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Sample records for acidic protein assay

  1. Interference of N-hydroxysuccinimide with bicinchoninic acid protein assay.

    PubMed

    Vashist, Sandeep Kumar; Dixit, Chandra Kumar

    2011-07-29

    We report here substantial interference from N-hydroxysuccinimide (NHS) in the bicinchoninic acid (BCA) protein assay. NHS is one of the most commonly used crosslinking agents in bioanalytical sciences, which can lead to serious potential errors in the BCA protein assay based protein estimation if it is present in the protein analyte solution. It was identified to be a reducing substance, which interferes with the BCA protein assay by reducing Cu(2+) in the BCA working reagent. The absorbance peak and absorbance signal of NHS were very similar to those of bovine serum albumin (BSA), thereby indicating a similar BCA reaction mechanism for NHS and protein. However, the combined absorbance of NHS and BSA was not additive. The time-response measurements of the BCA protein assay showed consistent single-phase kinetics for NHS and gradually decreasing kinetics for BSA. The error in protein estimation due to the presence of NHS was counteracted effectively by plotting additional BCA standard curve for BSA with a fixed concentration of NHS. The difference between the absorbance values of BSA and BSA with a fixed NHS concentration provided the absorbance contributed by NHS, which was then subtracted from the total absorbance of analyte sample to determine the actual absorbance of protein in the analyte sample. PMID:21762678

  2. Standard in vitro assays for protein-nucleic acid interactions--gel shift assays for RNA and DNA binding.

    PubMed

    Mitchell, Sarah F; Lorsch, Jon R

    2014-01-01

    The characterization of protein-nucleic acid interactions is necessary for the study of a wide variety of biological processes. One straightforward and widely used approach to this problem is the electrophoretic mobility shift assay (EMSA), in which the binding of a nucleic acid to one or more proteins changes its mobility through a nondenaturing gel matrix. Usually, the mobility of the nucleic acid is reduced, but examples of increased mobility do exist. This type of assay can be used to investigate the affinity of the interaction between the protein and nucleic acid, the specificity of the interaction, the minimal binding site, and the kinetics of the interaction. One particular advantage of EMSA is the ability to analyze multiple proteins, or protein complexes, binding to nucleic acids. This assay is relatively quick and easy and utilizes equipment available in most laboratories; however, there are many variables that can only be determined empirically; therefore, optimization is necessary and can be highly dependent upon the system. The protocol described here is for the poly(A)-binding protein (PABP) binding to an unstructured RNA probe of 43 bases. While this may be a useful protocol for some additional assays, it is recommended that both reaction conditions and gel running conditions be tailored to the individual interaction to be probed. PMID:24674072

  3. Electrophoretic Mobility Shift Assay (EMSA) for Detecting Protein-Nucleic Acid Interactions

    PubMed Central

    Hellman, Lance M.; Fried, Michael G.

    2009-01-01

    The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this article, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided. PMID:17703195

  4. Melamine and Cyanuric Acid do not interfere with Bradford and Ninhydrin assays for protein determination

    PubMed Central

    Field, Anjalie; Field, Jeffrey

    2010-01-01

    In the fall of 2007 pet food contaminated with melamine and cyanuric acid caused kidney stones in thousands of animals. In the summer of 2008, a more serious outbreak of adulterated dairy food caused the deaths of six infants and sickened about 290,000 children in China. In all cases, melamine was likely added to inflate the apparent protein content of the foods. To determine if we could measure protein without interference from melamine and cyanuric acid we tested these compounds in the Bradford and Ninhydrin assays, two common dye-based assays for protein, as well as by ammonia release, the most common assay used in the food industry. Neither compound was detected in the Ninhydrin and Bradford assays at concentrations of >100 μg/ml. The ammonia assay detected melamine but was inconclusive with respect to cyanuric acid. To develop an accurate test for food that would not detect either chemical as a protein, assays were run on cat food and reconstituted milk powder. The Bradford assay readily measured the protein content of each food, and importantly, the addition of melamine or cyanuric acid to reconstituted milk did not affect the readings. The protein concentrations obtained for reconstituted milk powder were as expected, but those for the cat food were 10 to 30-fold lower, due to its low solubility. We conclude that dye-binding assays can be employed to detect protein in food without interference from melamine and cyanuric acid, thus reducing the incentive to use them as additives. PMID:20228949

  5. Development of a Quantitative BRET Affinity Assay for Nucleic Acid-Protein Interactions

    PubMed Central

    Vickers, Timothy A.; Crooke, Stanley T.

    2016-01-01

    Protein-nucleic acid interactions play a crucial role in the regulation of diverse biological processes. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination, and RNA processing continues to be a crucial aspect of understanding of cell biology and the mechanisms of disease. In addition, proteins have been demonstrated to interact with antisense oligonucleotide therapeutics in a sequence and chemistry dependent manner, influencing ASO potency and distribution in cells and in vivo. While many assays have been developed to measure protein-nucleic acid interactions, many suffer from lack of throughput and sensitivity, or challenges with protein purification and scalability. In this report we present a new BRET assay for the analysis of DNA-protein interactions which makes use of an extremely bright luciferase as a tag for the binding protein, along with a long-wavelength fluorophore conjugated to the nucleic acid. The resulting assay is high throughput, sensitive, does not require protein purification, and even allows for quantitative characterization of these interactions within the biologically relevant context of whole cells. PMID:27571227

  6. Development of a Quantitative BRET Affinity Assay for Nucleic Acid-Protein Interactions.

    PubMed

    Vickers, Timothy A; Crooke, Stanley T

    2016-01-01

    Protein-nucleic acid interactions play a crucial role in the regulation of diverse biological processes. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination, and RNA processing continues to be a crucial aspect of understanding of cell biology and the mechanisms of disease. In addition, proteins have been demonstrated to interact with antisense oligonucleotide therapeutics in a sequence and chemistry dependent manner, influencing ASO potency and distribution in cells and in vivo. While many assays have been developed to measure protein-nucleic acid interactions, many suffer from lack of throughput and sensitivity, or challenges with protein purification and scalability. In this report we present a new BRET assay for the analysis of DNA-protein interactions which makes use of an extremely bright luciferase as a tag for the binding protein, along with a long-wavelength fluorophore conjugated to the nucleic acid. The resulting assay is high throughput, sensitive, does not require protein purification, and even allows for quantitative characterization of these interactions within the biologically relevant context of whole cells. PMID:27571227

  7. 40 CFR 79.67 - Glial fibrillary acidic protein assay.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... addition to information meeting the requirements stated under 40 CFR 79.60, the following specific...), 5 mM Hepes, pH 7.4, 0.7 percent Triton X-100) to a final concentration of 0.25 mg total protein per... the desired dilution in blocking solution containing 0.1 percent Triton X-100). Serum anti-bovine...

  8. 40 CFR 79.67 - Glial fibrillary acidic protein assay.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... addition to information meeting the requirements stated under 40 CFR 79.60, the following specific...), 5 mM Hepes, pH 7.4, 0.7 percent Triton X-100) to a final concentration of 0.25 mg total protein per... the desired dilution in blocking solution containing 0.1 percent Triton X-100). Serum anti-bovine...

  9. 40 CFR 79.67 - Glial fibrillary acidic protein assay.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... addition to information meeting the requirements stated under 40 CFR 79.60, the following specific...), 5 mM Hepes, pH 7.4, 0.7 percent Triton X-100) to a final concentration of 0.25 mg total protein per... the desired dilution in blocking solution containing 0.1 percent Triton X-100). Serum anti-bovine...

  10. 40 CFR 79.67 - Glial fibrillary acidic protein assay.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... addition to information meeting the requirements stated under 40 CFR 79.60, the following specific...), 5 mM Hepes, pH 7.4, 0.7 percent Triton X-100) to a final concentration of 0.25 mg total protein per... the desired dilution in blocking solution containing 0.1 percent Triton X-100). Serum anti-bovine...

  11. 40 CFR 79.67 - Glial fibrillary acidic protein assay.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... addition to information meeting the requirements stated under 40 CFR 79.60, the following specific...), 5 mM Hepes, pH 7.4, 0.7 percent Triton X-100) to a final concentration of 0.25 mg total protein per... the desired dilution in blocking solution containing 0.1 percent Triton X-100). Serum anti-bovine...

  12. Bioprocess monitoring: minimizing sample matrix effects for total protein quantification with bicinchoninic acid assay.

    PubMed

    Reichelt, Wieland N; Waldschitz, Daniel; Herwig, Christoph; Neutsch, Lukas

    2016-09-01

    Determining total protein content is a routine operation in many laboratories. Despite substantial work on assay optimization interferences, the widely used bicinchoninic acid (BCA) assay remains widely recognized for its robustness. Especially in the field of bioprocess engineering the inaccuracy caused by interfering substances remains hardly predictable and not well understood. Since the introduction of the assay, sample pre-treatment by trichloroacetic acid (TCA) precipitation has been indicated as necessary and sufficient to minimize interferences. However, the sample matrix in cultivation media is not only highly complex but also dynamically changing over process time in terms of qualitative and quantitative composition. A significant misestimation of the total protein concentration of bioprocess samples is often observed when following standard work-up schemes such as TCA precipitation, indicating that this step alone is not an adequate means to avoid measurement bias. Here, we propose a modification of the BCA assay, which is less influenced by sample complexity. The dynamically changing sample matrix composition of bioprocessing samples impairs the conventional approach of compensating for interfering substances via a static offset. Hence, we evaluated the use of a correction factor based on an internal spike measurement for the respective samples. Using protein spikes, the accuracy of the BCA protein quantification could be improved fivefold, taking the BCA protein quantification to a level of accuracy comparable to other, more expensive methods. This will allow reducing expensive iterations in bioprocess development to due inaccurate total protein analytics. PMID:27314233

  13. Fluorometric assay for quantitation of biotin covalently attached to proteins and nucleic acids.

    PubMed

    Batchelor, Robert H; Sarkez, Adam; Cox, W Gregory; Johnson, Iain

    2007-10-01

    As a component of the (strept)avidin affinity system, biotin is often covalently linked to proteins or nucleic acids. We describe here a microplate-based high-throughput fluorometric assay for biotin linked to either proteins or nucleic acids based on fluorescence resonance energy transfer (FRET). This assay utilizes a complex of Alexa Fluoro 488 dye-labeled avidin with a quencher dye, 2-(4'-hydroxyazobenzene) benzoic acid (HABA), occupying the biotin binding sites of the avidin. In the absence of biotin, HABA quenches the fluorescence emission of the Alexa Fluor 488 dyes via FRET HABA is displaced when biotin binds to the Alexa Fluor 488 dye-labeled avidin, resulting in decreased FRET efficiency. This mechanism results in an increase in fluorescence intensity directly related to the amount of biotin present in the sample. The assay is able to detect as little as 4 pmol biotin in a 0.1 mL volume within 15 min of adding sample to the reagent, with a Z-factor > 0.9. PMID:18019342

  14. QUANTIFICATION OF GLIAL FIBRILLARY ACIDIC PROTEIN: COMPARISON OF SLOT-IMMUNOBINDING ASSAYS WITH A NOVEL SANDWICH ELISA

    EPA Science Inventory

    Detailed protocols are presented for assaying glial fibrillary acidic protein (GFAP), an astrocyte localized protein rich serves as a quantitative marker of toxicant- induced injury to the central nervous system. wo different solid-phase assay procedures are described: 1) a nitro...

  15. Total protein quantitation using the bicinchoninic acid assay and gradient elution moving boundary electrophoresis.

    PubMed

    Kralj, Jason G; Munson, Matthew S; Ross, David

    2014-07-01

    We investigated the ability of gradient elution moving boundary electrophoresis (GEMBE) with capacitively coupled contactless conductivity detection (C(4) D) to assay total protein concentration using the bicinchoninic acid (BCA) reaction. We chose this format because GEMBE-C(4) D behaves as a concentration dependent detection system, unlike optical methods that also rely on pathlength (due to Beer's law). This system tolerates proteins well compared with other capillary electrophoretic methods, allowing the capillary to be reused without coatings or additional hydroxide wash steps. The typical reaction protocol was modified by reducing the pH slightly from 11.25 to 9.4, which enabled elimination of tartrate from the reagents. We estimated that copper (I) could be detected at approximately 3.0 μmol/L, which agrees with similar GEMBE and CZE systems utilizing C(4) D. Under conditions similar to the BCA "micro method" assay, we determined the LOD for three common proteins (insulin, BSA, and bovine gamma globulin) and found that they agree well with the existing spectroscopic detection methods. Further, we investigated how long reaction times impact the LOD and found that the conversion was proportional to log(time). This indicated that little sensitivity is gained by extending the reaction past 1 h. Hence, GEMBE provides an alternative platform for total protein assays while maintaining the excellent sensitivity of the optical-based methods. PMID:24648165

  16. Target-driven self-assembly of stacking deoxyribonucleic acids for highly sensitive assay of proteins.

    PubMed

    Cao, Ya; Chen, Weiwei; Han, Peng; Wang, Zhuxin; Li, Genxi

    2015-08-26

    In this paper, we report a new signal amplification strategy for highly sensitive and enzyme-free method to assay proteins based on the target-driven self-assembly of stacking deoxyribonucleic acids (DNA) on an electrode surface. In the sensing procedure, binding of target protein with the aptamer probe is used as a starting point for a scheduled cycle of DNA hairpin assembly, which consists of hybridization, displacement and target regeneration. Following numbers of the assembly repeats, a great deal of DNA duplexes can accordingly be formed on the electrode surface, and then switch on a succeeding propagation of self-assembled DNA concatemers that provide further signal enhancement. In this way, each target binding event can bring out two cascaded DNA self-assembly processes, namely, stacking DNA self-assembly, and therefore can be converted into remarkably intensified electrochemical signals by associating with silver nanoparticle-based readout. Consequently, highly sensitive detection of target proteins can be achieved. Using interferon-gamma as a model, the assay method displays a linear range from 1 to 500 pM with a detection limit of 0.57 pM, which is comparable or even superior to other reported amplified assays. Moreover, the proposed method eliminates the involvement of any enzymes, thereby enhancing the feasibility in clinical diagnosis. PMID:26347164

  17. Paper-based fluorescence resonance energy transfer assay for directly detecting nucleic acids and proteins.

    PubMed

    Li, Hua; Fang, Xueen; Cao, Hongmei; Kong, Jilie

    2016-06-15

    Paper-based fluorescence resonance energy transfer assay (FRET) is gaining great interest in detecting macro-biological molecule. It is difficult to achieve conveniently and fast detection for macro-biological molecule. Herein, a graphene oxide (GO)-based paper chip (glass fiber) integrated with fluorescence labeled single-stranded DNA (ssDNA) for fast, inexpensive and direct detection of biological macromolecules (proteins and nucleic acids) has been developed. In this paper, we employed the Cy3/FAM-labeled ssDNA as the reporter and the GO as quencher and the original glass fiber paper as data acquisition substrates. The chip which was designed and fabricated by a cutting machine is a miniature biosensor that monitors fluorescence recovery from resonance energy transfer. The hybridization assays and fluorescence detection were all simplified, and the surface of the chip did not require immobilization or washing. A Nikon Eclipse was employed as excited resource and a commercial digital camera was employed for capturing digital images. This paper-based microfluidics chip has been applied in the detection of proteins and nucleic acids. The biosensing capability meets many potential requirements for disease diagnosis and biological analysis. PMID:26807518

  18. Assays for Determination of Protein Concentration.

    PubMed

    Olson, Bradley J S C

    2016-01-01

    Biochemical analysis of proteins relies on accurate quantification of protein concentration. Detailed in this appendix are some commonly used methods for protein analysis, e.g., Lowry, Bradford, bicinchoninic acid (BCA), UV spectroscopic, and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) assays. The primary focus of this report is assay selection, emphasizing sample and buffer compatibility. The fundamentals of generating protein assay standard curves and of data processing are considered, as are high-throughput adaptations of the more commonly used protein assays. Also included is a rapid, inexpensive, and reliable BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels. © 2016 by John Wiley & Sons, Inc. PMID:27248579

  19. Nucleic acid detection assays

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann; Dahlberg, James E.

    2005-04-05

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  20. Methods and devices for protein assays

    DOEpatents

    Chhabra, Swapnil; Cintron, Jose M.; Shediac, Renee

    2009-11-03

    Methods and devices for protein assays based on Edman degradation in microfluidic channels are disclosed herein. As disclosed, the cleaved amino acid residues may be immobilized in an array format and identified by detectable labels, such as antibodies, which specifically bind given amino acid residues. Alternatively, the antibodies are immobilized in an array format and the cleaved amino acids are labeled identified by being bound by the antibodies in the array.

  1. Protein binding assay for hyaluronate

    SciTech Connect

    Lacy, B.E.; Underhill, C.B.

    1986-11-01

    A relatively quick and simple assay for hyaluronate was developed using the specific binding protein, hyaluronectin. The hyaluronectin was obtained by homogenizing the brains of Sprague-Dawley rats, and then centrifuging the homogenate. The resulting supernatant was used as a source of crude hyaluronectin. In the binding assay, the hyaluronectin was mixed with (/sup 3/H)hyaluronate, followed by an equal volume of saturated (NH/sub 4/)/sub 2/SO/sub 4/, which precipitated the hyaluronectin and any (/sup 3/H)hyaluronate associated with it, but left free (/sup 3/H)hyaluronate in solution. The mixture was then centrifuged, and the amount of bound (/sup 3/H)hyaluronate in the precipitate was determined. Using this assay, the authors found that hyaluronectin specifically bound hyaluronate, since other glycosaminoglycans failed to compete for the binding protein. In addition, the interaction between hyaluronectin and hyaluronate was of relatively high affinity, and the size of the hyaluronate did not appear to substantially alter the amount of binding. To determine the amount of hyaluronate in an unknown sample, they used a competition assay in which the binding of a set amount of (/sup 3/H)hyaluronate was blocked by the addition of unlabeled hyaluronate. By comparing the degree of competition of the unknown samples with that of known amounts of hyaluronate, it was possible to determine the amount of hyaluronate in the unknowns. They have found that this method is sensitive to 1 ..mu..g or less of hyaluronate, and is unaffected by the presence of proteins.

  2. Automated Protein Assay Using Flow Injection Analysis

    NASA Astrophysics Data System (ADS)

    Wolfe, Carrie A. C.; Oates, Matthew R.; Hage, David S.

    1998-08-01

    The technique of flow injection analysis (FIA) is a common instrumental method used in detecting a variety of chemical and biological agents. This paper describes an undergraduate laboratory that uses FIA to perform a bicinchoninic acid (BCA) colorimetric assay for quantitating protein samples. The method requires less than 2 min per sample injection and gives a response over a broad range of protein concentrations. This method can be used in instrumental analysis labs to illustrate the principles and use of FIA, or as a means for introducing students to common methods employed in the analysis of biological agents.

  3. A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay.

    PubMed

    Fleming, Jonathan K; Glass, Thomas R; Lackie, Steve J; Wojciak, Jonathan M

    2016-09-01

    Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are bioactive signaling lysophospholipids that activate specific G protein-coupled receptors on the cell surface triggering numerous biological events. In circulation, S1P and LPA associate with specific carrier proteins or chaperones; serum albumin binds both S1P and LPA while HDL shuttles S1P via interactions with apoM. We used a series of kinetic exclusion assays in which monoclonal anti-S1P and anti-LPA antibodies competed with carrier protein for the lysophospholipid to measure the equilibrium dissociation constants (Kd) for these carrier proteins binding S1P and the major LPA species. Fatty acid-free (FAF)-BSA binds these lysophospholipids with the following Kd values: LPA(16:0), 68 nM; LPA(18:1), 130 nM; LPA(18:2), 350 nM; LPA(20:4), 2.2 μM; and S1P, 41 μM. FAF human serum albumin binds each lysophospholipid with comparable affinities. By measuring the apoM concentration and expanding the model to include endogenous ligand, we were able to resolve the Kd values for S1P binding apoM in the context of human HDL and LDL particles (21 nM and 2.4 nM, respectively). The novel competitive assay and analysis described herein enables measurement of Kd values of completely unmodified lysophospholipids binding unmodified carrier proteins in solution, and thus provide insights into S1P and LPA storage in the circulation system and may be useful in understanding chaperone-dependent receptor activation and signaling. PMID:27444045

  4. Parallel Force Assay for Protein-Protein Interactions

    PubMed Central

    Aschenbrenner, Daniela; Pippig, Diana A.; Klamecka, Kamila; Limmer, Katja; Leonhardt, Heinrich; Gaub, Hermann E.

    2014-01-01

    Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay. PMID:25546146

  5. Proximity assays for sensitive quantification of proteins.

    PubMed

    Greenwood, Christina; Ruff, David; Kirvell, Sara; Johnson, Gemma; Dhillon, Harvinder S; Bustin, Stephen A

    2015-06-01

    Proximity assays are immunohistochemical tools that utilise two or more DNA-tagged aptamers or antibodies binding in close proximity to the same protein or protein complex. Amplification by PCR or isothermal methods and hybridisation of a labelled probe to its DNA target generates a signal that enables sensitive and robust detection of proteins, protein modifications or protein-protein interactions. Assays can be carried out in homogeneous or solid phase formats and in situ assays can visualise single protein molecules or complexes with high spatial accuracy. These properties highlight the potential of proximity assays in research, diagnostic, pharmacological and many other applications that require sensitive, specific and accurate assessments of protein expression. PMID:27077033

  6. Proximity assays for sensitive quantification of proteins

    PubMed Central

    Greenwood, Christina; Ruff, David; Kirvell, Sara; Johnson, Gemma; Dhillon, Harvinder S.; Bustin, Stephen A.

