compared with young men. In conclusion, β-actin, GAPDH, and α-tubulin may not be used for normalization in studies that include subjects with a large age difference. In contrast, the RPs may not be affected in studies that include muscle wasting and differences in muscle fiber type. The novel SF technology adds lower variation to the results compared with the existing methods for correcting for loading inaccuracy in Western blotting of human skeletal muscle in applied physiology.

  14. Refinement of the high-resolution physical and genetic map of Rhodobacter capsulatus and genome surveys using blots of the cosmid encyclopedia.

    PubMed Central

    Fonstein, M; Koshy, E G; Nikolskaya, T; Mourachov, P; Haselkorn, R

    1995-01-01

    Cosmids from a library containing Rhodobacter capsulatus DNA fragments were previously ordered in two contigs: one corresponding to the chromosome and one to a 134 kb plasmid. This map contained 40 regions connected only by colony hybridization. To confirm the linkage and correct the map, the actual sizes of the overlaps were determined by blot-hybridization with Rhodobacter chromosomal DNA and by mapping of additional cosmids. Several revisions of the earlier map include single cosmid shifts and inversions. One additional gap in a cosmid contig was also found, raising the possibility that the chromosome is not a contiguous circle. About 2500 additional EcoRI,BamHI and HindIII restriction sites were added to the 560 EcoRV sites previously mapped onto the Rhodobacter chromosome, increasing the resolution of the physical map to the size of individual genes. Twenty-five new markers were located on the genetic map. The 48 markers now mapped represent nearly 300 genes and ORFs cloned from different species of Rhodobacter. The orientation of transcription of the four rrn operons was established using 16S rRNA- and 23S rRNA-specific probes and digestion with the rare-cutting enzyme, CeuI. Gel blots of 192 cosmids of the miniset of R.capsulatus digested with EcoRV were prepared. Such a hybridization template represents the whole genome cut into 560 DNA fragments varying in size from 0.4 to 25 kb. This template was used for high-resolution mapping of single genes, analysis of total genomic DNAs from related Rhodobacter strains and differentially expressed RNAs. Images PMID:7737133

  15. A Secondary Antibody-Detecting Molecular Weight Marker with Mouse and Rabbit IgG Fc Linear Epitopes for Western Blot Analysis

    PubMed Central

    Cheng, Ta-Chun; Tung, Yi-Ching; Chu, Pei-Yu; Chuang, Chih-Hung; Hsieh, Yuan-Chin; Huang, Chien-Chiao; Wang, Yeng-Tseng; Kao, Chien-Han; Roffler, Steve R.; Cheng, Tian-Lu

    2016-01-01

    Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. Here, we describe M&R LE protein markers which contain linear epitopes derived from the heavy chain constant regions of mouse and rabbit immunoglobulin G (IgG Fc LE). These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. We selected three mouse (M1, M2 and M3) linear IgG1 and three rabbit (R1, R2 and R3) linear IgG heavy chain epitope candidates based on their respective crystal structures. Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. We fused the M2 and R2 epitopes (M&R LE) and incorporated the polypeptide in a range of 15–120 kDa auto-detecting markers (M&R LE protein marker). The M&R LE protein marker can be auto-detected by anti-mouse and anti-rabbit IgG secondary antibodies in standard immunoblots. Linear regression analysis of the M&R LE protein marker plotted as gel mobility versus the log of the marker molecular weights revealed good linearity with a correlation coefficient R2 value of 0.9965, indicating that the M&R LE protein marker displays high accuracy for determining protein molecular weights. This accurate, regular and auto-detected M&R LE protein marker may provide a simple, efficient and economical tool for protein analysis. PMID:27494183

  16. Localization and Distribution of 'Candidatus Liberibacter asiaticus’ in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody

    PubMed Central

    Ding, Fang; Duan, Yongping; Paul, Cristina; Brlansky, Ronald H.; Hartung, John S.

    2015-01-01

    ‘Candidatus Liberibacter asiaticus’ (CaLas), a non-cultured member of the α-proteobacteria, is the causal agent of citrus Huanglongbing (HLB). Due to the difficulties of in vitro culture, antibodies against CaLas have not been widely used in studies of this pathogen. We have used an anti-OmpA polyclonal antibody based direct tissue blot immunoassay to localize CaLas in different citrus tissues and in periwinkle leaves. In citrus petioles, CaLas was unevenly distributed in the phloem sieve tubes, and tended to colonize in phloem sieve tubes on the underside of petioles in preference to the upper side of petioles. Both the leaf abscission zone and the junction of the petiole and leaf midrib had fewer CaLas bacteria compared to the main portions of the petiole and the midribs. Colonies of CaLas in phloem sieve tubes were more frequently found in stems with symptomatic leaves than in stems with asymptomatic leaves with an uneven distribution pattern. In serial sections taken from the receptacle to the peduncle, more CaLas were observed in the peduncle sections adjacent to the stem. In seed, CaLas was located in the seed coat. Many fewer CaLas were found in the roots, as compared to the seeds and petioles when samples were collected from trees with obvious foliar symptoms. The direct tissue blot immuno assay was adapted to whole periwinkle leaves infected by CaLas. The pathogen was distributed throughout the lateral veins and the results were correlated with results of qPCR. Our data provide direct spatial and anatomical information for CaLas in planta. This simple and scalable method may facilitate the future research on the interaction of CaLas and host plant. PMID:25946013

  17. Localization and Distribution of 'Candidatus Liberibacter asiaticus' in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody.

    PubMed

    Ding, Fang; Duan, Yongping; Paul, Cristina; Brlansky, Ronald H; Hartung, John S

    2015-01-01

    'Candidatus Liberibacter asiaticus' (CaLas), a non-cultured member of the α-proteobacteria, is the causal agent of citrus Huanglongbing (HLB). Due to the difficulties of in vitro culture, antibodies against CaLas have not been widely used in studies of this pathogen. We have used an anti-OmpA polyclonal antibody based direct tissue blot immunoassay to localize CaLas in different citrus tissues and in periwinkle leaves. In citrus petioles, CaLas was unevenly distributed in the phloem sieve tubes, and tended to colonize in phloem sieve tubes on the underside of petioles in preference to the upper side of petioles. Both the leaf abscission zone and the junction of the petiole and leaf midrib had fewer CaLas bacteria compared to the main portions of the petiole and the midribs. Colonies of CaLas in phloem sieve tubes were more frequently found in stems with symptomatic leaves than in stems with asymptomatic leaves with an uneven distribution pattern. In serial sections taken from the receptacle to the peduncle, more CaLas were observed in the peduncle sections adjacent to the stem. In seed, CaLas was located in the seed coat. Many fewer CaLas were found in the roots, as compared to the seeds and petioles when samples were collected from trees with obvious foliar symptoms. The direct tissue blot immuno assay was adapted to whole periwinkle leaves infected by CaLas. The pathogen was distributed throughout the lateral veins and the results were correlated with results of qPCR. Our data provide direct spatial and anatomical information for CaLas in planta. This simple and scalable method may facilitate the future research on the interaction of CaLas and host plant.

  18. Structural features offshore northern Taiwan

    NASA Astrophysics Data System (ADS)

    Yicheng Yang, Eason; Liu, Char-Shine; Chang, Jih-Hsin; Chiu, Chien-Hsuan

    2016-04-01

    The area offshore northern Taiwan is the place where East China Sea Shelf extends into the Southern Okinawa Trough, and where pre-Pleistocene arc-continental collision had occurred. Comparison between fault distribution in the area with previously published results suggests that the fault distribution and regional structural framework are still controversial. Using marine multichannel seismic reflection data collected in 3 marine geophysical survey cruises, we remapped the fault distribution in the northern offshore area of Taiwan. By analyzing all the seismic profiles using the KINGDOM suite (a seismic interpretation software), a new fault distribution map is presented, and a subsurface unconformity PRSB (Pliocene reflection sequence boundary) is identified. Six major NE-SW trending high-angle normal faults cut the PRSB can be traced to the fault systems on land northernmost Taiwan. These normal faults are located between the Southern Okinawa Trough and the East China Sea continental shelf basin, and have been suggested to be reactivated from pre-existing reverse faults. The offsets of fault ramps in PRSB increase toward southeast. The isopach map of the study area compiled shows that sediment strata overlying PRSB thin toward northwest.

  19. Groundwater management in northern Iraq

    NASA Astrophysics Data System (ADS)

    Stevanovic, Zoran; Iurkiewicz, Adrian

    2009-03-01

    Groundwater is vital and the sole resource in most of the studied region of northern Iraq. It has a significant role in agriculture, water supply and health, and the elimination of poverty in rural areas. Although Iraq is currently dramatically disturbed by complex political and socio-economic problems, in its northern part, i.e. the Kurdish-inhabited region, fast urbanization and economic expansion are visible everywhere. Monitoring and water management schemes are necessary to prevent aquifer over-exploitation in the region. Artificial recharge with temporary runoff water, construction of subsurface dams and several other aquifer management and regulation measures have been designed, and some implemented, in order to improve the water situation. Recommendations, presented to the local professionals and decision-makers in water management, include creation of Water Master Plans and Water User Associations, synchronization of drilling programmes, rehabilitation of the existing well fields, opening of new well fields, and the incorporation of new spring intakes in some areas with large groundwater reserves, as well as construction of numerous small-scale schemes for initial in situ water treatment where saline groundwater is present.

  20. Climate modelling: Northern Hemisphere circulation.

    PubMed

    Gillett, Nathan P

    2005-09-22

    Air pressure at sea level during winter has decreased over the Arctic and increased in the Northern Hemisphere subtropics in recent decades, a change that has been associated with 50% of the Eurasian winter warming observed over the past 30 years, with 60% of the rainfall increase in Scotland and with 60% of the rainfall decrease in Spain. This trend is inconsistent with the simulated response to greenhouse-gas and sulphate-aerosol changes, but it has been proposed that other climate influences--such as ozone depletion--could account for the discrepancy. Here I compare observed Northern Hemisphere sea-level pressure trends with those simulated in response to all the major human and natural climate influences in nine state-of-the-art coupled climate models over the past 50 years. I find that these models all underestimate the circulation trend. This inconsistency suggests that we cannot yet simulate changes in this important property of the climate system or accurately predict regional climate changes.

  1. Illuminating Northern California's Active Faults

    NASA Astrophysics Data System (ADS)

    Prentice, Carol S.; Crosby, Christopher J.; Whitehill, Caroline S.; Arrowsmith, J. Ramón; Furlong, Kevin P.; Phillips, David A.

    2009-02-01

    Newly acquired light detection and ranging (lidar) topographic data provide a powerful community resource for the study of landforms associated with the plate boundary faults of northern California (Figure 1). In the spring of 2007, GeoEarthScope, a component of the EarthScope Facility construction project funded by the U.S. National Science Foundation, acquired approximately 2000 square kilometers of airborne lidar topographic data along major active fault zones of northern California. These data are now freely available in point cloud (x, y, z coordinate data for every laser return), digital elevation model (DEM), and KMZ (zipped Keyhole Markup Language, for use in Google Earth™ and other similar software) formats through the GEON OpenTopography Portal (http://www.OpenTopography.org/data). Importantly, vegetation can be digitally removed from lidar data, producing high-resolution images (0.5- or 1.0-meter DEMs) of the ground surface beneath forested regions that reveal landforms typically obscured by vegetation canopy (Figure 2).

  2. Comparison of northern goshawk nesting habitat in Appalachian oak and northern hardwood forests of Pennsylvania

    Treesearch

    J.T. Kimmel; R.H. Yahner

    1991-01-01

    The Northern Goshawk (Accipiter gentilis) is a rare to uncommon woodland raptor in Pennsylvania. Although it is primarily a boreal species, the goshawk nests in Northern Hardwoods and Appalachian oak forests along the southern margin of its range in Pennsylvania. This study compared the nesting habitat of goshawks in Appalachian oak and Northern...

  3. Bullying in Schools: A Northern Ireland Study

    ERIC Educational Resources Information Center

    Collins, Katrina; McAleavy, Gerry; Adamson, Gary

    2004-01-01

    Northern Ireland, unlike the Republic of Ireland or England, has no province-wide information on bullying in schools. This study provided baseline information on this complex issue across 120 schools in all five Education and Library Boards in Northern Ireland, comprising 60 primary and 60 post-primary schools, 1079 primary pupils (Year 6) and…

  4. Meeting Northern Arizona's Supported Employment Training Needs.

    ERIC Educational Resources Information Center

    Martin, William E., Jr.; And Others

    In 1989 Northern Arizona University established a Supported Employment Training Center (SETC) to increase the number of trained job coaches in northern Arizona and provide knowledge and skills in supported employment to personnel from cooperating schools and agencies. First-year SETC activities focused on assessment of the training needs of…

  5. Harvesting systems for the northern forest hardwoods

    Treesearch

    Chris B. LeDoux

    2011-01-01

    This monograph is a summary of research results and environmental compliance measures for timber harvesting operations. Data are presented from the Northern Research Station's forest inventory and analysis of 20 states in the northern forest hardwoods. Harvesting systems available in the region today are summarized. Equations for estimating harvesting costs are...