    2015-01-01

    Proximity assays are immunohistochemical tools that utilise two or more DNA-tagged aptamers or antibodies binding in close proximity to the same protein or protein complex. Amplification by PCR or isothermal methods and hybridisation of a labelled probe to its DNA target generates a signal that enables sensitive and robust detection of proteins, protein modifications or protein–protein interactions. Assays can be carried out in homogeneous or solid phase formats and in situ assays can visualise single protein molecules or complexes with high spatial accuracy. These properties highlight the potential of proximity assays in research, diagnostic, pharmacological and many other applications that require sensitive, specific and accurate assessments of protein expression. PMID:27077033

  7. Bicinchoninic acid (BCA) assay in low volume.

    PubMed

    Bainor, Anthony; Chang, Lyra; McQuade, Thomas J; Webb, Brian; Gestwicki, Jason E

    2011-03-15

    The BCA assay is a colorimetric method for estimating protein concentration. In 96-well plates, the relationship between protein content and absorbance is nearly linear over a wide range; however, performance is reduced in lower volume. To overcome this limitation, we performed the BCA assays in opaque, white 384-well plates. These plates emit fluorescence between 450-600 nm when excited at 430 nm; thus, their fluorescence is quenched by the BCA chromophore (λ(max) 562 nm). This arrangement allowed accurate determination of protein content using only 2 μL of sample. Moreover, soluble flourescein could replace the white plates, creating a homogenous format. PMID:21078286

  8. Protein immobilization techniques for microfluidic assays

    PubMed Central

    Kim, Dohyun; Herr, Amy E.

    2013-01-01

    Microfluidic systems have shown unequivocal performance improvements over conventional bench-top assays across a range of performance metrics. For example, specific advances have been made in reagent consumption, throughput, integration of multiple assay steps, assay automation, and multiplexing capability. For heterogeneous systems, controlled immobilization of reactants is essential for reliable, sensitive detection of analytes. In most cases, protein immobilization densities are maximized, while native activity and conformation are maintained. Immobilization methods and chemistries vary significantly depending on immobilization surface, protein properties, and specific assay goals. In this review, we present trade-offs considerations for common immobilization surface materials. We overview immobilization methods and chemistries, and discuss studies exemplar of key approaches—here with a specific emphasis on immunoassays and enzymatic reactors. Recent “smart immobilization” methods including the use of light, electrochemical, thermal, and chemical stimuli to attach and detach proteins on demand with precise spatial control are highlighted. Spatially encoded protein immobilization using DNA hybridization for multiplexed assays and reversible protein immobilization surfaces for repeatable assay are introduced as immobilization methods. We also describe multifunctional surface coatings that can perform tasks that were, until recently, relegated to multiple functional coatings. We consider the microfluidics literature from 1997 to present and close with a perspective on future approaches to protein immobilization. PMID:24003344

  9. Proteins and Amino Acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the most abundant substances in living organisms and cells. All proteins are constructed from the same twenty amino acids that are linked together by covalent bonds. Shorter chains of two or more amino acids can be linked by covalent bonds to form polypeptides. There are twenty amino...

  10. Protein subcellular localization assays using split fluorescent proteins

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2009-09-08

    The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

  11. Identification of DNA-binding proteins that interact with the 5'-flanking region of the human D-amino acid oxidase gene by pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry.

    PubMed

    Tran, Diem Hong; Shishido, Yuji; Chung, Seong Pil; Trinh, Huong Thi Thanh; Yorita, Kazuko; Sakai, Takashi; Fukui, Kiyoshi

    2015-12-10

    D-Amino acid oxidase (DAO) is a flavoenzyme that metabolizes D-amino acids and is expected to be a promising therapeutic target of schizophrenia and glioblastoma. The study of DNA-binding proteins has yielded much information in the regulation of transcription and other biological processes. However, proteins interacting with DAO gene have not been elucidated. Our assessment of human DAO promoter activity using luciferase reporter system indicated the 5'-flanking region of this gene (-4289 bp from transcription initiation site) has a regulatory sequence for gene expression, which is regulated by multi-protein complexes interacting with this region. By using pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry, we identified six proteins binding to the 5'-flanking region of the human DAO gene (zinc finger C2HC domain-containing protein 1A; histidine-tRNA ligase, cytoplasmic; molybdenum cofactor biosynthesis protein; 60S ribosomal protein L37; calponin-1; calmodulin binding protein and heterogeneous nuclear ribonucleoprotein A2/B1). These preliminary results will contribute to the advance in the understanding of the potential factors associated with the regulatory mechanism of DAO expression. PMID:25749303

  12. Digital microfluidic assay for protein detection.

    PubMed

    Mok, Janine; Mindrinos, Michael N; Davis, Ronald W; Javanmard, Mehdi

    2014-02-11

    Global studies of the human proteome have revealed a plethora of putative protein biomarkers. However, their application for early disease detection remains at a standstill without suitable methods to realize their utility in the clinical setting. There thus continues to be tremendous interest in developing new technology for sensitive protein detection that is both low in cost and carries a small footprint to be able to be used at the point of care. The current gold standard method for protein biomarker detection is the ELISA, which measures protein abundance using bulky fluorescent scanners that lack portability. Here, we present a digital microfluidic platform for protein biomarker detection that is low in cost compared with standard optical detection methods, without any compromise in sensitivity. This platform furthermore makes use of simple electronics, enabling its translation into a portable handheld device, and has been developed in a manner that can easily be adapted to assay different types of proteomic biomarkers. We demonstrate its utility in quantifying not only protein abundance, but also activity. Interleukin-6 abundance could be assayed from concentrations as low as 50 pM (an order of magnitude lower than that detectable by a comparable laboratory designed ELISA) using less than 5 μL of sample, and Abelson tyrosine kinase activity was detectable in samples containing 100 pM of kinase. PMID:24449893

  13. Competitive protein binding assay for piritrexim

    SciTech Connect

    Woolley, J.L. Jr.; Ringstad, J.L.; Sigel, C.W. )

    1989-09-01

    A competitive protein binding assay for piritrexim (PTX, 1) that makes use of a commercially available radioassay kit for methotrexate has been developed. After it is selectively extracted from plasma, PTX competes with ({sup 125}I)methotrexate for binding to dihydrofolate reductase isolated from Lactobacillus casei. Free drug is separated from bound drug by adsorption to dextran-coated charcoal. Piritrexim is measurable over a range of 0.01 to 10.0 micrograms/mL in plasma with a coefficient of variation less than 15%. The limit of sensitivity of the assay is approximately 2 ng/mL. An excellent correlation between this assay and a previously published HPLC method was found.

  14. An assay for measurement of protein adsorption to glass vials.

    PubMed

    Varmette, Elizabeth; Strony, Brianne; Haines, Daniel; Redkar, Rajendra

    2010-01-01

    Protein adsorption to primary packaging is one of the problems faced by biopharmaceutical drug companies. An assay was developed to quantify loss of proteins to glass vial surfaces. The assay involves the labeling of protein with a fluorescent dye, incubation of the labeled protein with the vial surface, elution of the adsorbed protein using a stripping buffer, and determination of fluorescence of the adsorbed protein using a fluorometer. The assay is simple to set up, accurate, sensitive, and flexible. The assay can be modified for indirect measurement of protein adsorption and offers an attractive alternative for researchers to quantify protein adsorption to glass vials and syringes. PMID:21502031

  15. The highly abundant urinary metabolite urobilin interferes with the bicinchoninic acid assay.

    PubMed

    Sampson, D L; Chng, Y L; Upton, Z; Hurst, C P; Parker, A W; Parker, T J

    2013-11-01

    Estimation of total protein concentration is an essential step in any protein- or peptide-centric analysis pipeline. This study demonstrates that urobilin, a breakdown product of heme and a major constituent of urine, interferes considerably with the bicinchoninic acid (BCA) assay. This interference is probably due to the propensity of urobilin to reduce cupric ions (Cu(2+)) to cuprous ions (Cu(1+)), thus mimicking the reduction of copper by proteins, which the assay was designed to do. In addition, it is demonstrated that the Bradford assay is more resistant to the influence of urobilin and other small molecules. As such, urobilin has a strong confounding effect on the estimate of total protein concentrations obtained by BCA assay and thus this assay should not be used for urinary protein quantification. It is recommended that the Bradford assay be used instead. PMID:23911526

  16. Evaluation of colorimetric assays for analyzing reductively methylated proteins: Biases and mechanistic insights.

    PubMed

    Brady, Pamlea N; Macnaughtan, Megan A

    2015-12-15

    Colorimetric protein assays, such as the Coomassie blue G-250 dye-binding (Bradford) and bicinchoninic acid (BCA) assays, are commonly used to quantify protein concentration. The accuracy of these assays depends on the amino acid composition. Because of the extensive use of reductive methylation in the study of proteins and the importance of biological methylation, it is necessary to evaluate the impact of lysyl methylation on the Bradford and BCA assays. Unmodified and reductively methylated proteins were analyzed using the absorbance at 280 nm to standardize the concentrations. Using model compounds, we demonstrate that the dimethylation of lysyl ε-amines does not affect the proteins' molar extinction coefficients at 280 nm. For the Bradford assay, the responses (absorbance per unit concentration) of the unmodified and reductively methylated proteins were similar, with a slight decrease in the response upon methylation. For the BCA assay, the responses of the reductively methylated proteins were consistently higher, overestimating the concentrations of the methylated proteins. The enhanced color formation in the BCA assay may be due to the lower acid dissociation constants of the lysyl ε-dimethylamines compared with the unmodified ε-amine, favoring Cu(II) binding in biuret-like complexes. The implications for the analysis of biologically methylated samples are discussed. PMID:26342307

  17. A Transgenic Mouse Assay for Agouti Protein Activity

    PubMed Central

    Perry, W. L.; Hustad, C. M.; Swing, D. A.; Jenkins, N. A.; Copeland, N. G.

    1995-01-01

    The mouse agouti gene encodes an 131 amino acid paracrine signaling molecule that instructs hair follicle melanocytes to switch from making black to yellow pigment. Expression of agouti during the middle part of the hair growth cycle in wild-type mice produces a yellow band on an otherwise black hair. The ubiquitous unregulated expression of agouti in mice carrying dominant yellow alleles is associated with pleiotropic effects including increased yellow pigment in the coat, obesity, diabetes and increased tumor susceptibility. Agouti shows no significant homology to known genes, and the molecular analysis of agouti alleles has shed little new light on the important functional elements of the agouti protein. In this paper, we show that agouti expression driven by the human β-ACTIN promoter produces obese yellow transgenic mice and that this can be used as an assay for agouti activity. We used this assay to evaluate a point mutation associated with the a(16H) allele within the region encoding agouti's putative signal sequence and our results suggest that this mutation is sufficient to cause the a(16H) phenotype. Thus, in vitro mutagenesis followed by the generation of transgenic mice should allow us to identify important functional elements of the agouti protein. PMID:7635291

  18. Simple Bulk Readout of Digital Nucleic Acid Quantification Assays.

    PubMed

    Morinishi, Leanna S; Blainey, Paul

    2015-01-01

    Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays. We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout. Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology. PMID:26436576

  19. Ultrasensitive isolation, identification and quantification of DNA-protein adducts by ELISA-based RADAR assay.

    PubMed

    Kiianitsa, Kostantin; Maizels, Nancy

    2014-07-01

    Enzymes that form transient DNA-protein covalent complexes are targets for several potent classes of drugs used to treat infectious disease and cancer, making it important to establish robust and rapid procedures for analysis of these complexes. We report a method for isolation of DNA-protein adducts and their identification and quantification, using techniques compatible with high-throughput screening. This method is based on the RADAR assay for DNA adducts that we previously developed (Kiianitsa and Maizels (2013) A rapid and sensitive assay for DNA-protein covalent complexes in living cells. Nucleic Acids Res., 41:e104), but incorporates three key new steps of broad applicability. (i) Silica-assisted ethanol/isopropanol precipitation ensures reproducible and efficient recovery of DNA and DNA-protein adducts at low centrifugal forces, enabling cell culture and DNA precipitation to be carried out in a single microtiter plate. (ii) Rigorous purification of DNA-protein adducts by a procedure that eliminates free proteins and free nucleic acids, generating samples suitable for detection of novel protein adducts (e.g. by mass spectroscopy). (iii) Identification and quantification of DNA-protein adducts by direct ELISA assay. The ELISA-based RADAR assay can detect Top1-DNA and Top2a-DNA adducts in human cells, and gyrase-DNA adducts in Escherichia coli. This approach will be useful for discovery and characterization of new drugs to treat infectious disease and cancer, and for development of companion diagnostics assays for individualized medicine. PMID:24914050

  20. The Exchangeability of Amino Acids in Proteins

    PubMed Central

    Yampolsky, Lev Y.; Stoltzfus, Arlin

    2005-01-01

    The comparative analysis of protein sequences depends crucially on measures of amino acid similarity or distance. Many such measures exist, yet it is not known how well these measures reflect the operational exchangeability of amino acids in proteins, since most are derived by methods that confound a variety of effects, including effects of mutation. In pursuit of a pure measure of exchangeability, we present (1) a compilation of data on the effects of 9671 amino acid exchanges engineered and assayed in a set of 12 proteins; (2) a statistical procedure to combine results from diverse assays of exchange effects; (3) a matrix of “experimental exchangeability” values EXij derived from applying this procedure to the compiled data; and (4) a set of three tests designed to evaluate the power of an exchangeability measure to (i) predict the effects of amino acid exchanges in the laboratory, (ii) account for the disease-causing potential of missense mutations in the human population, and (iii) model the probability of fixation of missense mutations in evolution. EX not only captures useful information on exchangeability while remaining free of other effects, but also outperforms all measures tested except for the best-performing alignment scoring matrix, which is comparable in performance. PMID:15944362

  1. Analyzing Protein-Phosphoinositide Interactions with Liposome Flotation Assays.

    PubMed

    Busse, Ricarda A; Scacioc, Andreea; Schalk, Amanda M; Krick, Roswitha; Thumm, Michael; Kühnel, Karin

    2016-01-01

    Liposome flotation assays are a convenient tool to study protein-phosphoinositide interactions. Working with liposomes resembles physiological conditions more than protein-lipid overlay assays, which makes this method less prone to detect false positive interactions. However, liposome lipid composition must be well-considered in order to prevent nonspecific binding of the protein through electrostatic interactions with negatively charged lipids like phosphatidylserine. In this protocol we use the PROPPIN Hsv2 (homologous with swollen vacuole phenotype 2) as an example to demonstrate the influence of liposome lipid composition on binding and show how phosphoinositide binding specificities of a protein can be characterized with this method. PMID:26552682

  2. The clinical utility of mass spectrometry based protein assays.

    PubMed

    Lassman, Michael E; McAvoy, Thomas; Chappell, Derek L; Lee, Anita Y; Zhao, Xuemei X; Laterza, Omar F

    2016-08-01

    Reports of mass spectrometry based assays for peptides and proteins have become increasingly common in the literature. The growing interest of mass spectrometry for use in clinical laboratories has been primarily driven by the inherent selectivity of the platform relative to more traditional platforms such as immunoassays. However, the adoption of mass spectrometry for peptide and protein analysis in the clinic has been relatively slow compared its adoption in non-clinical laboratories such as in biomarker discovery efforts or within laboratories that support pharmaceutical and academic research. Here, we review some of the successful reports of MS based assays for human proteins in multiple stages of assay research, and describe how and why the platform was employed in order to demonstrate where and when mass spectrometry based assays will have value in the future. PMID:27259466

  3. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Enterovirus nucleic acid assay. 866.3225 Section 866.3225 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3225...

  4. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Enterovirus nucleic acid assay. 866.3225 Section 866.3225 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3225...

  5. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Enterovirus nucleic acid assay. 866.3225 Section 866.3225 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3225...

  6. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Enterovirus nucleic acid assay. 866.3225 Section 866.3225 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3225...

  7. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Enterovirus nucleic acid assay. 866.3225 Section 866.3225 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3225...

  8. Optimization of protein samples for NMR using thermal shift assays.

    PubMed

    Kozak, Sandra; Lercher, Lukas; Karanth, Megha N; Meijers, Rob; Carlomagno, Teresa; Boivin, Stephane

    2016-04-01

    Maintaining a stable fold for recombinant proteins is challenging, especially when working with highly purified and concentrated samples at temperatures >20 °C. Therefore, it is worthwhile to screen for different buffer components that can stabilize protein samples. Thermal shift assays or ThermoFluor(®) provide a high-throughput screening method to assess the thermal stability of a sample under several conditions simultaneously. Here, we describe a thermal shift assay that is designed to optimize conditions for nuclear magnetic resonance studies, which typically require stable samples at high concentration and ambient (or higher) temperature. We demonstrate that for two challenging proteins, the multicomponent screen helped to identify ingredients that increased protein stability, leading to clear improvements in the quality of the spectra. Thermal shift assays provide an economic and time-efficient method to find optimal conditions for NMR structural studies. PMID:26984476

  9. Digestion Assays in Allergenicity Assessment of Transgenic Proteins

    PubMed Central

    Herman, Rod A.; Storer, Nicholas P.; Gao, Yong

    2006-01-01

    The food-allergy risk assessment for transgenic proteins expressed in crops is currently based on a weight-of-evidence approach that holistically considers multiple lines of evidence. This approach recognizes that no single test or property is known to distinguish allergens from nonallergens. The stability of a protein to digestion, as predicted by an in vitro simulated gastric fluid assay, currently is used as one element in the risk assessment process. A review of the literature on the use of the simulated gastric fluid assay to predict the allergenic status of proteins suggests that more extensive kinetic studies with well-characterized reference proteins are required before the predictive value of this assay can be adequately judged. PMID:16882518

  10. Gel mobility shift assays to detect protein-RNA interactions.

    PubMed

    Yakhnin, Alexander V; Yakhnin, Helen; Babitzke, Paul

    2012-01-01

    The gel mobility shift assay is a powerful technique for detecting and quantifying protein-RNA interactions. While other techniques such as filter binding and isothermal titration calorimetry (ITC) are available for quantifying protein-RNA interactions, gel shift analysis provides the added advantage that you can visualize the protein-RNA complexes. In the gel shift assay, protein-RNA complexes are typically separated from the unbound RNA using native polyacrylamide gels in Tris/borate/EDTA buffer, although an alternative Tris-glycine buffering system is superior in many situations. Here, we describe both gel shift methods, along with strategies to improve separation of protein-RNA complexes from free RNA, which can be a particular challenge for small RNA binding proteins. PMID:22736005

  11. Real-time quantification of fatty acid uptake using a novel fluorescence assay.

    PubMed

    Liao, Jinfang; Sportsman, Richard; Harris, Jeff; Stahl, Andreas

    2005-03-01

    Uptake of nonesterified long-chain fatty acids (LCFAs) into many cell types and organs such as liver, heart, intestine, and skeletal muscle occurs primarily through a saturable, protein-mediated mechanism. Membrane proteins that increase the uptake of LCFAs, such as FAT/CD36 and fatty acid transport proteins, represent significant therapeutic targets for the treatment of metabolic disorders, including type 2 diabetes. However, currently available methods for the quantification of LCFA uptake neither allow for real-time measurements of uptake kinetics nor are ideally suited for the development of LCFA uptake inhibitors in high-throughput screens. To address both problems, we developed a LCFA uptake assay using a fluorescently labeled fatty acid and a nontoxic cell-impermeable quenching agent that allows fatty acid transport to be measured in real time using fluorescence plate readers or standard fluorescence microscopy. With this assay, we faithfully reproduced known differentiation- and hormone-induced changes in LCFA uptake by 3T3-L1 cells and determined LCFA uptake kinetics with previously unobtainable temporal resolution. Applications of this novel assay should facilitate new insights into the biology of fatty acid uptake and provide new means for obesity-related drug discovery. PMID:15547301

  12. Direct dye binding--a quantitative assay for solid-phase immobilized protein.

    PubMed

    Bonde, M; Pontoppidan, H; Pepper, D S

    1992-01-01

    A direct dye-binding procedure was established for the quantification of protein after its immobilization on a solid phase, using IgG and BSA as model proteins. The assay, which in the range 0-5 mg protein/ml gel correlates well with indirect protein determination by A280 as well as determination of protein hydrolyzed from the gel, is based on a modified Bradford dye-binding assay. As the protein coupled to the gel binds the dye, a decrease in A465 of the supernatant is measured. Three solid supports commonly used for protein immobilization (Sepharose, Sephadex, Sephacryl) were found to be compatible with the dye-binding assay while nonspecific dye binding was found to HEMA gels. Protein was coupled to Sephacryl S-1000 using three different activation methods (aldehyde, hydrazine, and adipic acid dihydrazide). Artifactual dye-binding was not observed using any of the three different "linkers." The assay is easily carried out and represents a useful tool, e.g., when optimizing procedures for protein immobilization. PMID:1595895

  13. Bioanalytical advances in assays for C-reactive protein.

    PubMed

    Vashist, Sandeep Kumar; Venkatesh, A G; Marion Schneider, E; Beaudoin, Christopher; Luppa, Peter B; Luong, John H T

    2016-01-01

    This review presents advances in assays for human C-reactive protein (CRP), the most important biomarker of infection and inflammation for a plethora of diseases and pathophysiological conditions. Routine assays in clinical settings are based on analyzers, enzyme-linked immunosorbent assays and lateral flow assays. However, assays encompassing novel sensing schemes, improved chemistry, signal enhancement, lab-on-a-chip, microfluidics and smartphone detection, have emerged in recent years. The incorporation of immune-transducing chips or sensing interfaces with nanomaterials enables multiplexing analysis of CRP with co-existing biomarkers. However, there are still considerable challenges in the development of rapid diagnostics for both pentameric and monomeric CRP forms. PMID:26717866

  14. Quantification Assays for Total and Polyglutamine-Expanded Huntingtin Proteins

    PubMed Central

    Boogaard, Ivette; Smith, Melanie; Pulli, Kristiina; Szynol, Agnieszka; Albertus, Faywell; Lamers, Marieke B. A. C.; Dijkstra, Sipke; Kordt, Daniel; Reindl, Wolfgang; Herrmann, Frank; McAllister, George; Fischer, David F.; Munoz-Sanjuan, Ignacio

    2014-01-01

    The expansion of a CAG trinucleotide repeat in the huntingtin gene, which produces huntingtin protein with an expanded polyglutamine tract, is the cause of Huntington's disease (HD). Recent studies have reported that RNAi suppression of polyglutamine-expanded huntingtin (mutant HTT) in HD animal models can ameliorate disease phenotypes. A key requirement for such preclinical studies, as well as eventual clinical trials, aimed to reduce mutant HTT exposure is a robust method to measure HTT protein levels in select tissues. We have developed several sensitive and selective assays that measure either total human HTT or polyglutamine-expanded human HTT proteins on the electrochemiluminescence Meso Scale Discovery detection platform with an increased dynamic range over other methods. In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions. We demonstrate the application of these assays to measure HTT protein in several HD in vitro cellular and in vivo animal model systems as well as in HD patient biosamples. Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples. PMID:24816435

  15. A Luciferase Reporter Gene Assay to Measure Ebola Virus Viral Protein 35-Associated Inhibition of Double-Stranded RNA-Stimulated, Retinoic Acid-Inducible Gene 1-Mediated Induction of Interferon β.