  6. Logging truck noise near nesting northern goshawks

    Treesearch

    Teryl G. Grubb; Larry L. Pater; David K. Delaney

    1998-01-01

    We measured noise levels of four logging trucks as the trucks passed within approximately 500 m of two active northern goshawk (Accipiter gentilis) nests on the Kaibab Plateau in northern Arizona in 1997. Neither a brooding adult female nor a lone juvenile exhibited any discernable behavioral response to logging truck noise, which peaked at 53.4 and...

  7. Environmental overview of geothermal development: northern Nevada

    SciTech Connect

    Slemmons, D.B.; Stroh, J.M.; Whitney, R.A.

    1980-08-01

    Regional environmental problems and issues associated with geothermal development in northern Nevada are studied to facilitate environmental assessment of potential geothermal resources. The various issues discussed are: environmental geology, seismicity of northern Nevada, hydrology and water quality, air quality, Nevada ecosystems, noise effects, socio-economic impacts, and cultural resources and archeological values. (MHR)

  8. The Alcoholism Situation in a Northern City

    ERIC Educational Resources Information Center

    Martynov, M. Iu.; Martynova, D. Iu.

    2012-01-01

    Alcohol abuse in Russia has been increasing in recent years, especially in northern regions, as has the incidence of alcohol-related disease rates. A survey was conducted in Surgut (the Khanty-Mansi autonomous okrug) that determined the factors lending to the prevalence of alcohol abuse among the population of the northern city and assessed the…

  9. Northern Desegregation: A Tale of Two Cities

    ERIC Educational Resources Information Center

    Danns, Dionne

    2011-01-01

    Studies on northern desegregation have focused on political strategies, the role of the courts, the responsibility of the federal government (HEW), and barriers to northern desegregation. Some have conducted individual case studies and comparative studies, and others have examined a number of cities. This article examines the way school…

  10. Woodcock nesting habitat in northern Wisconsin

    USGS Publications Warehouse

    Gregg, L.E.; Hale, J.B.

    1977-01-01

    Of 32 woodcock nests studied in northern Wisconsin, 29 were in forest stands dominated by aspen, and 3 were in northern hardwoods. Well-drained, upland nest sites near the brushy edges of poorly stocked poletimber stands were apparently preferred. More than 30 woody plant species were found at the 32 nest sites. Hazel was the most important shrub species noted.

  11. Bullying in Schools: A Northern Ireland Study

    ERIC Educational Resources Information Center

    Collins, Katrina; McAleavy, Gerry; Adamson, Gary

    2004-01-01

    Northern Ireland, unlike the Republic of Ireland or England, has no province-wide information on bullying in schools. This study provided baseline information on this complex issue across 120 schools in all five Education and Library Boards in Northern Ireland, comprising 60 primary and 60 post-primary schools, 1079 primary pupils (Year 6) and…

  12. Success of Underplanting Northern Red Oaks

    Treesearch

    Martin A. Spetich; Daniel C. Dey; Paul S. Johnson; David L. Graney

    2004-01-01

    We summarize results of the growth and survival of northern red oak (Quercus rubra L.) seedlings 11 years after planting in shelterwoods in the Boston Mountains of Arkansas. Shelterwood overstories were harvested 3 years after underplanting > 4,000 northern red oak seedlings. Woody vegetation that was competing with planted seedlings received two...

  13. The Alcoholism Situation in a Northern City

    ERIC Educational Resources Information Center

    Martynov, M. Iu.; Martynova, D. Iu.

    2012-01-01

    Alcohol abuse in Russia has been increasing in recent years, especially in northern regions, as has the incidence of alcohol-related disease rates. A survey was conducted in Surgut (the Khanty-Mansi autonomous okrug) that determined the factors lending to the prevalence of alcohol abuse among the population of the northern city and assessed the…

  14. Northern Parkway PTA Makes Health a Habit

    ERIC Educational Resources Information Center

    Ferdinand, Marilyn

    2011-01-01

    Health and fitness have been on the agenda of Northern Parkway Elementary School for quite some time, thanks to the concerted efforts of its involved and active PTA officers and members. For the past five years, the Northern Parkway PTA has held a popular and well-attended Family Fun and Fitness Night and has complemented the activities and…

  15. Increasing Northern Hemisphere water deficit

    USGS Publications Warehouse

    McCabe, Gregory J.; Wolock, David M.

    2015-01-01

    A monthly water-balance model is used with CRUTS3.1 gridded monthly precipitation and potential evapotranspiration (PET) data to examine changes in global water deficit (PET minus actual evapotranspiration) for the Northern Hemisphere (NH) for the years 1905 through 2009. Results show that NH deficit increased dramatically near the year 2000 during both the cool (October through March) and warm (April through September) seasons. The increase in water deficit near 2000 coincides with a substantial increase in NH temperature and PET. The most pronounced increases in deficit occurred for the latitudinal band from 0 to 40°N. These results indicate that global warming has increased the water deficit in the NH and that the increase since 2000 is unprecedented for the 1905 through 2009 period. Additionally, coincident with the increase in deficit near 2000, mean NH runoff also increased due to increases in P. We explain the apparent contradiction of concurrent increases in deficit and increases in runoff.

  16. Electrical Fatalities in Northern Ireland

    PubMed Central

    Lucas, James

    2009-01-01

    A review of autopsy reports in cases of electrocution in Northern Ireland revealed that there were 50 accidental electrocutions and 9 suicidal electrocutions over a 22 year period (1982 – 2003). No cases of homicidal electrocution were detected in this jurisdiction. Analysis of the cohort of accidental electrocutions showed that there was a clear skew towards young and middle-aged male adults with deaths occurring more frequently in the summer months. Almost 60% of individuals were engaged in occupational tasks when they were accidentally electrocuted. High and low voltage-related deaths occurred with similar frequency and electrical appliances were found to be responsible for approximately one third of accidental electrocutions. The potential hazards of electricity must continue to be stressed in public safety campaigns if these relatively uncommon but tragic deaths are to be prevented. PMID:19252729

  17. Farmer's lung in Northern Ireland.

    PubMed Central

    Stanford, C F; Hall, G; Chivers, A; Martin, B; Nicholls, D P; Evans, J

    1990-01-01

    A total of 381 farmers in Northern Ireland were studied using a questionnaire, pulmonary function tests, and antibody levels to Micropolyspora faena to assess the incidence of farmer's lung. Twenty (4.9%) had a history of a previous diagnosis of farmer's lung by their doctor. Forty four (10.4%) had delayed onset symptoms compatible with farmer's lung, 32 (7.9%) had precipitant antibody, and 61 (15%) had raised antibody by the enzyme linked immunosorbent (ELISA) method. Restricted lungs were present physiologically in 40 (9.8%). A confirmation of delayed symptoms and precipitant antibody was present in seven (1.7%) whereas delayed symptoms and ELISA antibody was present in nine (2.2%). Using either antibody method only two (0.5%) had a combination of antibody to M faenae, delayed onset symptoms, and restricted pulmonary physiology. PMID:2357452

  18. Farmer's lung in Northern Ireland.

    PubMed

    Stanford, C F; Hall, G; Chivers, A; Martin, B; Nicholls, D P; Evans, J

    1990-05-01

    A total of 381 farmers in Northern Ireland were studied using a questionnaire, pulmonary function tests, and antibody levels to Micropolyspora faena to assess the incidence of farmer's lung. Twenty (4.9%) had a history of a previous diagnosis of farmer's lung by their doctor. Forty four (10.4%) had delayed onset symptoms compatible with farmer's lung, 32 (7.9%) had precipitant antibody, and 61 (15%) had raised antibody by the enzyme linked immunosorbent (ELISA) method. Restricted lungs were present physiologically in 40 (9.8%). A confirmation of delayed symptoms and precipitant antibody was present in seven (1.7%) whereas delayed symptoms and ELISA antibody was present in nine (2.2%). Using either antibody method only two (0.5%) had a combination of antibody to M faenae, delayed onset symptoms, and restricted pulmonary physiology.

  19. Ultraviolet resources over Northern Eurasia.

    PubMed

    Chubarova, Natalia; Zhdanova, Yekaterina

    2013-10-05

    We propose a new climatology of UV resources over Northern Eurasia, which includes the assessments of both detrimental (erythema) and positive (vitamin D synthesis) effects of ultraviolet radiation on human health. The UV resources are defined by using several classes and subclasses - UV deficiency, UV optimum, and UV excess - for 6 different skin types. To better quantifying the vitamin D irradiance threshold we accounted for an open body fraction S as a function of effective air temperature. The spatial and temporal distribution of UV resources was estimated by radiative transfer (RT) modeling (8 stream DISORT RT code) with 1×1° grid and monthly resolution. For this purpose special datasets of main input geophysical parameters (total ozone content, aerosol characteristics, surface UV albedo, UV cloud modification factor) have been created over the territory of Northern Eurasia. The new approaches were used to retrieve aerosol parameters and cloud modification factor in the UV spectral region. As a result, the UV resources were obtained for clear-sky and mean cloudy conditions for different skin types. We show that the distribution of UV deficiency, UV optimum and UV excess is regulated by various geophysical parameters (mainly, total ozone, cloudiness and open body fraction) and can significantly deviate from latitudinal dependence. We also show that the UV optimum conditions can be simultaneously observed for people with different skin types (for example, for 4-5 skin types at the same time in spring over Western Europe). These UV optimum conditions for different skin types occupy a much larger territory over Europe than that over Asia.

  20. Anti-Peptide Monoclonal Antibodies Generated for Immuno-Multiple Reaction Monitoring-Mass Spectrometry Assays Have a High Probability of Supporting Western blot and ELISA*

    PubMed Central

    Schoenherr, Regine M.; Saul, Richard G.; Whiteaker, Jeffrey R.; Yan, Ping; Whiteley, Gordon R.; Paulovich, Amanda G.

    2015-01-01

    Immunoaffinity enrichment of peptides coupled to targeted, multiple reaction monitoring-mass spectrometry (immuno-MRM) has recently been developed for quantitative analysis of peptide and protein expression. As part of this technology, antibodies are generated to short, linear, tryptic peptides that are well-suited for detection by mass spectrometry. Despite its favorable analytical performance, a major obstacle to widespread adoption of immuno-MRM is a lack of validated affinity reagents because commercial antibody suppliers are reluctant to commit resources to producing anti-peptide antibodies for immuno-MRM while the market is much larger for conventional technologies, especially Western blotting and ELISA. Part of this reluctance has been the concern that affinity reagents generated to short, linear, tryptic peptide sequences may not perform well in traditional assays that detect full-length proteins. In this study, we test the feasibility and success rates of generating immuno-MRM monoclonal antibodies (mAbs) (targeting tryptic peptide antigens) that are also compatible with conventional, protein-based immuno-affinity technologies. We generated 40 novel, peptide immuno-MRM assays and determined that the cross-over success rates for using immuno-MRM monoclonals for Western blotting is 58% and for ELISA is 43%, which compare favorably to cross-over success rates amongst conventional immunoassay technologies. These success rates could most likely be increased if conventional and immuno-MRM antigen design strategies were combined, and we suggest a workflow for such a comprehensive approach. Additionally, the 40 novel immuno-MRM assays underwent fit-for-purpose analytical validation, and all mAbs and assays have been made available as a resource to the community via the Clinical Proteomic Tumor Analysis Consortium's (CPTAC) Antibody (http://antibodies.cancer.gov) and Assay Portals (http://assays.cancer.gov), respectively. This study also represents the first

  1. Middle Cretaceous dinosaur assemblages from northern Brazil and northern Africa and their implications for northern Gondwanan composition

    NASA Astrophysics Data System (ADS)

    Candeiro, Carlos Roberto A.

    2015-08-01

    Dinosaurs are one of the most dominant groups in Cretaceous reptilian faunas. A summary of their record in northern Brazil and northern Africa during the middle of the Cretaceous Period (Aptian-Cenomanian) is presented here. Dinosaurs are represented by 32 species (three ornithischians, six sauropods and 23 theropods) from Brazil, Egypt, Lybia, Morocco, Niger, Sudan and Tunisia. These dinosaur assemblages provide fundamental data about distribution and composition of sauropods and theropods in northern Gondwana during the middle of the Cretaceous Period and confirm these assemblages to be among the most important dinosaur faunas in the north Gondwana areas.

  2. The northern light. From mythology to space research.

    NASA Astrophysics Data System (ADS)

    Brekke, A.; Egeland, A.

    Contents: The northern light in folklore and mythology. The northern light in Norse literature. The northern light - a source of inspiration. Accounts of northern lights in Scandinavia - from the Viking era to the Renaissance. The northern light in Scandinavia during the eighteenth century. Scientific auroral experiments beginning in the nineteenth century. Norwegian auroral pioneers in the dawn of our century. The northern lights as weather signs - and the auroral sound. Northern lights and geomagnetic disturbances - their influence on daily life. Auroral research as a tool to study the upper atmosphere and near space. The first systematic observations of the northern light in Norway. Summary and concluding remarks.

  3. Western blot seroindeterminate individuals for human T-lymphotropic virus I/II (HTLV-I/II) in Fortaleza (Brazil): a serological and molecular diagnostic and epidemiological approach.