    PubMed

    Cannas, Valeria; Daino, Gian Luca; Corona, Angela; Esposito, Francesca; Tramontano, Enzo

    2015-10-01

    During Ebola virus (EBOV) infection, the type I interferon α/β (IFN-α/β) innate immune response is suppressed by EBOV viral protein 35 (VP35), a validated drug target. Identification of EBOV VP35 inhibitors requires a cellular system able to assess the VP35-based inhibitory functions of viral double-stranded RNA (dsRNA) IFN-β induction. We established a miniaturized luciferase gene reporter assay in A549 cells that measures IFN-β induction by viral dsRNA and is dose-dependently inhibited by VP35 expression. When compared to influenza A virus NS1 protein, EBOV VP35 showed improved inhibition of viral dsRNA-based IFN-β induction. This assay can be used to screen for EBOV VP35 inhibitors. PMID:25926684

  16. A potent and highly selective peptide substrate for protein kinase C assay.

    PubMed Central

    Toomik, R; Ek, P

    1997-01-01

    Protein kinases exhibit substrate specificities that are often primarily determined by the amino acids around the phosphorylation sites. Peptides corresponding to protein kinase C phosphorylation sites in several different proteins were synthesized on SPOTs membrane which has recently been found to be applicable for studies of protein kinase specificity. After phosphorylation with protein kinase C, we chose the best phosphorylated peptides for the investigation of the importance of amino acids immediately adjacent to the phosphorylation site. The selectivity of the best protein kinase C substrates from this study was analysed with protein kinases A, CK1 and CK2. According to these tests, the most favourable characteristics of SPOTs-membrane-associated peptides were demonstrated by peptide KRAKRKTAKKR. Kinetic analysis of peptide phosphorylation with protein kinase C revealed an apparent Km of 0.49 +/- 0.13 microM and Vmax of 10.0 +/- 0.5 nmol/min per mg with soluble peptide KRAKRKTAKKR. In addition, we assayed several other soluble peptides commonly used as protein kinase C substrates. Peptide KRAKRKTAKKR showed the lowest Km and the highest Vmax/Km value in comparison with peptides FKKSFKL, pEKRPSQRSKYL and KRAKRKTTKKR. Furthermore, of the peptides tested, KRAKRKTAKKR was the most selective substrate for protein kinase C. The favourable kinetic parameters combined with the selectivity should make the KRAKRKTAKKR peptide useful as a substrate for protein kinase C in the assays of both purified enzyme and in crude cell extracts. PMID:9065763

  17. Fluorimetric screening assay for protein carbonyl evaluation in biological samples.

    PubMed

    Stocker, P; Ricquebourg, E; Vidal, N; Villard, C; Lafitte, D; Sellami, L; Pietri, S

    2015-08-01

    Many assays are available for the detection of protein carbonyls (PCs). Currently, the measurement of PC groups after their derivatization with 2,4-dinitrophenol hydrazine (DNPH) is widely used for measuring protein oxidation in biological samples. However, this method includes several washing steps. In this context, we have developed a rapid, sensitive, and accurate fluorimetric method adapted to 96-well microplates for the convenient assessment of protein carbonyl level in biological samples. The method reported here is based on the reaction of carbonyl content in proteins with 7-hydrazino-4-nitrobenzo-2,1,3-oxadiazole (NBDH) to form highly fluorescent derivatives via hydrazone formation. PCs were determined using the DNPH and NBDH assays in fully reduced bovine serum albumin (BSA) and plasma and liver homogenates obtained from healthy control rats up the addition of various amounts of HOCl-oxidized BSA (OxBSA). Using the NBDH assay, PC concentrations as low as 0.2 nmol/mg were detected with precision as low as 5%. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy was used to successfully identify the formation of the NBDH adducts after derivatization with standard oxidized peptides. Finally, the two methods were further used for PC determination in plasma and liver samples from diabetic and normal rats, showing that the NBDH assay can be reliably used in biological experiments. PMID:25933703

  18. Real-time Measurements of Amino Acid and Protein Hydroperoxides Using Coumarin Boronic Acid*

    PubMed Central

    Michalski, Radoslaw; Zielonka, Jacek; Gapys, Ewa; Marcinek, Andrzej; Joseph, Joy; Kalyanaraman, Balaraman

    2014-01-01

    Hydroperoxides of amino acid and amino acid residues (tyrosine, cysteine, tryptophan, and histidine) in proteins are formed during oxidative modification induced by reactive oxygen species. Amino acid hydroperoxides are unstable intermediates that can further propagate oxidative damage in proteins. The existing assays (oxidation of ferrous cation and iodometric assays) cannot be used in real-time measurements. In this study, we show that the profluorescent coumarin boronic acid (CBA) probe reacts with amino acid and protein hydroperoxides to form the corresponding fluorescent product, 7-hydroxycoumarin. 7-Hydroxycoumarin formation was catalase-independent. Based on this observation, we have developed a fluorometric, real-time assay that is adapted to a multiwell plate format. This is the first report showing real-time monitoring of amino acid and protein hydroperoxides using the CBA-based assay. This approach was used to detect protein hydroperoxides in cell lysates obtained from macrophages exposed to visible light and photosensitizer (rose bengal). We also measured the rate constants for the reaction between amino acid hydroperoxides (tyrosyl, tryptophan, and histidine hydroperoxides) and CBA, and these values (7–23 m−1 s−1) were significantly higher than that measured for H2O2 (1.5 m−1 s−1). Using the CBA-based competition kinetics approach, the rate constants for amino acid hydroperoxides with ebselen, a glutathione peroxidase mimic, were also determined, and the values were within the range of 1.1–1.5 × 103 m−1 s−1. Both ebselen and boronates may be used as small molecule scavengers of amino acid and protein hydroperoxides. Here we also show formation of tryptophan hydroperoxide from tryptophan exposed to co-generated fluxes of nitric oxide and superoxide. This observation reveals a new mechanism for amino acid and protein hydroperoxide formation in biological systems. PMID:24928516

  19. Development of a simple assay system for protein-stabilizing efficiency based on hemoglobin protection against denaturation and measurement of the cooperative effect of mixing protein stabilizers.

    PubMed

    Chen, Siyu; Manabe, Yoshiyuki; Minamoto, Naoya; Saiki, Naoka; Fukase, Koichi

    2016-10-01

    We have elucidated the cooperative stabilization of proteins by sugars, amino acids, and other protein-stabilizing agents using a new and simple assay system. Our system determines the protein-stabilizing ability of various compounds by measuring their ability to protect hemoglobin from denaturation. Hemoglobin denaturation was readily measured by quantitative changes in its ultraviolet-visible absorption spectrum. The efficiency of our assay was confirmed using various sugars such as trehalose and sucrose that are known to be good protein stabilizers. We have also found that mixtures of two different types of protein stabilizers resulted in a cooperative stabilizing effect on protein. PMID:27253914

  20. Development of a stable isotope dilution assay for tenuazonic acid.

    PubMed

    Asam, Stefan; Liu, Yang; Konitzer, Katharina; Rychlik, Michael

    2011-04-13

    A stable isotope dilution assay (SIDA) for the Alternaria mycotoxin tenuazonic acid was developed. Therefore, [(13)C(6),(15)N]-tenuazonic acid was synthesized from [(13)C(6),(15)N]-isoleucine by Dieckmann intramolecular cyclization after acetoacetylation with diketene. The synthesized [(13)C(6),(15)N]-tenuazonic acid was used as the internal standard for determination of tenuazonic acid in tomato products by liquid chromatography tandem mass spectrometry after derivatization with 2,4-dinitrophenylhydrazine. Method validation revealed a limit of detection of 0.1 μg/kg and a limit of quantitation of 0.3 μg/kg. Recovery was close to 100% in the range of 3-300 μg/kg. Determination of tenuazonic acid in two samples of different tomato ketchups (naturally contaminated) was achieved with a coefficient of variation of 2.3% and 4.7%. Different tomato products (n = 16) were analyzed for their content of tenuazonic acid using the developed SIDA. Values were between 15 and 195 μg/kg (tomato ketchup, n = 9), 363 and 909 μg/kg (tomato paste, n = 2), and 8 and 247 μg/kg (pureed tomatoes and comparable products, n = 5). PMID:21370870

  1. Dot-blot assay for heparin-binding proteins

    SciTech Connect

    Hirose, N.; Krivanek, M.; Jackson, R.L.; Cardin, A.D.

    1986-08-01

    A method for the detection and quantitation of picomole amounts of heparin-binding proteins is described. Proteins are first spotted on nitrocellulose and then incubated with /sup 125/I-heparin. Binding of heparin to the proteins is detected by radioautography and quantitated by scanning densitometry; proteins are quantitated by densitometric analysis of the amido black stained nitrocellulose. Heparin-binding was time-dependent and sensitive to the presence of metal ions, urea, and detergents (anionic, nonionic, and zwitterionic). The divalent cations Ca/sup 2 +/ and Mg/sup 2 +/ and the zwitterionic detergent 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate increased heparin binding whereas NaCl, urea, sodium dodecylsulfate, and La3+ decreased binding. This assay is applicable to the identification and characterization of a variety of heparin-binding proteins.

  2. A step-by-step protocol for assaying protein carbonylation in biological samples.

    PubMed

    Colombo, Graziano; Clerici, Marco; Garavaglia, Maria Elisa; Giustarini, Daniela; Rossi, Ranieri; Milzani, Aldo; Dalle-Donne, Isabella

    2016-04-15

    Protein carbonylation represents the most frequent and usually irreversible oxidative modification affecting proteins. This modification is chemically stable and this feature is particularly important for storage and detection of carbonylated proteins. Many biochemical and analytical methods have been developed during the last thirty years to assay protein carbonylation. The most successful method consists on protein carbonyl (PCO) derivatization with 2,4-dinitrophenylhydrazine (DNPH) and consequent spectrophotometric assay. This assay allows a global quantification of PCO content due to the ability of DNPH to react with carbonyl giving rise to an adduct able to absorb at 366 nm. Similar approaches were also developed employing chromatographic separation, in particular HPLC, and parallel detection of absorbing adducts. Subsequently, immunological techniques, such as Western immunoblot or ELISA, have been developed leading to an increase of sensitivity in protein carbonylation detection. Currently, they are widely employed to evaluate change in total protein carbonylation and eventually to highlight the specific proteins undergoing selective oxidation. In the last decade, many mass spectrometry (MS) approaches have been developed for the identification of the carbonylated proteins and the relative amino acid residues modified to carbonyl derivatives. Although these MS methods are much more focused and detailed due to their ability to identify the amino acid residues undergoing carbonylation, they still require too expensive equipments and, therefore, are limited in distribution. In this protocol paper, we summarise and comment on the most diffuse protocols that a standard laboratory can employ to assess protein carbonylation; in particular, we describe step-by-step the different protocols, adding suggestions coming from our on-bench experience. PMID:26706659

  3. Simultaneous assay of pigments, carbohydrates, proteins and lipids in microalgae.

    PubMed

    Chen, Yimin; Vaidyanathan, Seetharaman

    2013-05-01

    Biochemical compositional analysis of microbial biomass is a useful tool that can provide insight into the behaviour of an organism and its adaptational response to changes in its environment. To some extent, it reflects the physiological and metabolic status of the organism. Conventional methods to estimate biochemical composition often employ different sample pretreatment strategies and analytical steps for analysing each major component, such as total proteins, carbohydrates, and lipids, making it labour-, time- and sample-intensive. Such analyses when carried out individually can also result in uncertainties of estimates as different pre-treatment or extraction conditions are employed for each of the component estimations and these are not necessarily standardised for the organism, resulting in observations that are not easy to compare within the experimental set-up or between laboratories. We recently reported a method to estimate total lipids in microalgae (Chen, Vaidyanathan, Anal. Chim. Acta, 724, 67-72). Here, we propose a unified method for the simultaneous estimation of the principal biological components, proteins, carbohydrates, lipids, chlorophyll and carotenoids, in a single microalgae culture sample that incorporates the earlier published lipid assay. The proposed methodology adopts an alternative strategy for pigment assay that has a high sensitivity. The unified assay is shown to conserve sample (by 79%), time (67%), chemicals (34%) and energy (58%) when compared to the corresponding assay for each component, carried out individually on different samples. The method can also be applied to other microorganisms, especially those with recalcitrant cell walls. PMID:23601278

  4. Biotoxicity assays for fruiting body lectins and other cytoplasmic proteins.

    PubMed

    Künzler, Markus; Bleuler-Martinez, Silvia; Butschi, Alex; Garbani, Mattia; Lüthy, Peter; Hengartner, Michael O; Aebi, Markus

    2010-01-01

    Recent studies suggest that a specific class of fungal lectins, commonly referred to as fruiting body lectins, play a role as effector molecules in the defense of fungi against predators and parasites. Hallmarks of these fungal lectins are their specific expression in reproductive structures, fruiting bodies, and/or sclerotia and their synthesis on free ribosomes in the cytoplasm. Fruiting body lectins are released upon damage of the fungal cell and bind to specific carbohydrate structures of predators and parasites, which leads to deterrence, inhibition of growth, and development or even killing of these organisms. Here, we describe assays to assess the toxicity of such lectins and other cytoplasmic proteins toward three different model organisms: the insect Aedes aegypti, the nematode Caenorhabditis elegans, and the amoeba Acanthamoeba castellanii. All three assays are based on heterologous expression of the examined proteins in the cytoplasm of Escherichia coli and feeding of these recombinant bacteria to omnivorous and bacterivorous organisms. PMID:20816208

  5. Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

    PubMed Central

    Abrams, Jennifer L.; Morano, Kevin A.

    2013-01-01

    Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach. PMID:23892247

  6. RNA Whole-Mount In situ Hybridisation Proximity Ligation Assay (rISH-PLA), an Assay for Detecting RNA-Protein Complexes in Intact Cells

    PubMed Central

    Roussis, Ioannis M.; Guille, Matthew; Myers, Fiona A.; Scarlett, Garry P.

    2016-01-01

    Techniques for studying RNA-protein interactions have lagged behind those for DNA-protein complexes as a consequence of the complexities associated with working with RNA. Here we present a method for the modification of the existing In Situ Hybridisation–Proximity Ligation Assay (ISH-PLA) protocol to adapt it to the study of RNA regulation (rISH-PLA). As proof of principle we used the well-characterised interaction of the Xenopus laevis Staufen RNA binding protein with Vg1 mRNA, the complex of which co-localises to the vegetal pole of Xenopus oocytes. The applicability of both the Stau1 antibody and the Locked Nucleic Acid probe (LNA) recognising Vg1 mRNA were independently validated by whole-mount Immunohistochemistry and whole-mount in situ hybridisation assays respectively prior to combining them in the rISH-PLA assay. The rISH-PLA assay allows the identification of a given RNA-protein complex at subcellular and single cell resolution, thus avoiding the lack of spatial resolution and sensitivity associated with assaying heterogenous cell populations from which conventional RNA-protein interaction detection techniques suffer. This technique will be particularly usefully for studying the activity of RNA binding proteins (RBPs) in complex mixtures of cells, for example tissue sections or whole embryos. PMID:26824753

  7. A rapid assay for affinity and kinetics of molecular interactions with nucleic acids.

    PubMed

    Donaldson, Gregory P; Roelofs, Kevin G; Luo, Yiling; Sintim, Herman O; Lee, Vincent T

    2012-04-01

    The Differential Radial Capillary Action of Ligand Assay (DRaCALA) allows detection of protein interactions with low-molecular weight ligands based on separation of the protein-ligand complex by differential capillary action. Here, we present an application of DRaCALA to the study of nucleic acid-protein interactions using the Escherichia coli cyclic AMP receptor protein (CRP). CRP bound in DRaCALA specifically to (32)P-labeled oligonucleotides containing the consensus CRP binding site, but not to oligonucleotides with point mutations known to abrogate binding. Affinity and kinetic studies using DRaCALA yielded a dissociation constant and dissociation rate similar to previously reported values. Because DRaCALA is not subject to ligand size restrictions, whole plasmids with a single CRP-binding site were used as probes, yielding similar results. DNA can also function as an easily labeled carrier molecule for a conjugated ligand. Sequestration of biotinylated nucleic acids by streptavidin allowed nucleic acids to take the place of the protein as the immobile binding partner. Therefore, any molecular interactions involving nucleic acids can be tested. We demonstrate this principle utilizing a bacterial riboswitch that binds cyclic-di-guanosine monophosphate. DRaCALA is a flexible and complementary approach to other biochemical methods for rapid and accurate measurements of affinity and kinetics at near-equilibrium conditions. PMID:22210888

  8. Cell-Binding Assays for Determining the Affinity of Protein-Protein Interactions: Technologies and Considerations.

    PubMed

    Hunter, S A; Cochran, J R

    2016-01-01

    Determining the equilibrium-binding affinity (Kd) of two interacting proteins is essential not only for the biochemical study of protein signaling and function but also for the engineering of improved protein and enzyme variants. One common technique for measuring protein-binding affinities uses flow cytometry to analyze ligand binding to proteins presented on the surface of a cell. However, cell-binding assays require specific considerations to accurately quantify the binding affinity of a protein-protein interaction. Here we will cover the basic assumptions in designing a cell-based binding assay, including the relevant equations and theory behind determining binding affinities. Further, two major considerations in measuring binding affinities-time to equilibrium and ligand depletion-will be discussed. As these conditions have the potential to greatly alter the Kd, methods through which to avoid or minimize them will be provided. We then outline detailed protocols for performing direct- and competitive-binding assays against proteins displayed on the surface of yeast or mammalian cells that can be used to derive accurate Kd values. Finally, a comparison of cell-based binding assays to other types of binding assays will be presented. PMID:27586327

  9. Comparison of amino acid digestibility of feedstuffs determined with the precision-fed cecectomized rooster assay and the standardized ileal amino acid digestibility assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to evaluate and compare amino acid digestibility of several feedstuffs using 2 commonly accepted methods: the precision-fed cecectomized rooster assay (PFR) and the standardized ileal amino acid assay (SIAAD). Six corn, 6 corn distillers dried grains with or without s...

  10. Bioluminescence regenerative cycle (BRC) system for nucleic acid quantification assays

    NASA Astrophysics Data System (ADS)

    Hassibi, Arjang; Lee, Thomas H.; Davis, Ronald W.; Pourmand, Nader

    2003-07-01

    A new label-free methodology for nucleic acid quantification has been developed where the number of pyrophosphate molecules (PPi) released during polymerization of the target nucleic acid is counted and correlated to DNA copy number. The technique uses the enzymatic complex of ATP-sulfurylase and firefly luciferase to generate photons from PPi. An enzymatic unity gain positive feedback is also implemented to regenerate the photon generation process and compensate any decay in light intensity by self regulation. Due to this positive feedback, the total number of photons generated by the bioluminescence regenerative cycle (BRC) can potentially be orders of magnitude higher than typical chemiluminescent processes. A system level kinetic model that incorporates the effects of contaminations and detector noise was used to show that the photon generation process is in fact steady and also proportional to the nucleic acid quantity. Here we show that BRC is capable of detecting quantities of DNA as low as 1 amol (10-18 mole) in 40μlit aqueous solutions, and this enzymatic assay has a controllable dynamic range of 5 orders of magnitude. The sensitivity of this technology, due to the excess number of photons generated by the regenerative cycle, is not constrained by detector performance, but rather by possible PPi or ATP (adenosine triphosphate) contamination, or background bioluminescence of the enzymatic complex.