    PubMed

    Santos, Terezinha de Jesus Teixeira; Costa, Carlos Maurício de Castro; Goubau, Patrick; Vandamme, Anne-Mieke; Desmyter, Jan; Van Doren, Sonia; Mota, Rosa M S; de Castro Costa, Francine Bovy; Oliveira, Ana C S; Barreto, Vania; Gomes, A F; Carneiro-Proietti, Anna B; de Bruin, Veralice Meireles Sales; de Sousa, Francisca C F; Oriá, Reinaldo Barreto

    2003-06-01

    How to handle Western blot (WB) seroindeterminate individuals for Human T-lymphotropic Virus 1/2 (HTLV-1/2) constitutes a challenge for blood banks and families. We made a cross-sectional study of 191 enzyme linked immunoassay (EIA) reactive individuals from the hematological center (HEMOCE) of Fortaleza (Brazil), examining their serological (WB) and molecular (PCR) diagnosis, and demographic profiles, as well as a possible association of their condition with other infectious pathologies and risk factors. Ethical institutional approval and personal consent were obtained. Out of 191 EIA reactive individuals, 118 were WB seroindeterminate and 73 were seropositive for HTLV-1/2. In the PCR analysis of 41 WB seroindeterminate individuals, 9 (22%) were positive and 32 (78%) were negative for HTLV-1/2. The demographic analysis indicated a trend towards a predominance of males among the seroindeterminate individuals and females in the seropositive ones. The seroindeterminate individuals were younger than the seropositive ones. We did not find any association of these conditions with syphilis, Chagas disease or HIV or hepatitis, and with risk factors such as breast-feeding, blood transfusion, STD (syphilis) and IDU.

  4. An optimized xylene-free protein extraction method adapted to formalin-fixed paraffin embedded tissue sections for western blot analysis.

    PubMed

    Mansour, Anthony G; Khalil, Pamela Abou; Bejjani, Noha; Chatila, Rajaa; Dagher-Hamalian, Carole; Faour, Wissam H

    2017-03-01

    Deparaffinization of formalin-fixed paraffin embedded (FFPE) tissues with xylene currently remains a major challenge to the biomedical community. We developed an efficient xylene-free protocol to isolate proteins from archived FFPE human tissue sections. A total of 79 different types of FFPE tissue sections of 8 µm thickness were obtained from various archived FFPE specimens. Deparaffinization was conducted by gently washing each section with around 1 ml of hot distilled water (≈80°C). The deparaffinized tissues were homogenized in lysis buffer, and the isolated proteins were quantified and efficiently resolved using western blot analysis for the presence of Protein kinase B (PKB/AKT) and β-actin. Moreover, a significant amount of proteins was successfully isolated with an average of 2.31 µg/µl. The migration pattern of AKT and β-actin obtained from the specimens was similar to the positive control obtained from protein lysates prepared from in vitro cultured MDA231 cancer cell lines. AKT was successfully identified in all specimens, and β-actin protein was resolved with an efficiency higher than 80%. The entire extraction procedure requires only 20 minutes. This newly developed technique is an efficient, safe, cost-effective, and rapid method to isolate proteins from FFPE tissue sections adequate for molecular analysis.

  5. [The comparison in double immunodiffusion and western blotting methods of anti-SS-A/B antibodies in patients with Sjögren's syndrome].

    PubMed

    Xu, Zhiquan; Takeuchi, Ken; Matsudaira, Ran; Kanai, Yoshinori; Tokano, Yoshiaki; Takasaki, Yoshinari; Hashimoto, Hiroshi

    2003-04-01

    To investigate the autoimmune responses against SS-A/B antigens by double immunodiffusion (DID) and western blotting (WB) in primary and secondary Sjögren's syndrome (SS). Forty-nine patients with primary SS (PSS), 28 patients with secondary SS (SSS) and control group that couldn't be diagnosed as SS were included in this study. In DID analysis, Anti-SS-A antibody was detected in 69% of PSS and 86% of SSS, and anti-SS-B antibody was found in 22% of PSS and 39% of SSS. No significant difference could be demonstrated between PSS and SSS concerning anti-SS-A/B antibodies. Conversely, WB studies disclosed evidences that 18% of PSS and no SSS reacted only with the 52 kD protein, and there was significantly increased in PSS. Sera reacting with the 60 kD antigen were found in 37% of PSS, 71% of SSS, and 75% of SSS with SLE, 63% of SSS with RA. The ratio of SSS, and SSS with SLE were particularly significantly higher than PSS. Our results revealed data that there are the difference of reactivity against SS-A/B antigens in WB between PSS and SSS.

  6. Human T lymphotropic virus types I and II western blot seroindeterminate status and its association with exposure to prototype HTLV-I.

    PubMed

    Yao, Karen; Hisada, Michie; Maloney, Elizabeth; Yamano, Yoshihisa; Hanchard, Barrie; Wilks, Rainford; Rios, Maria; Jacobson, Steven

    2006-02-01

    Human T lymphotropic virus types I and II (HTLV-I/II) Western blot (WB) seroindeterminate status, which is defined as an incomplete banding pattern of HTLV protein Gag (p19 or p24) or Env (GD21 or rgp46), is commonly observed. To investigate the significance of this finding, we examined HTLV-I/II serostatus and HTLV-I proviral load in 2 groups of individuals with WB seroindeterminate status. Low proviral loads were detected in 42% of patients with neurologic symptoms and 44% of voluntary blood donors. These data suggest that a subset of WB seroindeterminate individuals may be infected with prototype HTLV-I. To confirm this hypothesis, we evaluated HTLV-I/II serostatus and proviral load in prospectively collected specimens from 66 WB seronegative patients who had received HTLV-I-infected blood products by transfusion. Eight individuals developed WB seroindeterminate profiles after the transfusion. In addition, using a human leukocyte antigen type A*201-restricted HTLV-I Tax11-19 tetramer, we detected virus-specific CD8(+) T cells in peripheral blood mononuclear cells from WB seroindeterminate patients. These CD8(+) T cells were effective at targeting HTLV-I-infected cells. Collectively, the results suggest that HTLV-I/II WB seroindeterminate status may reflect a history of HTLV-I exposure. Our findings warrant further investigation of the possible clinical outcomes associated with WB seroindeterminate status.

  7. Estimation of hepatitis E virus (HEV) pig seroprevalence using ELISA and Western blot and comparison between human and pig HEV sequences in Belgium.

    PubMed

    Thiry, Damien; Mauroy, Axel; Saegerman, Claude; Thomas, Isabelle; Wautier, Magali; Miry, Cora; Czaplicki, Guy; Berkvens, Dirk; Praet, Nicolas; van der Poel, Wim; Cariolet, Roland; Brochier, Bernard; Thiry, Etienne

    2014-08-27

    Zoonotic transmission of hepatitis E virus (HEV) is of special concern, particularly in high income countries were waterborne infections are less frequent than in developing countries. High HEV seroprevalences can be found in European pig populations. The aims of this study were to obtain prevalence data on HEV infection in swine in Belgium and to phylogenetically compare Belgian human HEV sequences with those obtained from swine. An ELISA screening prevalence of 73% (95% CI 68.8-77.5) was determined in Belgian pigs and a part of the results were re-evaluated by Western blot (WB). A receiver operating characteristic curve analysis was performed and scenarios varying the ELISA specificity relative to WB were analysed. The seroprevalences estimated by the different scenarios ranged between 69 and 81% and are in agreement with the high exposure of the European pig population to HEV. Pig HEV sequences were genetically compared to those detected in humans in Belgium and a predominance of genotype 3 subtype f was shown in both swine and humans. The high HEV seroprevalence in swine and the close phylogenetic relationships between pig and human HEV sequences further support the risk for zoonotic transmission of HEV between humans and pigs.

  8. Rapid detection of six common Mediterranean and three non-Mediterranean alpha-thalassemia point mutations by reverse dot blot analysis.

    PubMed

    Foglietta, Enrica; Bianco, Ida; Maggio, Aurelio; Giambona, Antonino

    2003-11-01

    We describe the implementation of reverse dot blot (RDB) hybridization as a rapid nonradioactive method for the identification of six frequent globin gene point mutations in the Mediterranean population: alpha(Hph)alpha: alpha2 IVS I donor site GGTGAGG --> GG-----; alpha(NcoI)alpha: alpha2 initiation codon ATG --> ACG; alpha(TSaudi)alpha: alpha2Poly A signal AATAA --> AATAAG; alpha(Icaria)alpha: alpha2 termination codon TAA --> AAA (Ter --> LYS); alpha(CS)alpha: alpha2 termination codon TAA --> CAA (Ter --> gly); alphaalpha(NcoI): alpha1 initiation codon ATG --> GTG; and three alpha2 globin gene point mutations found in immigrants in Italy: alpha(T-Quongsze)alpha: alpha2 codon 12 CTG --> CCG (Leu --> Pro); alpha(Seal Rock)alpha: alpha2 termination codon TAA --> GAA (TER --> GLU); and alpha(Koyadora)alpha: alpha2 termination codon TAA --> TCA (TER --> SER). The method uses the principle of allele-specific oligonucleotide (ASO) hybridization, but it is a nonradioactive method and permits rapid and simultaneous typing of point mutations and small deletions.

  9. Effects of histoplasmin M antigen chemical and enzymatic deglycosylation on cross-reactivity in the enzyme-linked immunoelectrotransfer blot method.

    PubMed Central

    Zancopé-Oliveira, R M; Bragg, S L; Reiss, E; Wanke, B; Peralta, J M

    1994-01-01

    The enzyme-linked immunoelectrotransfer blot (EITB) method was evaluated as a suitable method for detecting antibodies against M antigen of Histoplasma capsulatum by use of both glycosylated and deglycosylated M protein of histoplasmin (HMIN). Sera from patients with histoplasmosis, paracoccidioidomycosis, blastomycosis, coccidioidomycosis, and aspergillosis were tested by the EITB with glycosylated M protein of HMIN. This assay demonstrated 100% sensitivity with histoplasmosis serum samples, all of which reacted with the 94-kDa glycoprotein (M antigen). Although the EITB is highly sensitive, it is not specific for histoplasmosis when glycosylated M protein is used as an antigen. A total of 81% of paracoccidioidomycosis, 25% of blastomycosis, 33% of coccidioidomycosis, 73% of aspergillosis, and 16% of tuberculosis serum samples cross-reacted with M protein of HMIN and yielded patterns indistinguishable from those obtained with histoplasmosis serum samples. The EITB reactions with both untreated M antigen and M antigen altered by periodate oxidation or by deglycosylation with endoglycosidases were compared. Cross-reactions with heterologous sera in the EITB could be attributed to periodate-sensitive carbohydrate epitopes, as reflected by the increase in the test specificity from 46.1 to 91.2% after periodate treatment of M protein. The EITB for the detection of antibodies to M antigen is a potential diagnostic test for histoplasmosis, provided that periodate-treated M protein is used as an antigen. Images PMID:8556474

  10. Comparison between the Skin Snip Test and Simple Dot Blot Assay as Potential Rapid Assessment Tools for Onchocerciasis in the Postcontrol Era in Ghana

    PubMed Central

    Guzmán, G. E.; Awadzi, K.; Opoku, N.; Narayanan, R. B.; Akuffo, H. O.

    2002-01-01

    Successful control of onchocerciasis through mass distribution of ivermectin needs to be coupled with reliable, sensitive, specific, yet affordable diagnostic methods to monitor and ensure the efficacy of such measures. The effort put into the development of diagnostic methods for onchocerciasis that can substitute for or work in combination with the present “gold standard,” the skin snip test, has resulted in the discovery of a number of immunogenic proteins with potential use as diagnostic tools in the postcontrol era. Most of these proteins have now been produced through recombinant DNA techniques. However, when costs are not a trivial issue, none of them have yet found their way into the areas where the disease still exists. In the present study, we have evaluated the performance of a simple dot blot assay which uses a mixture of native proteins designated PakF as a serious contender in the quest for a less invasive and more sensitive method to detect Onchocerca volvulus infection in areas with diverse endemicities. Our results indicate that the assay we propose is more sensitive than the skin snip test and shows high specificity, both characteristics required for a suitable tool for the monitoring of onchocerciasis in the postcontrol era. PMID:12204952

  11. Detection of antibodies against avian antigens in bronchoalveolar lavage from patients with pigeon breeder's disease: usefulness of enzyme-linked immunosorbent assay and enzyme immunotransfer blotting.

    PubMed

    Sandoval, J; Bañales, J L; Cortés, J J; Mendoza, F; Selman, M; Reyes, P A

    1990-01-01

    The study reported here evaluated the usefulness of the enzyme-linked immunosorbent assay (ELISA) in the detection of antibodies against pigeon antigens in the serum and bronchoalveolar lavage (BAL) of patients with clinical, radiological, and functional evidence of interstitial lung disease (ILD) with and without pigeon breeder's disease (PBD). The results were compared with those obtained by the simultaneous use of counterimmunoelectrophoresis (CIE) in the same patients. In PBD, ELISA detected antibodies against pigeon's sera in both serum and BAL in 100% of patients, while CIE failed to detect the antibodies in the serum of one patient and in most of the samples of BAL. In addition, we used enzyme immunotransfer blotting to determine the number of epitopes in pigeon serum recognized by antibodies present in serum and BAL. There was a heterogeneous response in both fluids, but the reaction pattern demonstrated that patient's sera recognize to-25 different pigeon epitopes. We conclude that ELISA is a highly sensitive and specific method for the detection of antibodies against pigeon antigens in the serum and BAL of patients with PBD and that the host response involves a great number of avian antigens.