  11. Human Protein and Amino Acid Requirements.

    PubMed

    Hoffer, L John

    2016-05-01

    Human protein and amino acid nutrition encompasses a wide, complex, frequently misunderstood, and often contentious area of clinical research and practice. This tutorial explains the basic biochemical and physiologic principles that underlie our current understanding of protein and amino acid nutrition. The following topics are discussed: (1) the identity, measurement, and essentiality of nutritional proteins; (2) the definition and determination of minimum requirements; (3) nutrition adaptation; (4) obligatory nitrogen excretion and the minimum protein requirement; (5) minimum versus optimum protein intakes; (6) metabolic responses to surfeit and deficient protein intakes; (7) body composition and protein requirements; (8) labile protein; (9) N balance; (10) the principles of protein and amino acid turnover, including an analysis of the controversial indicator amino acid oxidation technique; (11) general guidelines for evaluating protein turnover articles; (12) amino acid turnover versus clearance; (13) the protein content of hydrated amino acid solutions; (14) protein requirements in special situations, including protein-catabolic critical illness; (15) amino acid supplements and additives, including monosodium glutamate and glutamine; and (16) a perspective on the future of protein and amino acid nutrition research. In addition to providing practical information, this tutorial aims to demonstrate the importance of rigorous physiologic reasoning, stimulate intellectual curiosity, and encourage fresh ideas in this dynamic area of human nutrition. In general, references are provided only for topics that are not well covered in modern textbooks. PMID:26796095

  12. Visualizing repetitive diffusion activity of double-strand RNA binding proteins by single molecule fluorescence assays.

    PubMed

    Koh, Hye Ran; Wang, Xinlei; Myong, Sua

    2016-08-01

    TRBP, one of double strand RNA binding proteins (dsRBPs), is an essential cofactor of Dicer in the RNA interference pathway. Previously we reported that TRBP exhibits repetitive diffusion activity on double strand (ds)RNA in an ATP independent manner. In the TRBP-Dicer complex, the diffusion mobility of TRBP facilitates Dicer-mediated RNA cleavage. Such repetitive diffusion of dsRBPs on a nucleic acid at the nanometer scale can be appropriately captured by several single molecule detection techniques. Here, we provide a step-by-step guide to four different single molecule fluorescence assays by which the diffusion activity of dsRBPs on dsRNA can be detected. One color assay, termed protein induced fluorescence enhancement enables detection of unlabeled protein binding and diffusion on a singly labeled RNA. Two-color Fluorescence Resonance Energy Transfer (FRET) in which labeled dsRBPs is applied to labeled RNA, allows for probing the motion of protein along the RNA axis. Three color FRET reports on the diffusion movement of dsRBPs from one to the other end of RNA. The single molecule pull down assay provides an opportunity to collect dsRBPs from mammalian cells and examine the protein-RNA interaction at single molecule platform. PMID:27012177

  13. Multicenter evaluation of the Verigene Clostridium difficile nucleic acid assay.

    PubMed

    Carroll, Karen C; Buchan, Blake W; Tan, Sokha; Stamper, Paul D; Riebe, Katherine M; Pancholi, Preeti; Kelly, Cheryl; Rao, Arundhati; Fader, Robert; Cavagnolo, Robert; Watson, Wendy; Goering, Richard V; Trevino, Ernest A; Weissfeld, Alice S; Ledeboer, Nathan A

    2013-12-01

    The Verigene Clostridium difficile Nucleic Acid test (Verigene CDF test) (Nanosphere, Northbrook, IL) is a multiplex qualitative PCR assay that utilizes a nanoparticle-based array hybridization method to detect C. difficile tcdA and tcdB in fecal specimens. In addition, the assay detects binary toxin gene sequences and the single base pair deletion at nucleotide 117 (Δ 117) in tcdC to provide a presumptive identification of the epidemic strain 027/NAP1/BI (referred to here as ribotype 027). This study compared the Verigene CDF test with anaerobic direct and enriched toxigenic culture on stool specimens from symptomatic patients among five geographically diverse laboratories within the United States. The Verigene CDF test was performed according to the manufacturer's instructions, and the reference methods performed by a central laboratory included direct culture onto cycloserine cefoxitin fructose agar (CCFA) and enriched culture using cycloserine cefoxitin mannitol broth with taurocholate and lysozyme. Recovered isolates were identified as C. difficile using gas liquid chromatography and were tested for toxin using a cell culture cytotoxicity neutralization assay. Strains belonging to ribotype 027 were determined by PCR ribotyping and bidirectional sequencing for Δ 117 in tcdC. A total of 1,875 specimens were evaluable. Of these, 275 specimens (14.7%) were culture positive by either direct or enriched culture methods. Compared to direct culture alone, the overall sensitivity, specificity, positive predictive value, and negative predictive value for the Verigene CDF test were 98.7%, 87.5%, 42%, and 99.9%, respectively. Compared to combined direct and enriched culture results, the sensitivity, specificity, positive predictive value, and negative predictive values of the Verigene CDF test were 90.9%, 92.5%, 67.6%, and 98.3%, respectively. Of the 250 concordantly culture-positive specimens, 59 (23.6%) were flagged as "hypervirulent"; 53 were confirmed as ribotype

  14. Multicenter Evaluation of the Verigene Clostridium difficile Nucleic Acid Assay

    PubMed Central

    Buchan, Blake W.; Tan, Sokha; Stamper, Paul D.; Riebe, Katherine M.; Pancholi, Preeti; Kelly, Cheryl; Rao, Arundhati; Fader, Robert; Cavagnolo, Robert; Watson, Wendy; Goering, Richard V.; Trevino, Ernest A.; Weissfeld, Alice S.; Ledeboer, Nathan A.

    2013-01-01

    The Verigene Clostridium difficile Nucleic Acid test (Verigene CDF test) (Nanosphere, Northbrook, IL) is a multiplex qualitative PCR assay that utilizes a nanoparticle-based array hybridization method to detect C. difficile tcdA and tcdB in fecal specimens. In addition, the assay detects binary toxin gene sequences and the single base pair deletion at nucleotide 117 (Δ 117) in tcdC to provide a presumptive identification of the epidemic strain 027/NAP1/BI (referred to here as ribotype 027). This study compared the Verigene CDF test with anaerobic direct and enriched toxigenic culture on stool specimens from symptomatic patients among five geographically diverse laboratories within the United States. The Verigene CDF test was performed according to the manufacturer's instructions, and the reference methods performed by a central laboratory included direct culture onto cycloserine cefoxitin fructose agar (CCFA) and enriched culture using cycloserine cefoxitin mannitol broth with taurocholate and lysozyme. Recovered isolates were identified as C. difficile using gas liquid chromatography and were tested for toxin using a cell culture cytotoxicity neutralization assay. Strains belonging to ribotype 027 were determined by PCR ribotyping and bidirectional sequencing for Δ 117 in tcdC. A total of 1,875 specimens were evaluable. Of these, 275 specimens (14.7%) were culture positive by either direct or enriched culture methods. Compared to direct culture alone, the overall sensitivity, specificity, positive predictive value, and negative predictive value for the Verigene CDF test were 98.7%, 87.5%, 42%, and 99.9%, respectively. Compared to combined direct and enriched culture results, the sensitivity, specificity, positive predictive value, and negative predictive values of the Verigene CDF test were 90.9%, 92.5%, 67.6%, and 98.3%, respectively. Of the 250 concordantly culture-positive specimens, 59 (23.6%) were flagged as “hypervirulent”; 53 were confirmed as

  15. Determination of protein levels in soy and peanut oils by colorimetric assay and ELISA.

    PubMed

    Jablonski, Joseph E; Fu, Tong-Jen; Jackson, Lauren S; Gendel, Steven M

    2010-01-01

    Analytical methods are needed for measuring the levels of protein from allergenic food transferred into cooking oil. A simple method for determination of total protein in cooking oils was developed. Oil was extracted with phosphate-buffered saline with 0.05% Tween (PBST) and the extracts were partitioned with hexane to remove residual oil. Total protein in the PBST extracts was assayed with bicinchoninic acid (BCA), micro-BCA, reducing-agent compatible BCA and CB-XT kits. These methods were used to measure recovery of protein from peanut butter spikes of soy and peanut oil in the range of 50-1000 ppm. Recoveries were generally above 70%. However, the BCA and micro-BCA assays were subject to interference and enhanced color formation which were probably due to co-extracted antioxidants present in oil. The reducing agent-compatible BCA and CB-X protein assays reduced interference and gave lower protein values in crude, cold-pressed, and refined peanut oils. Heating oil to 180 degrees C before extraction also reduced interference-induced color enhancement. A commercial ELISA test kit was also used to measure peanut protein in oil spiked with peanut butter. Recovery of peanut residues measured by ELISA was significantly decreased when the peanut butter-spiked oil was heated to 180 degrees C compared to unheated oil. Recovery of spiked peanut butter protein measured by the buffer extraction-colorimetric method was not decreased in heated oil. The method developed here could be used to determine protein levels in crude and refined oil, and to assess the potential for allergen cross-contact from reused cooking oil. PMID:20334183

  16. Imaging Beads-Retained Prey Assay for Rapid and Quantitative Protein-Protein Interaction

    PubMed Central

    Zhou, Yan; Hong, Wanjin; Lu, Lei

    2013-01-01

    Conventional Western blot based pull-down methods involve lengthy and laborious work and the results are generally not quantitative. Here, we report the imaging beads-retained prey (IBRP) assay that is rapid and quantitative in studying protein-protein interactions. In this assay, the bait is immobilized onto beads and the prey is fused with a fluorescence protein. The assay takes advantage of the fluorescence of prey and directly quantifies the amount of prey binding to the immobilized bait under a microscope. We validated the assay using previously well studied interactions and found that the amount of prey retained on beads could have a relative linear relationship to both the inputs of bait and prey. IBRP assay provides a universal, fast, quantitative and economical method to study protein interactions and it could be developed to a medium- or high-throughput compatible method. With the availability of fluorescence tagged whole genome ORFs in several organisms, we predict IBRP assay should have wide applications. PMID:23555762

  17. U-2012: An improved Lowry protein assay, insensitive to sample color, offering reagent stability and enhanced sensitivity.

    PubMed

    Upreti, Girish C; Wang, Yanming; Finn, Alona; Sharrock, Abigail; Feisst, Nicholas; Davy, Marcus; Jordan, Robert B

    2012-03-01

    Traditional colorimetric protein assays such as Biuret, Lowry, and modified Lowry (U-1988) are unsuitable for colored biological samples. Here we describe an improved Lowry protein assay (U-2012), which utilizes stable reagents and offers enhanced sensitivity over the U-1988 assay. U-2012 circumvents interference from colored pigments and other substances (for example sugars) bound to perchloric acid (PCA) precipitated proteins by hydrogen peroxide (H2O2) induced oxidation at 50°C. Unused hydrogen peroxide is neutralized with sodium pyruvate before protein estimation for a stable end color. The U-2012 assay is carried out on the PCA precipitated protein pellet after neutralization (with Na2CO3 plus NaOH), solubilization (in Triton-NaCl), decolorization (by H2O2) and pyruvate treatment. Protein contents in red wine and homogenates of beetroot and blueberry are calculated from standard curves established for various proteins and generated using a rectangular hyperbola with parameters estimated with Microsoft Excel's Solver add-in. The U-2012 protein assay represents an improvement over U-1988 and gives a more accurate estimation of protein content. PMID:22401548

  18. A protein-protein binding assay using coated microtitre plates: increased throughput, reproducibility and speed compared to bead-based assays.

    PubMed

    Craig, Tim J; Ciufo, Leonora F; Morgan, Alan

    2004-07-30

    Protein-protein interactions, and the factors affecting them, are of fundamental importance to all biological systems. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITR) are powerful methods for assaying such interactions, but are expensive to implement. In contrast, bead-based pull-down assays using affinity tags such as glutathione-S-transferase (GST), require no specialist equipment. As a result, such assays are the most popular method for analysing protein-protein interactions, despite being time-consuming and prone to variability. In respect of these problems, we have modified this form of binding assay, using glutathione-coated 96-well plates rather than glutathione-Sepharose beads to bind the primary bait protein. Quantitation of bound protein utilises ELISA for purified proteins and scintillation counting for in vitro translated proteins, rather than the SDS-PAGE-based detection methods used in traditional bead-based assays. These modifications result in an approximately 10-fold increase in the number of samples that can be assayed daily, and allow results to be obtained within hours as opposed to days. We validate the modified assay by analysing the equilibrium binding of Munc18 and syntaxin, and also demonstrate that association and dissociation kinetics may be measured using this approach. The method we describe is generally applicable to any protein-protein interaction assay based on affinity tags and is amenable to automation, and so should benefit a wide range of biochemical research. PMID:15236910

  19. Staphylococcus aureus Cell Extract Transcription-Translation Assay: Firefly Luciferase Reporter System for Evaluating Protein Translation Inhibitors

    PubMed Central

    Murray, Robert W.; Melchior, Earline P.; Hagadorn, Jeanne C.; Marotti, Keith R.

    2001-01-01

    The promoter for the Staphylococcus aureus capsule polysaccharide synthesis gene (cap1A) was cloned in front of the firefly luciferase gene for use in a cell extract S. aureus transcription-translation system. The assay is rapid, reproducible, and sensitive and has a lower background level than the radiolabeled amino acid incorporation translation assays. We present data evaluating a transcription inhibitor and a number of protein translation inhibitors in this system. PMID:11353649

  20. An instantaneous colorimetric protein assay based on spontaneous formation of a protein corona on gold nanoparticles.

    PubMed

    Ho, Yan Teck; Poinard, Barbara; Yeo, Eugenia Li Ling; Kah, James Chen Yong

    2015-02-21

    Commercial protein assays used ubiquitously in laboratories typically require long incubation times due to the inherently slow protein-reagent reactions. In this study, we report a novel facile technique for the instantaneous measurement of total protein concentration by exploiting the rapid aggregation dynamics of gold nanoparticles (NPs). By adsorbing different amounts of proteins on their surface to form a protein corona, these NPs can be sterically stabilized to different degrees by aggregation, thus exhibiting a spectrum of color change which can be quantitatively characterized by UV-Vis absorption spectroscopy. We evaluated this technique on four model proteins with different structures: bovine serum albumin (BSA), normal mouse immunoglobulin G (IgG), fibrinogen (FBG) and apolipoprotein A-I (Apo-A1) using two approaches, sequential and simultaneous. We obtained an approach-dependent linear concentration range up to 80 μg mL(-1) and 400 μg mL(-1) for sequential and simultaneous approaches, respectively. This linear working range was wider than that of the commercial Bradford assay and comparable to the Micro BCA assay. The simultaneous approach was also able to produce a linear working range of 200 to 1000 μg mL(-1) (R(2) = 0.995) in human urine, while the sequential approach was non-functional in urine. Similar to Micro BCA, the NP-based protein assay was able to elicit a linear response (R(2) > 0.87) for all four proteins with different structures. However, unlike Micro BCA which requires up to 120 min of incubation, we were able to obtain the read-out almost instantaneously without the need for incubation. The NP-based technique using the simultaneous approach can thus be exploited as a novel assay for instantaneous protein quantification to increase the productivity of laboratory processes. PMID:25501998

  1. Myoglobin microplate assay to evaluate prevention of protein peroxidation.

    PubMed

    Marques, Sara S; Magalhães, Luís M; Mota, Ana I P; Soares, Tânia R P; Korsak, Barbara; Reis, Salette; Segundo, Marcela A

    2015-10-10

    The current therapeutic strategies are based on the design of multifunctional drug candidates able to interact with various disease related targets. Drugs that have the ability to scavenge reactive oxygen species (ROS), beyond their main therapeutic action, may prevent the oxidative damage of biomolecules. Therefore, analytical approaches that monitor in a continuous mode the ability of drugs to counteract peroxidation of physiologically relevant biotargets are required. In the present work, a microplate spectrophotometric assay is proposed to evaluate the ability of selected cardiovascular drugs, including angiotensin-converting enzyme (ACE) inhibitors, β -blockers and statins to prevent protein peroxidation. Myoglobin, which is a heme protein, and peroxyl radicals generated from thermolysis of 2,2'-azo-bis(2-amidinopropane) dihydrochloride at 37 °C, pH 7.4 were selected as protein model and oxidative species, respectively. Myoglobin peroxidation was continuously monitored by the absorbance decrease at 409 nm and the ability of drugs to counteract protein oxidation was determined by the calculation of the area under the curve upon the myoglobin oxidation. Fluvastatin (AUC₅₀=12.5 ± 1.2 μM) and enalapril (AUC₅₀=15.2 ± 1.8 μM) showed high ability to prevent myoglobin peroxidation, providing even better efficiency than endogenous antioxidants such as reduced glutathione. Moreover, labetalol, enalapril and fluvastatin prevent the autoxidation of myoglobin, while glutathione showed a pro-oxidant effect. PMID:26093510

  2. Identification of Cellular Proteins that Interact with Human Cytomegalovirus Immediate-Early Protein 1 by Protein Array Assay

    PubMed Central

    Puerta Martínez, Francisco; Tang, Qiyi

    2013-01-01

    Human cytomegalovirus (HCMV) gene expression during infection is characterized as a sequential process including immediate-early (IE), early (E), and late (L)-stage gene expression. The most abundantly expressed gene at the IE stage of infection is the major IE (MIE) gene that produces IE1 and IE2. IE1 has been the focus of study because it is an important protein, not only for viral gene expression but also for viral replication. It is believed that IE1 plays important roles in viral gene regulation by interacting with cellular proteins. In the current study, we performed protein array assays and identified 83 cellular proteins that interact with IE1. Among them, seven are RNA-binding proteins that are important in RNA processing; more than half are nuclear proteins that are involved in gene regulations. Tumorigenesis-related proteins are also found to interact with IE1, implying that the role of IE1 in tumorigenesis might need to be reevaluated. Unexpectedly, cytoplasmic proteins, such as Golgi autoantigen and GGA1 (both related to the Golgi trafficking protein), are also found to be associated with IE1. We also employed a coimmunoprecipitation assay to test the interactions of IE1 and some of the proteins identified in the protein array assays and confirmed that the results from the protein array assays are reliable. Many of the proteins identified by the protein array assay have not been previously reported. Therefore, the functions of the IE1-protein interactions need to be further explored in the future. PMID:24385082

  3. A Protein-Protein Interaction Assay FlimPIA Based on the Functional Complementation of Mutant Firefly Luciferases.

    PubMed

    Ohmuro-Matsuyama, Yuki; Ueda, Hiroshi

    2016-01-01

    There is a significant focus on detecting and assaying protein-protein interactions (PPIs) in biology and biotechnology. Protein-fragment complementation assay (PCA) is one of the most widely used methods to detect PPI by splitting the enzyme-coding or fluorescent protein-coding polypeptide, as well as Förster resonance energy transfer (FRET). Here, we describe a novel PPI assay FlimPIA (firefly luminescent intermediate-based protein-protein interaction assay) by a unique approach of splitting the two major catalytic steps (half reactions) of firefly luciferase (FLuc). PMID:27424900

  4. Surface plasmon resonance assay of inhibition by pharmaceuticals for thyroxine hormone binging to transport proteins.

    PubMed

    Kinouchi, Hiroki; Matsuyama, Keigo; Kitagawa, Hiroshi; Kamimori, Hiroshi

    2016-01-01

    We developed a surface plasmon resonance (SPR) assay to estimate the competitive inhibition by pharmaceuticals for thyroxine (T4) binding to thyroid hormone transport proteins, transthyretin (TTR) and thyroxine binding globulin (TBG). In this SPR assay, the competitive inhibition of pharmaceuticals for introducing T4 into immobilized TTR or TBG on the sensor chip can be estimated using a running buffer containing pharmaceuticals. The SPR assay showed reproducible immobilization of TTR and TBG, and the kinetic binding parameters of T4 to TTR or TBG were estimated. The equilibrium dissociation constants of TTR or TBG measured by SPR did not clearly differ from data reported for other binding assays. To estimate the competitive inhibition of tetraiodothyroacetic acid, diclofenac, genistein, ibuprofen, carbamazepine, and furosemide, reported to be competitive or noncompetitive pharmaceuticals for T4 binding to TTR or TBG, their 50% inhibition concentrations (IC50) (or 80% inhibition concentration, IC80) were calculated from the change of T4 responses in sensorgrams obtained with various concentrations of the pharmaceuticals. Our SPR method should be a useful tool for predicting the potential of thyroid toxicity of pharmaceuticals by evaluating the competitive inhibition of T4 binding to thyroid hormone binding proteins, TTR and TBG. PMID:26384643

  5. Protein biosynthesis with conformationally restricted amino acids

    SciTech Connect

    Mendel, D. Lawrence Berkeley Lab., CA ); Ellman, J.; Schultz, P.G. )

    1993-05-19

    The incorporation of conformationally constrained amino acids into peptides is a powerful approach for generating structurally defined peptides as conformational probes and bioactive agents. The ability to site-specifically introduce constrained amino acids into large polypeptide chains would provide a similar opportunity to probe the flexibility, conformation, folding and stability of proteins. To this end, we have examined the competence of the Escherichia coli protein biosynthetic machinery to incorporate a number of these unnatural amino acids into the 164 residue protein T4 lysozyme (T4L). Results clearly demonstrate that the protein biosynthetic machinery can accommodate a wide variety of conformationally constrained amino acids. The expansion of structural motifs that can be biosynthetically incorporated into proteins to include a large number of conformationally constrained amino acids significantly increases the power of mutagenesis methods as probes of protein structure and function and provides additional insights into the steric requirements of the translational machinery. 13 refs., 2 figs.