  12. Immunohistochemical and western blot analysis suggest that the soluble forms of FGF1-2 and FGFR1-2 sustain tail regeneration in the lizard.

    PubMed

    Alibardi, Lorenzo

    2017-08-18

    Fibroblast Growth Factors 1-2 (FGF1-2) stimulate tail regeneration in lizards and therefore the distribution of their receptors, FGFR1-2, in the regenerating tail of the lizard. Podarcis muralis has been studied using immunofluorescence and western blotting. Immunoreactive protein bands at 15-16kDa for FGF1-2 in addition to those at 50-65kDa are detected in the regenerating epidermis, but weak bands at 35, 45 and 50kDa appear from the regenerating connective tissues. Strongly immunolabeled bands for FGFR1 at 32, 60, and 80kDa and less intense for FGFR2 only appear in the regenerating tail. In normal tail epidermis and dermis, higher MW forms are present at 80 and 115-140kDa, respectively, but they disappear in the regenerating epidermis and dermis where low MW forms of FGFR1-2 are found at 50-70kDa. Immunolocalization confirms that most FGFR1-2 are present in the wound epidermis, Apical Epidermal Peg, ependymal tube while immunolabeling lowers in regenerating muscles, blastema cells, cartilage and connectives tissues. The likely release of FGFs from the Apical Epidermal Peg and ependyma and the presence of their receptors in these tissues may determine the autocrine stimulation of proliferation and a paracrine stimulation of the blastema cells through their FGF Receptors. Copyright © 2017 Elsevier GmbH. All rights reserved.

  13. Re-purposing of histological tissue sections for corroborative western blot analysis of hypothalamic metabolic neuropeptide expression following delineation of transactivated structures by Fos immuno-mapping.

    PubMed

    Alenazi, Fahaad S H; Ibrahim, Baher A; Briski, Karen P

    2015-04-01

    Fos immunocytochemistry is a valuable anatomical mapping tool for distinguishing cells within complex tissues that undergo genomic activation, but it is seldom paired with corroborative molecular analytical techniques. Due to preparatory requirements that include protein cross-linking for specimen sectioning, histological tissue sections are regarded as unsuitable for those methods. Our studies show that pharmacological activation of the hindbrain energy sensor AMPK by AICAR elicits estradiol (E)-dependent patterns of Fos immunolabeling of hypothalamic metabolic loci. Here, Western blotting was applied to hypothalamic tissue removed from histological sections of E- versus oil (O)-implanted ovariectomized (OVX) female rat brain to measure levels of metabolic transmitters associated with Fos-positive structures. In both E and O rats, AICAR treatment elicited alterations in pro-opiomelanocortin, neuropeptide Y, SF-1, and orexin-A neuropeptide expression that coincided with patterns of Fos labeling of structures containing neurons that synthesize these neurotransmitters, e.g. arcuate and ventromedial nuclei and lateral hypothalamic area. O, but not E animals also exhibited parallel augmentation of tissue corticotropin-releasing hormone neuropeptide levels and paraventricular nucleus Fos staining. Data demonstrate the utility of immunoblot analysis as a follow-through technique to capitalize on Fos mapping of transactivation sites in the brain. Findings that induction of Fos immunoreactivity coincides with adjustments in hypothalamic metabolic neuropeptide expression affirms that this functional indicator reflects changes in neurotransmission in pathways governing metabolic outflow. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Detection of genetically modified crops using multiplex asymmetric polymerase chain reaction and asymmetric hyperbranched rolling circle amplification coupled with reverse dot blot.

    PubMed

    Wang, Xiumin; Teng, Da; Guan, Qingfeng; Tian, Fang; Wang, Jianhua

    2015-04-15

    To meet the ever-increasing demand for detection of genetically modified crops (GMCs), low-cost, high-throughput and high-accuracy detection assays are needed. The new multiplex asymmetric polymerase chain reaction and asymmetric hyper-branched rolling circle amplification coupled with reverse dot blot (RDB) systems were developed to detect GMCs. Thirteen oligonucleotide probes were designed to identify endogenous targets (Lec1, Hmg and Sad1), event-specific targets (RRS-5C, RRS-3C, Bt176-3C and MON810-3C), screening targets (35S promoter and NOS terminator), and control targets (18S and PLX). Optimised conditions were as follows: tailed hybridization probes (1-2 pmol/l) were immobilized on a membrane by baking for 2h, and a 10:1 ratio of forward to reverse primers was used. The detection limits were 0.1 μg/l of 2% RRS and 0.5 ng/l of DNA from genetically modified (GM) soybean. These results indicate that the RDB assay could be used to detect multiplex target genes of GMCs rapidly and inexpensively.

  15. Utility of Schistosoma bovis adult worm antigens for diagnosis of human schistosomiasis by enzyme-linked immunosorbent assay and electroimmunotransfer blot techniques.

    PubMed

    Pardo, J; Carranza, C; Turrientes, M C; Pérez Arellano, J L; López Vélez, R; Ramajo, V; Muro, A

    2004-11-01

    Immunodiagnostic methods based on the detection of antibodies continue to be the most effective and practical methods for the diagnosis of imported schistosomiasis. Schistosoma bovis is a species whose final natural hosts are bovines, ovines, caprines, and small wild ruminants. Different studies have demonstrated the analogies existing between S. bovis and other Schistosoma species which affect humans. The objective of this work was to evaluate the utility of S. bovis adult worm antigens (AWA) for the diagnosis of imported human schistosomiasis by enzyme-linked immunosorbent assay (ELISA) and electroimmunotransfer blotting (EITB) techniques. By detecting eggs, the ELISA for S. bovis AWA was able to definitively detect imported cases with a sensitivity of 94%. The specificity of the ELISA for S. bovis AWA was 97%. There were no differences between the results of the S. bovis AWA ELISA for patients infected with Schistosoma mansoni and those infected with Schistosoma haematobium. The EITB technique showed bands of 85, 37, and 20 kDa, which are characteristic of infections with Schistosoma spp. Specific bands to indicate infection by different species of Schistosoma have not been detected. The combined use of the ELISA for S. bovis AWA and EITB increased the global sensitivity of the study to 97%. Our findings suggest that the ELISA for S. bovis AWA is a useful test for the immunodiagnosis of imported schistosomiasis and that EITB for detecting S. bovis AWA permits the confirmation of diagnosis when the ELISA for S. bovis AWA is positive.

  16. Utility of Schistosoma bovis Adult Worm Antigens for Diagnosis of Human Schistosomiasis by Enzyme-Linked Immunosorbent Assay and Electroimmunotransfer Blot Techniques

    PubMed Central

    Pardo, J.; Carranza, C.; Turrientes, M. C.; Arellano, J. L. Pérez; Vélez, R. López; Ramajo, V.; Muro, A.

    2004-01-01

    Immunodiagnostic methods based on the detection of antibodies continue to be the most effective and practical methods for the diagnosis of imported schistosomiasis. Schistosoma bovis is a species whose final natural hosts are bovines, ovines, caprines, and small wild ruminants. Different studies have demonstrated the analogies existing between S. bovis and other Schistosoma species which affect humans. The objective of this work was to evaluate the utility of S. bovis adult worm antigens (AWA) for the diagnosis of imported human schistosomiasis by enzyme-linked immunosorbent assay (ELISA) and electroimmunotransfer blotting (EITB) techniques. By detecting eggs, the ELISA for S. bovis AWA was able to definitively detect imported cases with a sensitivity of 94%. The specificity of the ELISA for S. bovis AWA was 97%. There were no differences between the results of the S. bovis AWA ELISA for patients infected with Schistosoma mansoni and those infected with Schistosoma haematobium. The EITB technique showed bands of 85, 37, and 20 kDa, which are characteristic of infections with Schistosoma spp. Specific bands to indicate infection by different species of Schistosoma have not been detected. The combined use of the ELISA for S. bovis AWA and EITB increased the global sensitivity of the study to 97%. Our findings suggest that the ELISA for S. bovis AWA is a useful test for the immunodiagnosis of imported schistosomiasis and that EITB for detecting S. bovis AWA permits the confirmation of diagnosis when the ELISA for S. bovis AWA is positive. PMID:15539523

  17. Identification of liver protein targets modified by tienilic acid metabolites using a two-dimensional Western blot-mass spectrometry approach

    NASA Astrophysics Data System (ADS)

    Methogo, Ruth Menque; Dansette, Patrick M.; Klarskov, Klaus

    2007-12-01

    A combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of tienilic acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase.

  18. Inverse PCR and Quantitative PCR as Alternative Methods to Southern Blotting Analysis to Assess Transgene Copy Number and Characterize the Integration Site in Transgenic Woody Plants.

    PubMed

    Stefano, Biricolti; Patrizia, Bogani; Matteo, Cerboneschi; Massimo, Gori

    2016-06-01

    One of the major unanswered questions with respect to the commercial use of genetic transformation in woody plants is the stability of the transgene expression over several decades within the same individual. Gene expression is strongly affected by the copy number which has been integrated into the plant genome and by the local DNA features close to the integration sites. Because woody plants cannot be subjected to selfing or backcrossing to modify the transgenic allelic structure without affecting the valuable traits of the cultivar, molecular characterization of the transformation event is therefore crucial. After assessing the transgene copy number of a set of apple transgenic clones with Southern blotting, we describe two alternative methods: the first is based on inverse PCR (i-PCR) and the second on the quantitative PCR (q-PCR). The methods produced comparable results with the exception of the data regarding a high copy number clone, but while the q-PCR-based system is rapid and easily adaptable to high throughput systems, the i-PCR-based method can provide information regarding the transformation event and the characteristics of the sequences flanking the transgenic construct.

  19. Differential staining of Western blots of human secreted glycoproteins from serum, milk, saliva, and seminal fluid using lectins displaying diverse sugar specificities.

    PubMed

    Gilboa-Garber, Nechama; Lerrer, Batya; Lesman-Movshovich, Efrat; Dgani, Orly

    2005-12-01

    Human milk, serum, saliva, and seminal fluid glycoproteins (gps) nourish and protect newborn and adult tissues. Their saccharides, which resemble cell membrane components, may block pathogen adhesion and infection. In the present study, they were examined by a battery of lectins from plants, animals, and bacteria, using hemagglutination inhibition and Western blot analyses. The lectins included galactophilic ones from Aplysia gonad, Erythrina corallodendron, Maclura pomifera (MPL), peanut, and Pseudomonas aeruginosa (PA-IL); fucose-binding lectins from Pseudomonas aeruginosa (PA-IIL), Ralstonia solanacearum (RSL), and Ulex europaeus (UEA-I), and mannose/glucose-binding Con A. The results demonstrated the chosen lectin efficiency for differential analysis of human secreted gps as compared to CBB staining. They unveiled the diversity of these body fluid gp glycans (those of the milk and seminal fluid being highest): the milk gps interacted most strongly with PA-IIL, followed by RSL; the saliva gps with RSL, followed by PA-IIL and MPL; the serum gps with Con A and MPL, followed by PA-IIL and RSL, and the seminal plasma gps with RSL and MPL, followed by UEA-I and PA-IIL. The potential usage of these lectins as probes for scientific, industrial, and medical purposes, and for quality control of the desired gps is clearly indicated.

  20. Controlled study of the frequency of anti-HSP 70 with the ELISA and the Western Blot methods in patients with Ménière Disease.

    PubMed

    Wiederkehr Baú, Anne-Rose L; Lavinsky, Luiz; Bonorino, Cristina; Munari, Leonardo; Lavinsky-Wolff, Michelle; Wolff, Fernando Herz

    2011-01-01

    To establish the frequency of auto-antibodies anti-HSP 70 using ELISA and Western Blot (WB) methods and to compare the results of each method among patients with the Ménière's Disease (MD) and internal ear diseases (IED) who do not fulfill criteria for MD. Sensibility, specificity and predictive values of anti-HSP70 test in diagnosis of MD were calculated. Prospective, case-control. Blood samples were collected from 31 patients with MD and 78 patients with non Ménière IED. Data regarding cochlear and vestibular symptoms were obtained and blood sample was tested. ELISA tests results were positive in 4(13%) patients and results of WB were positive in 8(26%) patients. Among patients with positive ELISA results, 1 patient presented active disease and in the remaining 3 patients the disease was inactive. Among the 8 WB positive patients, only 2 patients presented active disease. Statistical analyses did not establish any association between serologic findings and clinical factors of MD. The presence of anti-HSP70 using the ELISA and the WB methods did not demonstrate clinical value for the diagnosis of MD. We did not find association between idiopathic MD nor unspecific etiology MD and the presence of anti-HSP70 auto-antibodies.