  6. High-throughput protein analysis integrating bioinformatics and experimental assays.

    PubMed

    del Val, Coral; Mehrle, Alexander; Falkenhahn, Mechthild; Seiler, Markus; Glatting, Karl-Heinz; Poustka, Annemarie; Suhai, Sandor; Wiemann, Stefan

    2004-01-01

    The wealth of transcript information that has been made publicly available in recent years requires the development of high-throughput functional genomics and proteomics approaches for its analysis. Such approaches need suitable data integration procedures and a high level of automation in order to gain maximum benefit from the results generated. We have designed an automatic pipeline to analyse annotated open reading frames (ORFs) stemming from full-length cDNAs produced mainly by the German cDNA Consortium. The ORFs are cloned into expression vectors for use in large-scale assays such as the determination of subcellular protein localization or kinase reaction specificity. Additionally, all identified ORFs undergo exhaustive bioinformatic analysis such as similarity searches, protein domain architecture determination and prediction of physicochemical characteristics and secondary structure, using a wide variety of bioinformatic methods in combination with the most up-to-date public databases (e.g. PRINTS, BLOCKS, INTERPRO, PROSITE SWISSPROT). Data from experimental results and from the bioinformatic analysis are integrated and stored in a relational database (MS SQL-Server), which makes it possible for researchers to find answers to biological questions easily, thereby speeding up the selection of targets for further analysis. The designed pipeline constitutes a new automatic approach to obtaining and administrating relevant biological data from high-throughput investigations of cDNAs in order to systematically identify and characterize novel genes, as well as to comprehensively describe the function of the encoded proteins. PMID:14762202

  7. Distinguishing Proteins From Arbitrary Amino Acid Sequences

    PubMed Central

    Yau, Stephen S.-T.; Mao, Wei-Guang; Benson, Max; He, Rong Lucy

    2015-01-01

    What kinds of amino acid sequences could possibly be protein sequences? From all existing databases that we can find, known proteins are only a small fraction of all possible combinations of amino acids. Beginning with Sanger's first detailed determination of a protein sequence in 1952, previous studies have focused on describing the structure of existing protein sequences in order to construct the protein universe. No one, however, has developed a criteria for determining whether an arbitrary amino acid sequence can be a protein. Here we show that when the collection of arbitrary amino acid sequences is viewed in an appropriate geometric context, the protein sequences cluster together. This leads to a new computational test, described here, that has proved to be remarkably accurate at determining whether an arbitrary amino acid sequence can be a protein. Even more, if the results of this test indicate that the sequence can be a protein, and it is indeed a protein sequence, then its identity as a protein sequence is uniquely defined. We anticipate our computational test will be useful for those who are attempting to complete the job of discovering all proteins, or constructing the protein universe. PMID:25609314

  8. Identification and analysis of hepatitis C virus NS3 helicase inhibitors using nucleic acid binding assays

    PubMed Central

    Mukherjee, Sourav; Hanson, Alicia M.; Shadrick, William R.; Ndjomou, Jean; Sweeney, Noreena L.; Hernandez, John J.; Bartczak, Diana; Li, Kelin; Frankowski, Kevin J.; Heck, Julie A.; Arnold, Leggy A.; Schoenen, Frank J.; Frick, David N.

    2012-01-01

    Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF®; Cisbio) and AlphaScreen® (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma’s Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC50 = 1.4 μM), suramin sodium salt (IC50 = 3.6 μM), NF 023 hydrate (IC50 = 6.2 μM) and tyrphostin AG 538 (IC50 = 3.6 μM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye primuline, it revealed that some primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors. PMID:22740655

  9. Bimolecular Fluorescence Complementation (BiFC) Assay for Direct Visualization of Protein-Protein Interaction in vivo

    PubMed Central

    Lai, Hsien-Tsung; Chiang, Cheng-Ming

    2016-01-01

    Bimolecular Fluorescence Complementation (BiFC) assay is a method used to directly visualize protein-protein interaction in vivo using live-cell imaging or fixed cells. This protocol described here is based on our recent paper describing the functional association of human chromatin adaptor and transcription cofactor Brd4 with p53 tumor suppressor protein (Wu et al., 2013). BiFC was first described by Hu et al. (2002) using two non-fluorescent protein fragments of enhanced yellow fluorescent protein (EYFP), which is an Aequorea victoria GFP variant protein, fused respectively to a Rel family protein and a bZIP family transcription factor to investigate interactions between these two family members in living cells. The YFP was later improved by introducing mutations to reduce its sensitivity to pH and chloride ions, thus generating a super-enhanced YFP, named Venus fluorescent protein, without showing diminished fluorescence at 37 °C as typically observed with EYFP (Nagai et al., 2006). The fluorescence signal is regenerated by complementation of two non-fluorescent fragments (e.g., the Venus N-terminal 1–158 amino acid residues, called Venus-N, and its C-terminal 159–239 amino acid residues, named Venus-C; see Figure 1A and Gully et al., 2012; Ding et al., 2006; Kerppola, 2006) that are brought together by interaction between their respective fusion partners (e.g., Venus-N to p53, and Venus-C to the PDID domain of human Brd4; see Figure 1B and 1C). The intensity and cellular location of the regenerated fluorescence signals can be detected by fluorescence microscope. The advantages of the proximity-based BiFC assay are: first, it allows a direct visualization of spatial and temporal interaction between two partner proteins in vivo; second, the fluorescence signal provides a sensitive readout for detecting protein-protein interaction even at a low expression level comparable to that of the endogenous proteins; third, the intensity of the fluorescence signal is

  10. Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

    PubMed Central

    Filteau, Marie; Leducq, Jean-Baptiste; Dubé, Alexandre K.; Landry, Christian R.

    2015-01-01

    Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein’s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs. PMID:25867901

  11. A chromism-based assay (CHROBA) technique for in situ detection of protein kinase activity.

    PubMed

    Tomizaki, Kin-ya; Jie, Xu; Mihara, Hisakazu

    2005-03-15

    A unique chromism-based assay technique (CHROBA) using photochromic spiropyran-containing peptides has been firstly established for detection of protein kinase A-catalyzed phosphorylation. The alternative method has advantages that avoid isolation and/or immobilization of kinase substrates to remove excess reagents including nonreactive isotope-labeled ATP or fluorescently-labeled anti-phosphoamino acid antibodies from the reaction mixture. Such a novel protocol based on thermocoloration of the spiropyran moiety in the peptide can offer not only an efficient screening method of potent kinase substrates but also a versatile analytical tool for monitoring other post-translational modification activities. PMID:15745830

  12. Rapid agarose gel electrophoretic mobility shift assay for quantitating protein: RNA interactions.

    PubMed

    Ream, Jennifer A; Lewis, L Kevin; Lewis, Karen A

    2016-10-15

    Interactions between proteins and nucleic acids are frequently analyzed using electrophoretic mobility shift assays (EMSAs). This technique separates bound protein:nucleic acid complexes from free nucleic acids by electrophoresis, most commonly using polyacrylamide gels. The current study utilizes recent advances in agarose gel electrophoresis technology to develop a new EMSA protocol that is simpler and faster than traditional polyacrylamide methods. Agarose gels are normally run at low voltages (∼10 V/cm) to minimize heating and gel artifacts. In this study we demonstrate that EMSAs performed using agarose gels can be run at high voltages (≥20 V/cm) with 0.5 × TB (Tris-borate) buffer, allowing for short run times while simultaneously yielding high band resolution. Several parameters affecting band and image quality were optimized for the procedure, including gel thickness, agarose percentage, and applied voltage. Association of the siRNA-binding protein p19 with its target RNA was investigated using the new system. The agarose gel and conventional polyacrylamide gel methods generated similar apparent binding constants in side-by-side experiments. A particular advantage of the new approach described here is that the short run times (5-10 min) reduce opportunities for dissociation of bound complexes, an important concern in non-equilibrium nucleic acid binding experiments. PMID:27495142

  13. 21 CFR 866.5910 - Quality control material for cystic fibrosis nucleic acid assays.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... Test Systems § 866.5910 Quality control material for cystic fibrosis nucleic acid assays. (a... cystic fibrosis nucleic acid assays is a device intended to help monitor reliability of a test system by... testing. This type of device includes recombinant, synthetic, and cell line-based DNA controls....

  14. 21 CFR 866.5910 - Quality control material for cystic fibrosis nucleic acid assays.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Test Systems § 866.5910 Quality control material for cystic fibrosis nucleic acid assays. (a... cystic fibrosis nucleic acid assays is a device intended to help monitor reliability of a test system by... testing. This type of device includes recombinant, synthetic, and cell line-based DNA controls....

  15. 21 CFR 866.5910 - Quality control material for cystic fibrosis nucleic acid assays.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... Test Systems § 866.5910 Quality control material for cystic fibrosis nucleic acid assays. (a... cystic fibrosis nucleic acid assays is a device intended to help monitor reliability of a test system by... testing. This type of device includes recombinant, synthetic, and cell line-based DNA controls....

  16. 21 CFR 866.5910 - Quality control material for cystic fibrosis nucleic acid assays.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Test Systems § 866.5910 Quality control material for cystic fibrosis nucleic acid assays. (a... cystic fibrosis nucleic acid assays is a device intended to help monitor reliability of a test system by... testing. This type of device includes recombinant, synthetic, and cell line-based DNA controls....

  17. 21 CFR 866.5910 - Quality control material for cystic fibrosis nucleic acid assays.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... Test Systems § 866.5910 Quality control material for cystic fibrosis nucleic acid assays. (a... cystic fibrosis nucleic acid assays is a device intended to help monitor reliability of a test system by... testing. This type of device includes recombinant, synthetic, and cell line-based DNA controls....

  18. A Fluorescent Assay for Plant Caffeic Acid O-methyltransferases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have developed a facile, sensitive and continuous assay to measure the activities of plant COMTs using s-adenosyl homocysteine hydrolase as a coupling enzyme and and adeonsine a thiol-specific fluor, Thioglo1, as the detecting reagent. This assay was validated using recombinant sorghum COMT (BMR-...

  19. Protein assay structured on paper by using lithography

    NASA Astrophysics Data System (ADS)

    Wilhelm, E.; Nargang, T. M.; Al Bitar, W.; Waterkotte, B.; Rapp, B. E.

    2015-03-01

    There are two main challenges in producing a robust, paper-based analytical device. The first one is to create a hydrophobic barrier which unlike the commonly used wax barriers does not break if the paper is bent. The second one is the creation of the (bio-)specific sensing layer. For this proteins have to be immobilized without diminishing their activity. We solve both problems using light-based fabrication methods that enable fast, efficient manufacturing of paper-based analytical devices. The first technique relies on silanization by which we create a flexible hydrophobic barrier made of dimethoxydimethylsilane. The second technique demonstrated within this paper uses photobleaching to immobilize proteins by means of maskless projection lithography. Both techniques have been tested on a classical lithography setup using printed toner masks and on a lithography system for maskless lithography. Using these setups we could demonstrate that the proposed manufacturing techniques can be carried out at low costs. The resolution of the paper-based analytical devices obtained with static masks was lower due to the lower mask resolution. Better results were obtained using advanced lithography equipment. By doing so we demonstrated, that our technique enables fabrication of effective hydrophobic boundary layers with a thickness of only 342 μm. Furthermore we showed that flourescine-5-biotin can be immobilized on the non-structured paper and be employed for the detection of streptavidinalkaline phosphatase. By carrying out this assay on a paper-based analytical device which had been structured using the silanization technique we proofed biological compatibility of the suggested patterning technique.

  20. Lactobacillus casei microbiological assay of folic acid derivatives in 96-well microtiter plates.

    PubMed

    Horne, D W; Patterson, D

    1988-11-01

    Microbiological assay is still widely used for estimating folic acid derivatives in serum and other biological samples. We describe here a modification of this procedure involving use of 96-well microtiter plates. This procedure, used with modern, computer-interfaced microtiter-plate readers and data-reduction software, greatly shortens the time and minimizes reagent costs for this assay. Under the conditions of our assay procedures, all folic acid derivatives tested gave equal growth response for Lactobacillus casei. Results for assays of rat liver extracts showed excellent agreement between the standard bioassay and the 96-well procedure. PMID:3141087

  1. A Colorimetric Microplate Assay for DNA-Binding Activity of His-Tagged MutS Protein.

    PubMed

    Banasik, Michał; Sachadyn, Paweł

    2016-09-01

    A simple microplate method was designed for rapid testing DNA-binding activity of proteins. The principle of the assay involves binding of tested DNA by his-tagged protein immobilized on a nickel-coated ELISA plate, following colorimetric detection of biotinylated DNA with avidin conjugated to horseradish peroxidase. The method was used to compare DNA mismatch binding activities of MutS proteins from three bacterial species. The assay required relatively low amounts of tested protein (approximately 0.5-10 pmol) and DNA (0.1-10 pmol) and a relatively short time of analysis (up to 60 min). The method is very simple to apply and convenient to test different buffer conditions of DNA-protein binding. Sensitive colorimetric detection enables naked eye observations and quantitation with an ELISA reader. The performance of the assay, which we believe is a distinguishing trait of the method, is based on two strong and specific molecular interactions: binding of a his-tagged protein to a nickel-coated microplate and binding of biotinylated DNA to avidin. In the reported experiments, the solution was used to optimize the conditions for DNA mismatch binding by MutS protein; however, the approach could be implemented to test nucleic acids interactions with any protein of interest. PMID:27241123

  2. Lipid G Protein-coupled Receptor Ligand Identification Using β-Arrestin PathHunter™ Assay

    PubMed Central

    Yin, Hong; Chu, Alan; Li, Wei; Wang, Bin; Shelton, Fabiola; Otero, Francella; Nguyen, Deborah G.; Caldwell, Jeremy S.; Chen, Yu Alice

    2009-01-01

    A growing number of orphan G-protein-coupled receptors (GPCRs) have been reported to be activated by lipid ligands, such as lysophosphatidic acid, sphingosine 1-phosphate (S1P), and cannabinoids, for which there are already well established receptors. These new ligand claims are controversial due to either lack of independent confirmations or conflicting reports. We used the β-arrestin PathHunter™ assay system, a newly developed, generic GPCR assay format that measures β-arrestin binding to GPCRs, to evaluate lipid receptor and ligand pairing. This assay eliminates interference from endogenous receptors on the parental cells because it measures a signal that is specifically generated by the tagged receptor and is immediately downstream of receptor activation. We screened a large number of newly “deorphaned” receptors (GPR23, GPR92, GPR55, G2A, GPR18, GPR3, GPR6, GPR12, and GPR63) and control receptors against a collection of ∼400 lipid molecules to try to identify the receptor ligand in an unbiased fashion. GPR92 was confirmed to be a lysophosphatidic acid receptor with weaker responses to farnesyl pyrophosphate and geranylgeranyl diphosphate. The putative cannabinoid receptor GPR55 responded strongly to AM251, rimonabant, and lysophosphatidylinositol but only very weakly to endocannabinoids. G2A receptor was confirmed to be an oxidized free fatty acid receptor. In addition, we discovered that 3,3′-diindolylmethane, a dietary molecule from cruciferous vegetables, which has known anti-cancer properties, to be a CB2 receptor partial agonist, with binding affinity around 1 μm. The anti-inflammatory effect of 3,3′-diindolylmethane in RAW264.7 cells was shown to be partially mediated by CB2. PMID:19286662

  3. Probing interactions between plant virus movement proteins and nucleic acids.

    PubMed

    Tzfira, Tzvi; Citovsky, Vitaly

    2008-01-01

    Most plant viruses move between plant cells with the help of their movement proteins (MPs). MPs are multifunctional proteins, and one of their functions is almost invariably binding to nucleic acids. Presumably, the MP-nucleic acid interaction is directly involved in formation of nucleoprotein complexes that function as intermediates in the cell-to-cell transport of many plant viruses. Thus, when studying a viral MP, it is important to determine whether or not it binds nucleic acids, and to characterize the hallmark parameters of such binding, i.e., preference for single- or double-stranded nucleic acids and binding cooperativity and sequence specificity. Here, we present two major experimental approaches, native gel mobility shift assay and ultra violet (UV) light cross-linking, for detection and characterization of MP binding to DNA and RNA molecules. We also describe protocols for purification of recombinant viral MPs over-expressed in bacteria and production of different DNA and RNA probes for these binding assays. PMID:18370264

  4. Development of an assay to simultaneously measure orotic acid, amino acids, and acylcarnitines in dried blood spots

    PubMed Central

    Held, Patrice K.; Haynes, Christopher A.; De Jesús, Víctor R.; Baker, Mei W.

    2016-01-01

    Background Orotic aciduria in the presence of hyperammonemia is a key indicator for a defect in the urea cycle, specifically ornithine transcarbamylase (OTC) deficiency. Current newborn screening (NBS) protocols can detect several defects of the urea cycle, but screening for OTC deficiency remains a challenge due to the lack of a suitable assay. The purpose of this study was to develop a high-throughput assay to measure orotic acid in dried blood spot (DBS) specimens as an indicator for urea cycle dysfunction, which can be readily incorporated into routine NBS. Methods Orotic acid was extracted from DBS punches and analyzed using flow-injection analysis tandem mass spectrometry (FIA–MS/MS) with negative-mode ionization, requiring <2 min/sample run time. This method was then multiplexed into a conventional newborn screening assay for analysis of amino acids, acylcarnitines, and orotic acid. Results We describe 2 assays which can quantify orotic acid in DBS: a stand-alone method and a combined method for analysis of orotic acid, amino acids, and acylcarnitines. Both methods demonstrated orotic acid recovery of 75–85% at multiple levels of enrichment. Precision was also comparable to traditional FIA–MS/MS methods. Analysis of residual presumptively normal NBS specimens demonstrated a 5:1 signal to noise ratio and the average concentration of orotic acid was approximately 1.2 μmol/l. The concentration of amino acids and acylcarnitines as measured by the combined method showed no significant differences when compared to the conventional newborn screening assay. In addition, retrospective analysis of confirmed patients and presumptively normal newborn screening specimens suggests potential for the methods to identify patients with OTC deficiency, as well as other urea cycle defects. Conclusion The assays described here quantify orotic acid in DBS using a simple extraction and FIA–MS/MS analysis procedures that can be implemented into current NBS protocols. PMID

  5. Probing protein stability with unnatural amino acids

    SciTech Connect

    Mendel, D.; Ellman, J.A.; Zhiyuh Chang; Veenstra, D.L.; Kollman, P.A.; Schultz, P.G. )

    1992-06-26

    Unnatural amino acid mutagenesis, in combination with molecular modeling and simulation techniques, was used to probe the effect of side chain structure on protein stability. Specific replacements at position 133 in T4 lysozyme included (1) leucine (wt), norvaline, ethylglycine, and alanine to measure the cost of stepwise removal of methyl groups from the hydrophobic core, (2) norvaline and O-methyl serine to evaluate the effects of side chain solvation, and (3) leucine, S,S-2-amino-4-methylhexanoic acid, and S-2-amino-3-cyclopentylpropanoic acid to measure the influence of packing density and side chain conformational entropy on protein stability. All of these factors (hydrophobicity, packing, conformational entropy, and cavity formation) significantly influence protein stability and must be considered when analyzing any structural change to proteins.

  6. Development and Application of a High Throughput Protein Unfolding Kinetic Assay

    PubMed Central

    Wang, Qiang; Waterhouse, Nicklas; Feyijinmi, Olusegun; Dominguez, Matthew J.; Martinez, Lisa M.; Sharp, Zoey; Service, Rachel; Bothe, Jameson R.; Stollar, Elliott J.

    2016-01-01

    The kinetics of folding and unfolding underlie protein stability and quantification of these rates provides important insights into the folding process. Here, we present a simple high throughput protein unfolding kinetic assay using a plate reader that is applicable to the studies of the majority of 2-state folding proteins. We validate the assay by measuring kinetic unfolding data for the SH3 (Src Homology 3) domain from Actin Binding Protein 1 (AbpSH3) and its stabilized mutants. The results of our approach are in excellent agreement with published values. We further combine our kinetic assay with a plate reader equilibrium assay, to obtain indirect estimates of folding rates and use these approaches to characterize an AbpSH3-peptide hybrid. Our high throughput protein unfolding kinetic assays allow accurate screening of libraries of mutants by providing both kinetic and equilibrium measurements and provide a means for in-depth ϕ-value analyses. PMID:26745729

  7. Ileal amino acid digestibility assay for the growing meat chicken--comparison of ileal and excreta amino acid digestibility in the chicken.

    PubMed

    Kadim, I T; Moughan, P J; Ravindran, V

    2002-09-01

    1. The apparent and true amino acid digestibilities in sorghum, wheat, soyabean meal, meat-and-bone meal, fish meal and blood meal for growing meat chickens were determined using an assay based on the collection of digesta from the terminal ileum and comparison was made with digestibility values determined using an excreta-based assay. 2. Five-week-old meat chickens were given maize-soyabean meal basal diet or mixtures of the basal diet and test diets containing the 6 ingredients as the sole source of dietary protein (50:50 on weight basis). Apparent amino acid digestibility values of assay diets at ileal and excreta levels were calculated using chromic oxide as the indigestible marker. True digestibility values were calculated using endogenous outputs determined by feeding a protein-free diet. Amino acid digestibilities of the ingredients were calculated by difference. 3. The site of measurement had no influence on endogenous amino acid output, the exceptions being aspartic acid and glutamic acid. The output of these two amino acids was higher in the excreta. 4. Significant differences were found between ileal and excreta-based digestibility of certain amino acids in some ingredients, with excreta values being usually higher than the ileal values, indicating a net catabolism of amino acids in the large intestine. The degree of net amino acid disappearance was found to be variable among amino acids and ingredients. In general, threonine had the lowest digestibility at the ileal level and, compared with other amino acids, the highest degredation during passage through the hindgut. 5. The results showed that digestibility determination based on excreta collection will overestimate the uptake for some amino acids in some feeds. The degree of overestimation was often considerable, ranging from 8.9% (apparent digestibility of threonine in soyabean meal) to 56% (apparent digestibility of aspartic acid in wheat). It is concluded that digestibility values measured at the

  8. Detection of North American eastern and western equine encephalitis viruses by nucleic acid amplification assays.

    PubMed

    Lambert, Amy J; Martin, Denise A; Lanciotti, Robert S

    2003-01-01

    We have developed nucleic acid sequence-based amplification (NASBA), standard reverse transcription PCR (RT-PCR), and TaqMan nucleic acid amplification assays for the rapid detection of North American eastern equine encephalitis (EEE) and western equine encephalitis (WEE) viral RNAs from samples collected in the field and clinical samples. The sensitivities of these assays have been compared to that of virus isolation. While all three types of nucleic acid amplification assays provide rapid detection of viral RNAs comparable to the isolation of viruses in Vero cells, the TaqMan assays for North American EEE and WEE viral RNAs are the most sensitive. We have shown these assays to be specific for North American EEE and WEE viral RNAs by testing geographically and temporally distinct strains of EEE and WEE viruses along with a battery of related and unrelated arthropodborne viruses. In addition, all three types of nucleic acid amplification assays have been used to detect North American EEE and WEE viral RNAs from mosquito and vertebrate tissue samples. The sensitivity, specificity, and rapidity of nucleic acid amplification demonstrate the usefulness of NASBA, standard RT-PCR, and TaqMan assays, in both research and diagnostic settings, to detect North American EEE and WEE viral RNAs. PMID:12517876

  9. Established and emerging fluorescence-based assays for G-protein function: heterotrimeric G-protein alpha subunits and regulator of G-protein signaling (RGS) proteins.

    PubMed

    Kimple, Randall J; Jones, Miller B; Shutes, Adam; Yerxa, Benjamin R; Siderovski, David P; Willard, Francis S

    2003-06-01

    Heterotrimeric G-proteins are molecular switches that couple serpentine receptors to intracellular effector pathways and the regulation of cell physiology. Ligand-bound receptors cause G-protein alpha subunits to bind guanosine 5'-triphosphate (GTP) and activate effector pathways. Signal termination is facilitated by the intrinsic GTPase activity of G-protein alpha subunits. Regulators of G-protein signaling (RGS) proteins accelerate the GTPase activity of the G-protein alpha subunit, and thus negatively regulate G-protein-mediated signal transduction. In vitro biochemical assays of heterotrimeric G-proteins commonly include measurements of nucleotide binding, GTPase activity, and interaction with RGS proteins. However, the conventional assays for most of these processes involve radiolabeled guanine nucleotide analogues and scintillation counting. In this article, we focus on fluorescence-based methodologies to study heterotrimeric G-protein alpha subunit regulation in vitro. Furthermore, we consider the potential of such techniques in high-throughput screening and drug discovery. PMID:12769684

  10. Cholesterol-lowering effect of rice bran protein containing bile acid-binding proteins.

    PubMed

    Wang, Jilite; Shimada, Masaya; Kato, Yukina; Kusada, Mio; Nagaoka, Satoshi

    2015-01-01

    Dietary plant protein is well known to reduce serum cholesterol levels. Rice bran is a by-product of rice milling and is a good source of protein. The present study examined whether feeding rats a high-cholesterol diet containing 10% rice bran protein (RBP) for 10 d affected cholesterol metabolism. Rats fed dietary RBP had lower serum total cholesterol levels and increased excretion of fecal steroids, such as cholesterol and bile acids, than those fed dietary casein. In vitro assays showed that RBP strongly bound to taurocholate, and inhibited the micellar solubility of cholesterol, compared with casein. Moreover, the bile acid-binding proteins of the RBP were eluted by a chromatographic column conjugated with cholic acid, and one of them was identified as hypothetical protein OsJ_13801 (NCBI accession No. EAZ29742) using MALDI-TOF mass spectrometry analysis. These results suggest that the hypocholesterolemic action of the RBP may be caused by the bile acid-binding proteins. PMID:25374002

  11. Determining protein adducts of fipexide: mass spectrometry based assay for confirming the involvement of its reactive metabolite in covalent binding.

    PubMed

    Sleno, Lekha; Varesio, Emmanuel; Hopfgartner, Gérard

    2007-01-01

    Fipexide is a nootropic drug, withdrawn from the market due to its idiosyncratic drug reactions causing adverse effects in man. Previous work on its metabolites has identified several potential reactive metabolites which could be implicated in protein binding. Here, we investigated the formation of these metabolites in rat and human hepatocytes. Based on these results, the o-quinone of fipexide (FIP), formed via the demethylenation reaction through a catechol intermediate, was chosen for further investigation. Studies were then pursued in order to relate this metabolite to protein binding, and thus better understand potential mechanisms for the toxicity of the parent compound. An assay was developed for determining the fipexide catechol-cysteine adduct in the microsomal protein fractions following in vitro incubations. This method digests the entire protein fraction into amino acids, followed by the detection of the Cys-metabolite adduct by liquid chromatography/mass spectrometry (LC/MS). We have designed a strategy where drug metabolism taking place in microsomal incubations and involved in protein binding can be assessed after the proteins have been digested, with the detection of the specific amino acid adduct. In this study, the structure of the fipexide adduct was hypothesized using knowledge previously gained in glutathione and N-acetylcysteine trapping experiments. Acetaminophen was used as a positive control for detecting a drug metabolite-cysteine adduct by LC/MS. This approach has the potential to be applicable as a protein-binding assay in early drug discovery without the need for radioactive compounds. PMID:18022964

  12. Highly accurate boronimeter assay of concentrated boric acid solutions

    SciTech Connect

    Ball, R.M. )

    1992-01-01

    The Random-Walk Boronimeter has successfully been used as an on-line indicator of boric acid concentration in an operating commercial pressurized water reactor. The principle has been adapted for measurement of discrete samples to high accuracy and to concentrations up to 6000 ppm natural boron in light water. Boric acid concentration in an aqueous solution is a necessary measurement in many nuclear power plants, particularly those that use boric acid dissolved in the reactor coolant as a reactivity control system. Other nuclear plants use a high-concentration boric acid solution as a backup shutdown system. Such a shutdown system depends on rapid injection of the solution and frequent surveillance of the fluid to ensure the presence of the neutron absorber. The two methods typically used to measure boric acid are the chemical and the physical methods. The chemical method uses titration to determine the ionic concentration of the BO[sub 3] ions and infers the boron concentration. The physical method uses the attenuation of neutrons by the solution and infers the boron concentration from the neutron absorption properties. This paper describes the Random-Walk Boronimeter configured to measure discrete samples to high accuracy and high concentration.

  13. Functional Analysis of Mouse G6pc1 Mutations Using a Novel In Situ Assay for Glucose-6-Phosphatase Activity and the Effect of Mutations in Conserved Human G6PC1/G6PC2 Amino Acids on G6PC2 Protein Expression.

    PubMed

    Boortz, Kayla A; Syring, Kristen E; Pound, Lynley D; Wang, Yingda; Oeser, James K; O'Brien, Richard M

    2016-01-01

    Elevated fasting blood glucose (FBG) has been associated with increased risk for development of type 2 diabetes. Single nucleotide polymorphisms (SNPs) in G6PC2 are the most important common determinants of variations in FBG in humans. Studies using G6pc2 knockout mice suggest that G6pc2 regulates the glucose sensitivity of insulin secretion. G6PC2 and the related G6PC1 and G6PC3 genes encode glucose-6-phosphatase catalytic subunits. This study describes a functional analysis of 22 non-synonymous G6PC2 SNPs, that alter amino acids that are conserved in human G6PC1, mouse G6pc1 and mouse G6pc2, with the goal of identifying variants that potentially affect G6PC2 activity/expression. Published data suggest strong conservation of catalytically important amino acids between all four proteins and the related G6PC3 isoform. Because human G6PC2 has very low glucose-6-phosphatase activity we used an indirect approach, examining the effect of these SNPs on mouse G6pc1 activity. Using a novel in situ functional assay for glucose-6-phosphatase activity we demonstrate that the amino acid changes associated with the human G6PC2 rs144254880 (Arg79Gln), rs149663725 (Gly114Arg) and rs2232326 (Ser324Pro) SNPs reduce mouse G6pc1 enzyme activity without affecting protein expression. The Arg79Gln variant alters an amino acid mutation of which, in G6PC1, has previously been shown to cause glycogen storage disease type 1a. We also demonstrate that the rs368382511 (Gly8Glu), rs138726309 (His177Tyr), rs2232323 (Tyr207Ser) rs374055555 (Arg293Trp), rs2232326 (Ser324Pro), rs137857125 (Pro313Leu) and rs2232327 (Pro340Leu) SNPs confer decreased G6PC2 protein expression. In summary, these studies identify multiple G6PC2 variants that have the potential to be associated with altered FBG in humans. PMID:27611587

  14. Bioluminescent Ligand-Receptor Binding Assays for Protein or Peptide Hormones.

    PubMed

    Liu, Ya-Li; Guo, Zhan-Yun

    2016-01-01

    Bioluminescence has been widely used in biomedical research due to its high sensitivity, low background, and broad linear range. In recent studies, we applied bioluminescence to ligand-receptor binding assays for some protein or peptide hormones based on a newly developed small monomeric Nanoluciferase (NanoLuc) reporter that has the so far brightest bioluminescence. The conventional ligand-receptor binding assays rely on radioligands that have drawbacks, such as radioactive hazards and short shelf lives. In contrast, the novel bioluminescent binding assays use the NanoLuc-based protein or peptide tracers that are safe, stable, and ultrasensitive. Thus, the novel bioluminescent ligand-receptor binding assay would be applied to more and more protein or peptide hormones for ligand-receptor interaction studies in future. In the present article, we provided detailed protocols for setting up the novel bioluminescent ligand-receptor binding assays using two representative protein hormones as examples. PMID:27424896

  15. Determination of Chitin Based on the Colorimetric Assay of Glucosamine in Acidic Hydrolysate.

    PubMed

    Katano, Hajime; Takakuwa, Masahiro; Hayakawa, Hajime; Kimoto, Hisashi

    2016-01-01

    A colorimetric method for the glucosamine (GlcN) assay was applied for the determination of chitin, which can be hydrolyzed to produce GlcN. A 10-mg sample was mixed with 10 mL of a 5 mol/L HCl aqueous solution, and the mixture was kept at 100°C for 12 h. Under these conditions, chitin was completely depolymerized and deacetylated to produce GlcN, even when the sample was a crab shell. A 20-μL aliquot of the hydrolysate was mixed with 20 μL of a 5 mol/L NaOH aqueous solution and 200 μL of a 50 mmol/L Na2SiO3, 600 mmol/L Na2MoO4, 1.5 mol/L CH3COOH and 30% (v/v) dimethyl sulfoxide solution. The mixture was kept at 70°C for 30 min. In the mixture, GlcN reduced the Mo(VI) species to form a blue molybdosilicate anion, which gave an absorbance maximum at around 750 nm. Since N-acetylglucosamine and chitin oligosaccharides could not render the reaction mixture blue, GlcN in the hydrolysate could be assayed colorimetrically with high selectivity. When a standard chitin sample was examined, the GlcN concentration in the hydrolysate was determined to be 0.97 ± 0.02 g/L (as hydrochloride salt), indicating that the sample contained 10.0 ± 0.2 mg chitin (as an N-acetylglucosamine homopolymer). Calcium cation, amino acids, and proteins did not interfere with the GlcN assay. Thus, the proposed method was successfully applied to determine chitin in a crab shell sample. PMID:27302593

  16. Helicase Assays

    PubMed Central

    Wang, Xin; Li, Jing; Diaz, Jason; You, Jianxin

    2016-01-01

    Helicases are a class of enzymes which are motor proteins using energy derived from ATP hydrolysis to move directionally along a nucliec acid phosphodiester backbone (such as DNA, RNA and DNA-RNA hybrids) and separate two annealed nucleic acid strands. Many cellular processes, such as transcription, DNA replication, recombination and DNA repair involve helicase activity. Here, we provide a protocol to analyze helicase activities in vitro. In this protocol, the DNA helicase protein Merkel cell polyomavirus large T-antigen was expressed in the mammalian cell line HEK293 and immoblized on an IgG resin. The helicase assay is performing while the protein is immoblized on IgG resin.

  17. Photolabeling of brain membrane proteins by lysergic acid diethylamide.

    PubMed

    Mahon, A C; Hartig, P R

    1982-04-01

    3H-Lysergic acid diethylamide (3H-LSD) is irreversibly incorporated into bovine caudate membranes during ultraviolet light illumination. The incorporated radioligand apparently forms a covalent bond with a subpopulation of the membrane proteins. Although the photolabeling pattern differs significantly from the Coomassie blue staining pattern on SDS gels, the photolabeling is apparently not specific for LSD binding sites associated with neurotransmitter receptors. 3H-LSD photolabeling can occur during prolonged exposure of membrane samples to room lighting and thus may introduce artifacts into receptor binding assays. PMID:7087658

  18. Photolabeling of brain membrane proteins by lysergic acid diethylamide

    SciTech Connect

    Mahon, A.C.; Hartig, P.R.

    1982-04-05

    /sup 3/H-Lysergic acid diethylamide (/sup 3/H-LSD) is irreversibly incorporated into bovine caudate membranes during ultraviolet light illumination. The incorporated radioligand apparently forms a covalent bond with a sub-population of the membrane proteins. Although the photolabeling pattern differs significantly from the Coomassie blue staining pattern on SDS gels, the photolabeling is apparently not specific for LSD binding sites associated with neurotransmitter receptors. /sup 3/H-LSD photolabeling can occur during prolonged exposure of membrane samples to room lighting and thus may introduce artifacts into receptor binding assays.

  19. Detection of non-protein amino acids in the presence of protein amino acids. II.

    NASA Technical Reports Server (NTRS)

    Shapshak, P.; Okaji, M.

    1972-01-01

    Studies conducted with the JEOL 5AH amino acid analyzer are described. This instrument makes possible the programming of the chromatographic process. Data are presented showing the separations of seventeen non-protein amino acids in the presence of eighteen protein amino acids. It is pointed out that distinct separations could be obtained in the case of a number of chemically similar compounds, such as ornithine and lysine, N-amidino alanine and arginine, and iminodiacetic acid and S-carboxymethyl cysteine and aspartic acid.

  20. Recombinant HT.sub.m4 gene, protein and assays

    DOEpatents

    Lim, Bing; Adra, Chaker N.; Lelias, Jean-Michel

    1996-01-01

    The invention relates to a recombinant DNA molecule which encodes a HT.sub.m4 protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT.sub.m4 protein and a recombinant HT.sub.m4 protein. The invention also relates to a method for detecting the presence of a hereditary atopy.

  1. Protein and amino Acid supplementation in athletes.

    PubMed

    Armsey, Thomas D; Grime, Todd E

    2002-08-01

    Amino acid supplementation is practiced by numerous individuals with the hope of increasing muscle mass and function by increasing available proteins. Theoretically, this makes a great deal of sense; the scientific facts, however, fail to conclusively prove that ingesting more than the recommended dietary allowance of protein has any effect on otherwise healthy adults. Athletes may be the exception to this rule. This review examines the most current literature pertaining to amino acid supplementation, and reports on the potential benefits and risks of this common practice. PMID:12831703

  2. The natural non-protein amino acid N-β-methylamino-L-alanine (BMAA) is incorporated into protein during synthesis.

    PubMed

    Glover, W Broc; Mash, Deborah C; Murch, Susan J

    2014-11-01

    N-β-methylamino-L-alanine (BMAA) is an amino acid produced by cyanobacteria and accumulated through trophic levels in the environment and natural food webs. Human exposure to BMAA has been linked to progressive neurodegenerative diseases, potentially due to incorporation of BMAA into protein. The insertion of BMAA and other non-protein amino acids into proteins may trigger protein misfunction, misfolding and/or aggregation. However, the specific mechanism by which BMAA is associated with proteins remained unidentified. Such studies are challenging because of the complexity of biological systems and samples. A cell-free in vitro protein synthesis system offers an excellent approach for investigation of changing amino acid composition in protein. In this study, we report that BMAA incorporates into protein as an error in synthesis when a template DNA sequence is used. Bicinchoninic acid assay of total protein synthesis determined that BMAA effectively substituted for alanine and serine in protein product. LC-MS/MS confirmed that BMAA was selectively inserted into proteins in place of other amino acids, but isomers N-(2-aminoethyl)glycine (AEG) and 2,4-diaminobutyric acid (DAB) did not share this characteristic. Incorporation of BMAA into proteins was significantly higher when genomic DNA from post-mortem brain was the template. About half of BMAA in the synthetic proteins was released with denaturation with sodium dodecylsulfonate and dithiothreitol, but the remaining BMAA could only be released by acid hydrolysis. Together these data demonstrate that BMAA is incorporated into the amino acid backbone of proteins during synthesis and also associated with proteins through non-covalent bonding. PMID:25096519

  3. Nucleic acids, proteins, and chirality

    NASA Technical Reports Server (NTRS)

    Usher, D. A.; Profy, A. T.; Walstrum, S. A.; Needels, M. C.; Bulack, S. C.; Lo, K. M.

    1984-01-01

    The present investigation is concerned with experimental results related, in one case, to the chirality of nucleotides, and, in another case, to the possibility of a link between the chirality of nucleic acids, and that of peptides. It has been found that aminoacylation of the 'internal' hydroxyl group of a dinucleoside monophosphate can occur stereoselectively. However, this reaction has not yet been made a part of a working peptide synthesis scheme. The formation and cleavage of oligonucleotides is considered. In the event of the formation of a helical complex between the oligonucleotide and the polymer, 1-prime,5-prime-bonds in the oligomer are found to become more resistant towards cleavage. The conditions required for peptide bond formation are examined, taking into account the known structures of RNA and possible mechanisms for prebiotic peptide bond formation. The possibility is considered that the 2-prime,5-prime-internucleotide linkage could have played an important part in the early days of biological peptide synthesis.

  4. Development of betulinic acid as an agonist of TGR5 receptor using a new in vitro assay

    PubMed Central

    Lo, Shih-Hsiang; Cheng, Kai-Chung; Li, Ying-Xiao; Chang, Chin-Hong; Cheng, Juei-Tang; Lee, Kung-Shing

    2016-01-01

    Background G-protein-coupled bile acid receptor 1, also known as TGR5 is known to be involved in glucose homeostasis. In animal models, treatment with a TGR5 agonist induces incretin secretion to reduce hyperglycemia. Betulinic acid, a triterpenoid present in the leaves of white birch, has been introduced as a selective TGR5 agonist. However, direct activation of TGR5 by betulinic acid has not yet been reported. Methods Transfection of TGR5 into cultured Chinese hamster ovary (CHO-K1) cells was performed to establish the presence of TGR5. Additionally, TGR5-specific small interfering RNA was employed to silence TGR5 in cells (NCI-H716 cells) that secreted incretins. Uptake of glucose by CHO-K1 cells was evaluated using a fluorescent indicator. Amounts of cyclic adenosine monophosphate and glucagon-like peptide were quantified using enzyme-linked immunosorbent assay kits. Results Betulinic acid dose-dependently increases glucose uptake by CHO-K1 cells transfected with TGR5 only, which can be considered an alternative method instead of radioligand binding assay. Additionally, signals coupled to TGR5 activation are also increased by betulinic acid in cells transfected with TGR5. In NCI-H716 cells, which endogenously express TGR5, betulinic acid induces glucagon-like peptide secretion via increasing calcium levels. However, the actions of betulinic acid were markedly reduced in NCI-H716 cells that received TGR5-silencing treatment. Therefore, the present study demonstrates the activation of TGR5 by betulinic acid for the first time. Conclusion Similar to the positive control lithocholic acid, which is the established agonist of TGR5, betulinic acid has been characterized as a useful agonist of TGR5 and can be used to activate TGR5 in the future. PMID:27578964

  5. Enzyme-linked immunosorbent assay detection and bioactivity of Cry1Ab protein fragments

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzyme-linked immunosorbent assay (ELISA) has emerged as the preferred detection method for Cry proteins in environmental matrices. Concerns exist that ELISAs are capable of detecting fragments of Cry proteins, which may lead to an over-estimation of the concentration of these proteins in the enviro...

  6. Recombinant HT{sub m4} gene, protein and assays

    DOEpatents

    Lim, B.; Adra, C.N.; Lelias, J.M.

    1996-09-03

    The invention relates to a recombinant DNA molecule which encodes a HT{sub m4} protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT{sub m4} protein and a recombinant HT{sub m4} protein. The invention also relates to a method for detecting the presence of a hereditary atopy. 2 figs.

  7. Electrocatalytic assay for monitoring methylglyoxal-mediated protein glycation.

    PubMed

    Havlikova, Marika; Zatloukalova, Martina; Ulrichova, Jitka; Dobes, Petr; Vacek, Jan

    2015-02-01

    Protein glycation is a complex process that plays an important role in diabetes mellitus, aging, and the regulation of protein function in general. As a result, current methodological research on proteins is focused on the development of novel approaches for investigating glycation and the possibility of monitoring its modulation and selective inhibition. In this paper, a first sensing strategy for protein glycation is proposed, based on protein electroactivity measurement. Concretely, the label-free method proposed is based on the application of a constant-current chronopotentiometric stripping (CPS) analysis at Hg-containing electrodes. The glycation process was monitored as the decrease in the electrocatalytic protein signal, peak H, observed at highly negative potentials at around -1.8 V (vs Ag/AgCl3 M KCl), which was previously ascribed to a catalytic hydrogen evolution reaction (CHER). Using this method, a model protein bovine serum albumin was investigated over 3 days of incubation with the glycation agent methylglyoxal in the absence or presence of the glycation inhibitor aminoguanidine (pimagedine). The electrochemical methodology presented here could open up new possibilities in research on protein glycation and oxidative modification. The methodology developed also provides a new option for the analysis of protein intermolecular interactions using electrochemical sensors, which was demonstrated by the application of a silver solid amalgam electrode (AgSAE) for monitoring the glycation process in samples of bovine serum albumin, human serum albumin, and lysozyme. PMID:25539570

  8. Nonenzymatic catalytic signal amplification for nucleic acid hybridization assays

    NASA Technical Reports Server (NTRS)

    Fan, Wenhong (Inventor); Cassell, Alan M. (Inventor); Han, Jie (Inventor)

    2006-01-01

    Devices, methods, and kits for amplifying the signal from hybridization reactions between nucleic acid probes and their cognate targets are presented. The devices provide partially-duplexed, immobilized probe complexes, spatially separate from and separately addressable from immobilized docking strands. Cognate target acts catalytically to transfer probe from the site of probe complex immobilization to the site of immobilized docking strand, generating a detectable signal. The methods and kits of the present invention may be used to identify the presence of cognate target in a fluid sample.

  9. Development of a lectin binding assay to differentiate between recombinant and endogenous proteins in pharmacokinetic studies of protein-biopharmaceuticals.

    PubMed

    Weber, Alfred; Minibeck, Eva; Scheiflinger, Friedrich; Turecek, Peter L

    2015-04-10

    Human glycoproteins, expressed in hamster cell lines, show similar glycosylation patterns to naturally occurring human molecules except for a minute difference in the linkage of terminal sialic acid: both cell types lack α2,6-galactosyl-sialyltransferase, abundantly expressed in human hepatocytes and responsible for the α2,6-sialylation of circulating glycoproteins. This minute difference, which is currently not known to have any physiological relevance, was the basis for the selective measurement of recombinant glycoproteins in the presence of their endogenous counterparts. The assay is based on using the lectin Sambucus nigra agglutinin (SNA), selectively binding to α2,6-sialylated N-glycans. Using von Willebrand factor (VWF), factor IX (FIX), and factor VIIa (FVIIa), it was demonstrated that (i) the plasma-derived proteins, but not the corresponding recombinant proteins, specifically bind to SNA and (ii) this binding can be used to deplete the plasma-derived proteins. The feasibility of this approach was confirmed in spike-recovery studies for all three recombinant coagulation proteins in human plasma and for recombinant VWF (rVWF) in macaque plasma. Analysis of plasma samples from macaques after administration of recombinant and a plasma-derived VWF demonstrated the suitability and robustness of this approach. Data showed that rVWF could be selectively measured without changing the ELISAs and furthermore revealed the limitations of baseline adjustment using a single measurement of the predose concentration only. The SNA gel-based depletion procedure can easily be integrated in existing procedures as a specific sample pre-treatment step. While ELISA-based methods were used to measure the recombinant coagulation proteins in the supernatants obtained by depletion, this procedure is applicable for all biochemical analyses. PMID:25703236

  10. Dihydrolipoic acid reduces cytochrome b561 proteins.

    PubMed

    Bérczi, Alajos; Zimányi, László; Asard, Han

    2013-03-01

    Cytochrome b561 (Cyt-b561) proteins constitute a family of trans-membrane proteins that are present in a wide variety of organisms. Two of their characteristic properties are the reducibility by ascorbate (ASC) and the presence of two distinct b-type hemes localized on two opposite sides of the membrane. Here we show that the tonoplast-localized and the putative tumor suppressor Cyt-b561 proteins can be reduced by other reductants than ASC and dithionite. A detailed spectral analysis of the ASC-dependent and dihydrolipoic acid (DHLA)-dependent reduction of these two Cyt-b561 proteins is also presented. Our results are discussed in relation to the known antioxidant capability of DHLA as well as its role in the regeneration of other antioxidant compounds of cells. These results allow us to speculate on new biological functions for the trans-membrane Cyt-b561 proteins. PMID:22526465

  11. A reliable non-separation fluorescence quenching assay for total glycated serum protein: a simple alternative to nitroblue tetrazolium reduction.

    PubMed

    Blincko, S; Colbert, D; John, W G; Edwards, R

    2000-05-01

    A simple non-separation assay for the measurement of total glycated serum protein is described. It was found that the fluorescence intensity of a solution of a fluorescein-boronic acid derivative was quenched in proportion to the amount of serum added. This led to the development of an assay in which 10 microL of serum is added to 4 mL of a solution of the fluorescein-boronic acid derivative and the fluorescence intensity is measured after 15 min. The results, as measured by drop in fluorescence intensity, calibrated by a single standard, were compared with the results for nitroblue tetrazolium (NBT) reduction of fructosamine and showed good correlation (r=0.936, n=114). The intra-assay precision (seven samples each measured 10 times) was less than 2.1% (concentration range 190-660 micromol/L); inter-assay precision for seven samples in 10 assays was less than 2.5% (over the same concentration range). Dilution of serum that had a high concentration of total glycated protein showed the assay to be linear. Serum samples (with low, medium and high total glycated protein concentrations) showed less than 2.1% difference from base results with added glucose (up to 60 mmol/L), less than 9.7% difference with added bilirubin (up to 250 micromol/L) and less than 6.9% with added triglycerides (up to 50 mmol/L). Addition of haemoglobin (up to 0.9 g/dL) with high glycation (11.7% HbA1c) to plasma (298 micromol/L total glycated protein) showed less than 10% difference from the base result. Assays performed over a range of temperatures (12-34 degrees C) showed no significant differences in the results. The assay gives similar results to the currently used NTB method but with significantly less susceptibility to interferences. As such the method should be a useful aid in the management of diabetes. PMID:10817254

  12. Highly thermostable RadA protein from the archaeon Pyrococcus woesei enhances specificity of simplex and multiplex PCR assays.

    PubMed

    Stefanska, Aleksandra; Gaffke, Lidia; Kaczorowska, Anna-Karina; Plotka, Magdalena; Dabrowski, Slawomir; Kaczorowski, Tadeusz

    2016-05-01

    The radA gene of the hyperthermophilic archaeon Pyrococcus woesei (Thermococcales) was cloned and overexpressed in Escherichia coli. The 1050-bp gene codes for a 349-amino-acid polypeptide with an M r of 38,397 which shows 100 % positional amino acid identity to Pyrococcus furiosus RadA and 27.1 % to the E. coli RecA protein. Recombinant RadA was overproduced in Escherichia coli as a His-tagged fusion protein and purified to electrophoretic homogeneity using a simple procedure consisting of ammonium sulfate precipitation and metal-affinity chromatography. In solution RadA exists as an undecamer (11-mer). The protein binds both to ssDNA and dsDNA. RadA has been found to be highly thermostable, it remains almost unaffected by a 4-h incubation at 94 °C. The addition of the RadA protein to either simplex or multiplex PCR assays, significantly improves the specificity of DNA amplification by eliminating non-specific products. Among applications tested the RadA protein proved to be useful in allelic discrimination assay of HADHA gene associated with long-chain 3-hydroxylacyl-CoA dehydrogenase deficiency that in infancy may lead to hypotonia, serious heart and liver problems and even sudden death. PMID:26337425

  13. SOLID-PHASE ASSAY FOR THE PHOSPHORYLATION OF PROTEINS BLOTTED ON NITROCELLULOSE MEMBRANE FILTERS

    EPA Science Inventory

    A new procedure for the phosphorylation and assay of phosphoproteins is described. Proteins are solubilized from tissue samples, separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane filters and the blotted polypeptides are phosphorylated with ...

  14. Peptide-based protein capture agents with high affinity, selectivity, and stability as antibody replacements in biodetection assays

    NASA Astrophysics Data System (ADS)

    Coppock, Matthew B.; Farrow, Blake; Warner, Candice; Finch, Amethist S.; Lai, Bert; Sarkes, Deborah A.; Heath, James R.; Stratis-Cullum, Dimitra

    2014-05-01

    Current biodetection assays that employ monoclonal antibodies as primary capture agents exhibit limited fieldability, shelf life, and performance due to batch-to-batch production variability and restricted thermal stability. In order to improve upon the detection of biological threats in fieldable assays and systems for the Army, we are investigating protein catalyzed capture (PCC) agents as drop-in replacements for the existing antibody technology through iterative in situ click chemistry. The PCC agent oligopeptides are developed against known protein epitopes and can be mass produced using robotic methods. In this work, a PCC agent under development will be discussed. The performance, including affinity, selectivity, and stability of the capture agent technology, is analyzed by immunoprecipitation, western blotting, and ELISA experiments. The oligopeptide demonstrates superb selectivity coupled with high affinity through multi-ligand design, and improved thermal, chemical, and biochemical stability due to non-natural amino acid PCC agent design.

  15. Identification of new quinic acid derivatives as histone deacetylase inhibitors by fluorescence-based cellular assay.

    PubMed

    Son, Dohyun; Kim, Chung Sub; Lee, Kang Ro; Park, Hyun-Ju

    2016-05-01

    A fluorescence-based cellular assay system was established to identify potential epigenetic modulator ligands. This assay method is to detect the de-repression of an EGFP reporter in cancer cells by the treatment of HDAC (histone deacetylase) or DNMT (DNA methyltransferase) inhibitor. Using this system, we conducted a preliminary screening of in-house natural product library containing extracts and pure compounds, and identified several active compounds. Among them, novel quinic acid derivatives were recognized as excellent HDAC inhibitors by both enzymatic and cell-based HDAC assays. PMID:26996372

  16. Protein-based multiplex assays: mock presubmissions to the US Food and Drug Administration.

    PubMed

    Regnier, Fred E; Skates, Steven J; Mesri, Mehdi; Rodriguez, Henry; Tezak, Zivana; Kondratovich, Marina V; Alterman, Michail A; Levin, Joshua D; Roscoe, Donna; Reilly, Eugene; Callaghan, James; Kelm, Kellie; Brown, David; Philip, Reena; Carr, Steven A; Liebler, Daniel C; Fisher, Susan J; Tempst, Paul; Hiltke, Tara; Kessler, Larry G; Kinsinger, Christopher R; Ransohoff, David F; Mansfield, Elizabeth; Anderson, N Leigh

    2010-02-01

    As a part of ongoing efforts of the NCI-FDA Interagency Oncology Task Force subcommittee on molecular diagnostics, members of the Clinical Proteomic Technology Assessment for Cancer program of the National Cancer Institute have submitted 2 protein-based multiplex assay descriptions to the Office of In Vitro Diagnostic Device Evaluation and Safety, US Food and Drug Administration. The objective was to evaluate the analytical measurement criteria and studies needed to validate protein-based multiplex assays. Each submission described a different protein-based platform: a multiplex immunoaffinity mass spectrometry platform for protein quantification, and an immunological array platform quantifying glycoprotein isoforms. Submissions provided a mutually beneficial way for members of the proteomics and regulatory communities to identify the analytical issues that the field should address when developing protein-based multiplex clinical assays. PMID:20007858

  17. Solid-phase assay for the phosphorylation of proteins blotted on nitrocellulose membrane filters

    SciTech Connect

    Valtorta, F.; Schiebler, W.; Jahn, R.; Ceccarelli, B.; Greengard, P.

    1986-10-01

    A new procedure for the phosphorylation and assay of phosphoproteins is described. Proteins are solubilized from tissue samples, separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane filters, and the blotted polypeptides are phyosphorylated with the catalytic subunit of cyclic AMP (adenosine 3':5'-monophosphate)-dependent protein kinase. The method was developed for the assay of dephosphosynapsin I, but it has also proven suitable for the phosphorylation of other proteins. The patterns of phosphorylation of tissue samples phosphorylated using the new method are similar to those obtained using the conventional test tube assay. Once phosphorylated, the adsorbed proteins can be digested with proteases and subjected to phosphopeptide mapping. The phosphorylated blotted proteins can also be analyzed by overlay techniques for the immunological detection of polypeptides.

  18. Protein and Amino Acid Requirements during Pregnancy.

    PubMed

    Elango, Rajavel; Ball, Ronald O

    2016-07-01

    Protein forms an essential component of a healthy diet in humans to support both growth and maintenance. During pregnancy, an exceptional stage of life defined by rapid growth and development, adequate dietary protein is crucial to ensure a healthy outcome. Protein deposition in maternal and fetal tissues increases throughout pregnancy, with most occurring during the third trimester. Dietary protein intake recommendations are based on factorial estimates because the traditional method of determining protein requirements, nitrogen balance, is invasive and undesirable during pregnancy. The current Estimated Average Requirement and RDA recommendations of 0.88 and 1.1 g · kg(-1) · d(-1), respectively, are for all stages of pregnancy. The single recommendation does not take into account the changing needs during different stages of pregnancy. Recently, with the use of the minimally invasive indicator amino acid oxidation method, we defined the requirements to be, on average, 1.2 and 1.52 g · kg(-1) · d(-1) during early (∼16 wk) and late (∼36 wk) stages of pregnancy, respectively. Although the requirements are substantially higher than current recommendations, our values are ∼14-18% of total energy and fit within the Acceptable Macronutrient Distribution Range. Using swine as an animal model we showed that the requirements for several indispensable amino acids increase dramatically during late gestation compared with early gestation. Additional studies should be conducted during pregnancy to confirm the newly determined protein requirements and to determine the indispensable amino acid requirements during pregnancy in humans. PMID:27422521

  19. Development of fluorescent nanoparticle-labeled lateral flow assay for the detection of nucleic acids.

    PubMed

    Wang, Yuhong; Nugen, Sam R

    2013-10-01

    The rapid, specific and sensitive detection of nucleic acids is of utmost importance for the identification of infectious agents, diagnosis and treatment of genetic diseases, and the detection of pathogens related to human health and safety. Here we report the development of a simple and sensitive nucleic acid sequence-based and Ru(bpy)3 (2+)-doped silica nanoparticle-labeled lateral flow assay which achieves low limit of detection by using fluorescencent nanoparticles. The detection of the synthetic nucleic acid sequences representative of Trypanosoma mRNA, the causative agent for African sleeping sickness, was utilized to demonstrate this assay. The 30 nm spherical Ru(bpy)3 (2+)-doped silica nanoparticles were prepared in aqueous medium by a novel method recently reported. The nanoparticles were modified by 3-glycidoxypropyl trimethoxysilane in order to conjugate to amine-capped oligonucleotide reporter probes. The fluorescent intensities of the fluorescent assays were quantified on a mictrotiter plate reader using a custom holder. The experimental results showed that the lateral flow fluorescent assay developed was more sensitive compared with the traditional colloidal gold test strips. The limit of detection for the fluorescent lateral flow assay developed is approximately 0.066 fmols as compared to approximately 15 fmols for the colloidal gold. The limit of detection can further be reduced about one order of magnitude when "dipstick" format was used. PMID:23525961

  20. Identification of cellular proteins that interact with Newcastle Disease Virus and human Respiratory Syncytial Virus by a two-dimensional virus overlay protein binding assay (VOPBA).

    PubMed

    Holguera, Javier; Villar, Enrique; Muñoz-Barroso, Isabel

    2014-10-13

    Although it is well documented that the initial attachment receptors for Newcastle Disease Virus (NDV) and Respiratory Syncytial Virus (RSV) are sialic acid-containing molecules and glycosaminoglycans respectively, the exact nature of the receptors for both viruses remains to be deciphered. Moreover, additional molecules at the host cell surface might be involved in the entry mechanism. With the aim of identifying the cellular proteins that interact with NDV and RSV at the cell surface, we performed a virus overlay protein binding assay (VOPBA). Cell membrane lysates were separated by two dimensional (2D) gel electrophoresis and electrotransferred to PVDF membranes, after which they were probed with high viral concentrations. NDV interacted with a Protein Disulfide Isomerase from chicken fibroblasts. In the case of RSV, we detected 15 reactive spots, which were identified as six different proteins, of which nucleolin was outstanding. We discuss the possible role of PDI and nucleolin in NDV and RSV entry, respectively. PMID:25109545

  1. A robust assay to measure DNA topology-dependent protein binding affinity

    PubMed Central

    Litwin, Tamara R.; Solà, Maria; Holt, Ian J.; Neuman, Keir C.

    2015-01-01

    DNA structure and topology pervasively influence aspects of DNA metabolism including replication, transcription and segregation. However, the effects of DNA topology on DNA-protein interactions have not been systematically explored due to limitations of standard affinity assays. We developed a method to measure protein binding affinity dependence on the topology (topological linking number) of supercoiled DNA. A defined range of DNA topoisomers at equilibrium with a DNA binding protein is separated into free and protein-bound DNA populations using standard nitrocellulose filter binding techniques. Electrophoretic separation and quantification of bound and free topoisomers combined with a simple normalization procedure provide the relative affinity of the protein for the DNA as a function of linking number. Employing this assay we measured topology-dependent DNA binding of a helicase, a type IB topoisomerase, a type IIA topoisomerase, a non-specific mitochondrial DNA binding protein and a type II restriction endonuclease. Most of the proteins preferentially bind negatively supercoiled DNA but the details of the topology-dependent affinity differ among proteins in ways that expose differences in their interactions with DNA. The topology-dependent binding assay provides a robust and easily implemented method to probe topological influences on DNA-protein interactions for a wide range of DNA binding proteins. PMID:25552413

  2. Improved sensitivity of an acid sphingomyelinase activity assay using a C6:0 sphingomyelin substrate.

    PubMed

    Chuang, Wei-Lien; Pacheco, Joshua; Cooper, Samantha; Kingsbury, Jonathan S; Hinds, John; Wolf, Pavlina; Oliva, Petra; Keutzer, Joan; Cox, Gerald F; Zhang, Kate

    2015-06-01

    Short-chain C6-sphingomyelin is an artificial substrate that was used in an acid sphingomyelinase activity assay for a pilot screening study of patients with Niemann-Pick disease types A and B. Using previously published multiplex and single assay conditions, normal acid sphingomyelinase activity levels (i.e. false negative results) were observed in two sisters with Niemann-Pick B who were compound heterozygotes for two missense mutations, p.C92W and p.P184L, in the SMPD1 gene. Increasing the sodium taurocholate detergent concentration in the assay buffer lowered the activity levels of these two patients into the range observed with other patients with clear separation from normal controls. PMID:26937397

  3. Improved sensitivity of an acid sphingomyelinase activity assay using a C6:0 sphingomyelin substrate

    PubMed Central

    Chuang, Wei-Lien; Pacheco, Joshua; Cooper, Samantha; Kingsbury, Jonathan S.; Hinds, John; Wolf, Pavlina; Oliva, Petra; Keutzer, Joan; Cox, Gerald F.; Zhang, Kate

    2015-01-01

    Short-chain C6-sphingomyelin is an artificial substrate that was used in an acid sphingomyelinase activity assay for a pilot screening study of patients with Niemann–Pick disease types A and B. Using previously published multiplex and single assay conditions, normal acid sphingomyelinase activity levels (i.e. false negative results) were observed in two sisters with Niemann–Pick B who were compound heterozygotes for two missense mutations, p.C92W and p.P184L, in the SMPD1 gene. Increasing the sodium taurocholate detergent concentration in the assay buffer lowered the activity levels of these two patients into the range observed with other patients with clear separation from normal controls. PMID:26937397

  4. Development and Evaluation of a Calibrator Material for Nucleic Acid-Based Assays for Diagnosing Aspergillosis

    PubMed Central

    Abdul-Ali, Deborah; Loeffler, Juergen; White, P. Lewis; Wickes, Brian; Herrera, Monica L.; Alexander, Barbara D.; Baden, Lindsey R.; Clancy, Cornelius; Denning, David; Nguyen, M. Hong; Sugrue, Michele; Wheat, L. Joseph; Wingard, John R.; Donnelly, J. Peter; Barnes, Rosemary; Patterson, Thomas F.; Caliendo, Angela M.

    2013-01-01

    Twelve laboratories evaluated candidate material for an Aspergillus DNA calibrator. The DNA material was quantified using limiting-dilution analysis; the mean concentration was determined to be 1.73 × 1010 units/ml. The calibrator can be used to standardize aspergillosis diagnostic assays which detect and/or quantify nucleic acid. PMID:23616459

  5. Analysis of Citric Acid in Beverages: Use of an Indicator Displacement Assay

    ERIC Educational Resources Information Center

    Umali, Alona P.; Anslyn, Eric V.; Wright, Aaron T.; Blieden, Clifford R.; Smith, Carolyne K.; Tian, Tian; Truong, Jennifer A.; Crumm, Caitlin E.; Garcia, Jorge E.; Lee, Soal; Mosier, Meredith; Nguyen, Chester P.

    2010-01-01

    The use of an indicator displacement assay permits the visualization of binding events between host and guest molecules. An undergraduate laboratory experiment is described to demonstrate the technique in the determination of citric acid content in commercially available beverages such as soda pop and fruit juices. Through the technique, students…

  6. Biological Monitoring of 3-Phenoxybenzoic Acid in Urine by an Enzyme -Linked Immunosorbent Assay

    EPA Science Inventory

    An enzyme-linked immunosorbent assay (ELISA) method was employed for determination of the pyrethroid biomarker, 3-phenoxybenzoic acid (3-PBA) in human urine samples. The optimized coating antigen concentration was 0.5 ng/mL with a dilution of 1:4000 for the 3-PBA antibody and 1:6...

  7. Response to oxalic acid as a resistance assay for Sclerotinia minor in peanut

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Response to oxalic acid was evaluated as a potential assay for screening peanut breeding lines for resistance to Sclerotinia blight caused by Sclerotinia minor. Detached stems of seven Spanish- and six runner-type peanut cultivars and advanced breeding lines, varying in resistance to Sclerotinia bl...

  8. 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... detection and identification of a combination of the following viruses: (1) Influenza A and Influenza B; (2) Influenza A subtype H1 and Influenza A subtype H3; (3) Respiratory Syncytial Virus subtype A and Respiratory... Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and (3) For a device that detects and differentiates...

  9. 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... detection and identification of a combination of the following viruses: (1) Influenza A and Influenza B; (2) Influenza A subtype H1 and Influenza A subtype H3; (3) Respiratory Syncytial Virus subtype A and Respiratory... Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and (3) For a device that detects and differentiates...

  10. 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... detection and identification of a combination of the following viruses: (1) Influenza A and Influenza B; (2) Influenza A subtype H1 and Influenza A subtype H3; (3) Respiratory Syncytial Virus subtype A and Respiratory... Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and (3) For a device that detects and differentiates...

  11. 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... detection and identification of a combination of the following viruses: (1) Influenza A and Influenza B; (2) Influenza A subtype H1 and Influenza A subtype H3; (3) Respiratory Syncytial Virus subtype A and Respiratory... Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and (3) For a device that detects and differentiates...

  12. 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... detection and identification of a combination of the following viruses: (1) Influenza A and Influenza B; (2) Influenza A subtype H1 and Influenza A subtype H3; (3) Respiratory Syncytial Virus subtype A and Respiratory... Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and (3) For a device that detects and differentiates...

  13. A rapid and sensitive assay for DNA-protein covalent complexes in living cells.

    PubMed

    Kiianitsa, Kostantin; Maizels, Nancy

    2013-05-01

    A number of proteins form covalent bonds with DNA as obligatory transient intermediates in normal nuclear transactions. Drugs that trap these complexes have proven to be potent therapeutics in both cancer and infectious disease. Nonetheless, current assays for DNA-protein adducts are cumbersome, limiting both mechanistic studies and translational applications. We have developed a rapid and sensitive assay that enables quantitative immunodetection of protein-DNA adducts. This new 'RADAR' (rapid approach to DNA adduct recovery) assay accelerates processing time 4-fold, increases sample throughput 20-fold and requires 50-fold less starting material than the current standard. It can be used to detect topoisomerase 1-DNA adducts in as little as 60 ng of DNA, corresponding to 10 000 human cells. We apply the RADAR assay to demonstrate that expression of SLFN11 does not increase camptothecin sensitivity by promoting accumulation of topoisomerase 1-DNA adducts. The RADAR assay will be useful for analysis of the mechanisms of formation and resolution of DNA-protein adducts in living cells, and identification and characterization of reactions in which covalent DNA adducts are transient intermediates. The assay also has potential application to drug discovery and individualized medicine. PMID:23519618

  14. Identification of β-phenylalanine as a non-protein amino acid in cultivated rice, Oryza sativa

    PubMed Central

    Yokoo, Takayuki; Takata, Ryo; Yan, Jian; Matsumoto, Fuka; Teraishi, Masayoshi; Okumoto, Yutaka; Jander, Georg; Mori, Naoki

    2015-01-01

    Non-protein amino acids, often analogs of the standard 20 protein amino acids, have been discovered in many plant species. Recent research with cultivated rice (Oryza sativa) identified (3R)-β-tyrosine, as well as a tyrosine amino mutase that synthesizes (3R)-β-tyrosine from the protein amino acid (2S)-α-tyrosine. Gas chromatography-mass spectrometry (GC-MS) assays and comparison to an authentic standard showed that β-phenylalanine is also a relatively abundant non-protein amino acid in rice leaves and that its biosynthesis occurs independently from that of β-tyrosine. PMID:27066169

  15. Nucleic acid encoding DS-CAM proteins and products related thereto

    SciTech Connect

    Korenberg, Julie R.

    2005-11-01

    In accordance with the present invention, there are provided Down Syndrome-Cell Adhesion Molecule (DS-CAM) proteins. Nucleic acid sequences encoding such proteins and assays employing same are also disclosed. The invention DS-CAM proteins can be employed in a variety of ways, for example, for the production of anti-DS-CAM antibodies thereto, in therapeutic compositions and methods employing such proteins and/or antibodies. DS-CAM proteins are also useful in bioassays to identify agonists and antagonists thereto.

  16. A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins.

    PubMed

    Diao, Jiajie; Ishitsuka, Yuji; Lee, Hanki; Joo, Chirlmin; Su, Zengliu; Syed, Salman; Shin, Yeon-Kyun; Yoon, Tae-Young; Ha, Taekjip

    2012-05-01

    SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayers into one interconnected structure. This protocol describes our fluorescence resonance energy transfer (FRET)-based single vesicle-vesicle fusion assays for SNAREs and accessory proteins. Both lipid-mixing (with FRET pairs acting as lipophilic dyes in the membranes) and content-mixing assays (with FRET pairs present on a DNA hairpin that becomes linear via hybridization to a complementary DNA) are described. These assays can be used to detect substages such as docking, hemifusion, and pore expansion and full fusion. The details of flow cell preparation, protein-reconstituted vesicle preparation, data acquisition and analysis are described. These assays can be used to study the roles of various SNARE proteins, accessory proteins and effects of different lipid compositions on specific fusion steps. The total time required to finish one round of this protocol is 3–6 d. PMID:22582418

  17. A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins

    PubMed Central

    Diao, Jiajie; Ishitsuka, Yuji; Lee, Hanki; Joo, Chirlmin; Su, Zengliu; Syed, Salman; Shin, Yeon-Kyun; Yoon, Tae-Young; Ha, Taekjip

    2015-01-01

    SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayers into one interconnected structure. This protocol describes our fluorescence resonance energy transfer (FRET)-based single vesicle-vesicle fusion assays for SNAREs and accessory proteins. Both lipid-mixing (with FRET pairs acting as lipophilic dyes in the membranes) and content-mixing assays (with FRET pairs present on a DNA hairpin that becomes linear via hybridization to a complementary DNA) are described. These assays can be used to detect substages such as docking, hemifusion, and pore expansion and full fusion. The details of flow cell preparation, protein-reconstituted vesicle preparation, data acquisition and analysis are described. These assays can be used to study the roles of various SNARE proteins, accessory proteins and effects of different lipid compositions on specific fusion steps. The total time required to finish one round of this protocol is 3–6 d. PMID:22582418

  18. Versatile Apoferritin Nanoparticle Labels for Assay of Protein

    SciTech Connect

    Liu, Guodong; Wang, Jun; Wu, Hong; Lin, Yuehe

    2006-10-30

    A versatile bioassay label based on marker-loaded-apoferritin nanoparticles (MLAN) have been developed for sensitive protein detection. Dissociation and reconstitution characteristics at different pH as well as the special cavity structure of apoferritin provide a facile route to prepare nanoparticle labels, and avoid complicated and tedious synthesis process of conventional nanoparticle labels. The optical and electrochemical characteristics of the prepared nanoparticle labels are easily controlled by loading different optical or electrochemical markers. A fluorescence marker (fluorescein anion) and a redox marker [hexacyanoferrate (III)] were used as model markers to load into the cavity of apoferritin nanoparticle and developed for microscopic fluorescence immunoassay and electrochemical immunoassay, respectively. Detection limits of 0.06 ng mL-1 (0.39 pM) and 0.08 ng mL-1 (0.51 pM) of IgG were obtained with fluorescein MLAP and hexacyanoferrate MLAN, respectively. The new nanoparticle labels hold great promise for multiplex protein detection (in connection to nanoparticles loaded with different markers) and for enhancing the sensitivity of other bioassay.

  19. An aptamer assay using rolling circle amplification coupled with thrombin catalysis for protein detection.

    PubMed

    Guo, Limin; Hao, Lihua; Zhao, Qiang

    2016-07-01

    We describe a sensitive aptamer-based sandwich assay for protein detection on microplate by using rolling circle amplification (RCA) coupled with thrombin catalysis. This assay takes advantage of RCA generating long DNA oligonucleotides with repeat thrombin-binding aptamer sequence, specific aptamer affinity binding to achieve multiple thrombin labeling, and enzyme activity of thrombin for signal generation. Protein target is specifically captured by antibody-coated microplate. Then, an oligonucleotide containing an aptamer for protein and a primer sequence is added to form a typical sandwich structure. Following a template encoded with complementary sequence of aptamer for thrombin, RCA reaction extends the primer sequence into a long oligonucleotide. Many thrombin molecules bind with the RCA product. Thrombin catalyzes the conversion of its chromogenic or fluorogenic peptide substrates into detectable products for final quantification of protein targets. We applied this strategy to the detection of a model protein target, platelet-derived growth factor-BB (PDGF-BB). Due to double signal amplifications from RCA and thrombin catalysis, this assay enabled the detection of PDGF-BB as low as 3.1 pM when a fluorogenic peptide substrate was used. This assay provides a new way for signal generation in RCA-involved assay through direct thrombin labeling, circumventing time-consuming preparation of enzyme-conjugate and affinity probes. This method has promise for a variety of analytical applications. PMID:27108282

  20. A universal, high recovery assay for protein quantitation through temperature programmed liquid chromatography (TPLC).

    PubMed

    Orton, Dennis J; Doucette, Alan A

    2013-03-15

    As an alternative to direct UV absorbance measurements, estimation of total protein concentration is typically conducted through colorimetric reagent assays. However, for protein-limited applications, the proportion of the sample sacrificed to the assay becomes increasingly significant. This work demonstrates a method for quantitation of protein samples with high recovery. Temperature programmed liquid chromatography (TPLC) with absorbance detection at 214nm permits accurate estimation of total protein concentration from samples containing as little as 0.75μg. The method incorporates a temperature gradient from 25 to 80°C to facilitate elution of total protein into a single fraction. Analyte recovery, as measured from 1 and 10μg protein extracts of Escherichia coli, is shown to exceed 93%. Extinction coefficients at 214nm were calculated across the human proteome, providing a relative standard deviation of 21% (versus 42% at 280nm), suggesting absorbance values at 214nm provide a more consistent measure of protein concentration. These results translate to a universal protein detection strategy exhibiting a coefficient of variation below 10%. Together with the sensitivity and tolerance to contaminants, TPLC with UV detection is a favorable alternative to colorimetric assay for total protein quantitation, particularly in sample-limited applications. PMID:23435344

  1. Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials.

    PubMed

    Farris, M Heath; Ford, Kara A; Doyle, Richard C

    2016-01-01

    Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest. PMID:27166738

  2. Pyridine Hemochromagen Assay for Determining the Concentration of Heme in Purified Protein Solutions

    PubMed Central

    Barr, Ian; Guo, Feng

    2016-01-01

    Heme is a common cofactor in proteins, found in hemoglobin, myoglobin, cytochrome P450, DGCR8, and nitric oxide synthase, among others. This protocol describes a method for quantifying heme that works best in purified protein samples. This protocol might be used to, for example, determine whether a given heme-binding protein is fully occupied by heme, thus allowing correlation of heme content with activity. This requires the absolute heme concentration and an accurate protein concentration. Another use is to determine the extinction coefficients of a heme-bound protein. This assay is fast, easy, and reproducible if done correctly. PMID:27390766

  3. A simple and highly sensitive radioenzymatic assay for lysophosphatidic acid quantification

    PubMed Central

    Saulnier-Blache, Jean Sébastien; Girard, Alexia; Simon, Marie-Françoise; Lafontan, Max; Valet, Philippe

    2000-01-01

    The objective of the present work was to develop a simple and sensitive radioenzymatic assay to quantify lysophosphatidic acid (LPA). For that, a recombinant rat lysophosphatidic acid acyl transferase (LPAAT) produced in Escherichia coli was used. In the presence of [14C]oleoyl CoA, LPAAT selectively catalyzes the transformation of LPA and alkyl-LPA into [14C]phosphatidic acid. Acylation of LPA was complete and linear from 0 to 200 pmoles with a minimal detection of 0.2 pmoles. This method was used to quantify LPA in butanol- extracted lipids from bovine sera, as well as from human and mouse plasma. This radioenzymatic assay represents a new, simple, and highly sensitive method to quantify LPA in various biological fluids. PMID:11108727

  4. A modified enzyme-linked immunosorbent assay adapted for immunodetection of low amounts of water-insoluble proteins.

    PubMed

    Godfrin, Dominique; Sénéchal, Hélène; Sutra, Jean-Pierre; Busnel, Jean-Marc; Desvaux, François-Xavier; Peltre, Gabriel

    2007-09-30

    A mixture of thiourea, urea and CHAPS (TUC) is an excellent solvent compatible with isoelectrofocusing (IEF) separation of water-insoluble protein extracts, and their subsequent two-dimensional gel electrophoresis is an important step in proteomic studies. The main aim of this work was to quantify extremely low amounts of water-insoluble proteins contained, for instance, in samples collected in bio-aerosol samplers. High CHAPS concentrations solubilize many proteins. However, enzyme-linked immunosorbent assay (ELISA), which is the most popular immunodetection method of quantifying antigens, is unfortunately not compatible with these high CHAPS concentrations and with the low protein concentrations of TUC extracts. The most common mixture used to solubilize these proteins contains 2 mol l(-1) thiourea, 7 mol l(-1) urea and 5% w/v CHAPS. This paper shows that these components inhibit the adsorption and/or recognition of proteins on microtitration plates, preventing antigen quantification under classic ELISA conditions. We have tried several solvents (ethanol, isopropanol, acetonitrile and trichloroacetic acid) to make the TUC-soluble proteins stick to the ELISA plates, and ethanol was shown to be the most appropriate. In this study, we have defined a new ELISA protocol allowing rapid and sensitive detection of low concentrations (60-500 ng ml(-1)) of water-insoluble proteins extracted with high concentrations of TUC. PMID:17706662

  5. The uronic acids assay: a method for the determination of chemical activity on biofilm EPS.

    PubMed

    Mojica, Kristina D A; Cooney, Michael J

    2010-01-01

    In this work, the uronic acids assay was evaluated for its potential to function as a bioassay to screen for antagonistic activity against the production of microbial biofilm exopolysaccharide (EPS). The assay was first applied to biofilms produced in the presence of two universal disinfectants (sodium hypochlorite and sodium dodecyl sulfate) known to inhibit microbial growth and biofilm formation. The performance of the assay was then characterized through statistical assessment of threshold concentrations for disinfection efficiency and consistency relative to values reported in the literature. The assay was then evaluated for its utility in screening for enzymatic or chemical inhibitors of biofilm formation (eg glycosidases, halogenated furanones, and semi-crude fractions extracted from minimally fouled marine plants) and its ability to distinguish between true anti-biofilm activity and simple disinfection. Activity was characterized as (i) no effect, (ii) a true positive effect (ie increased biofilm EPS), (iii) anti-bacterial activity (ie decreased biofilm EPS and analogous decrease in planktonic growth), and (iv) anti-biofilm EPS activity (ie decreased biofilm EPS, without analogous decrease in planktonic growth). Results demonstrate that the uronic acids assay can augment existing biofilm characterization methods by providing a quantitative measure of biofilm EPS. PMID:20087802

  6. Nonradioactive GTP binding assay to monitor activation of g protein-coupled receptors.

    PubMed

    Frang, Heini; Mukkala, Veli-Matti; Syystö, Rita; Ollikka, Pia; Hurskainen, Pertti; Scheinin, Mika; Hemmilä, Ilkka

    2003-04-01

    GPCRs represent important targets for drug discovery because GPCRs participate in a wide range of cellular signaling pathways that play a role in a variety of pathological conditions. A large number of screening assays have been developed in HTS laboratories for the identification of hits or lead compounds acting on GPCRs. One type of assay that has found relatively widespread application, due to its at least in part generic nature, relies on the use of a radioactive GTP analogue, [(35)S]GTPgammaS. The G-protein alpha subunit is an essential part of the interaction between receptor and G proteins in transmembrane signaling, where the activated receptor catalyzes the release of GDP from Galpha, thereby enabling the subsequent binding of GTP or a GTP analogue. [(35)S]GTPgammaS allows the extent of this interaction to be followed quantitatively by determining the amount of radioactivity associated with cell membranes. However, with the increased desire to move assays to nonradioactive formats, there is a considerable need to develop a nonradioactive GTP binding assay to monitor ligand-induced changes in GPCR activity. The Eu-GTP binding assay described here is based on TRF that exploits the unique fluorescence properties of lanthanide chelates, and provides a powerful alternative to assays using radioisotopes. In this article, we have used the human alpha(2A)-AR as a model GPCR system to evaluate the usefulness of this Eu-GTP binding assay. PMID:15090192

  7. A facile assay to monitor secretory phospholipase A₂ using 8-anilino-1-naphthalenesulfonic acid.

    PubMed

    Vivek, Hamse K; Swamy, Supritha G; Priya, Babu S; Sethi, Gautam; Rangappa, Kanchugarakoppal S; Swamy, S Nanjunda

    2014-09-15

    Secretory phospholipases A2 (sPLA2s) are present in snake venoms, serum, and biological fluids of patients with various inflammatory, autoimmune and allergic disorders. Lipid mediators in the inflammatory processes have potential value for controlling phospholipid metabolism through sPLA2 inhibition. Thus, it demands the need for screening of potential leads for sPLA2 inhibition. To date, sPLA2 activity has been assayed using expensive radioactive or chromogenic substrates, thereby limiting a large number of assays. In this study, a simple and sensitive NanoDrop assay was developed using non-fluorogenic and non-chromogenic phospholipid substrate 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 8-anilino-1-naphthalenesulfonic acid (ANS) as interfacial hydrophobic probe. The modified assay required a 10ng concentration of sPLA2. ANS, as a strong anion, binds predominantly to cationic group of choline head of DMPC through ion pair formation, imparting hydrophobicity and lipophilicity and resulting in an increase in fluorescence. Triton X-100 imparts correct geometrical space during sPLA2 catalyzing DMPC, releasing lysophospholipid and acidic myristoyl acid, which in turn alters the hydrophobic environment prevailing around ANS-DMPC, which leads to weakening of the electrostatic ion pair interaction between DMPC and ANS ensuing decrease in fluorescence. These characteristic fluorescence changes between DMPC and ANS in response to sPLA2 catalysis are well documented and validated in this study. PMID:24915638

  8. Buffer optimization of thermal melt assays of Plasmodium proteins for detection of small-molecule ligands.

    PubMed

    Crowther, Gregory J; Napuli, Alberto J; Thomas, Andrew P; Chung, Diana J; Kovzun, Kuzma V; Leibly, David J; Castaneda, Lisa J; Bhandari, Janhavi; Damman, Christopher J; Hui, Raymond; Hol, Wim G J; Buckner, Frederick S; Verlinde, Christophe L M J; Zhang, Zhongsheng; Fan, Erkang; van Voorhis, Wesley C

    2009-07-01

    In the past decade, thermal melt/thermal shift assays have become a common tool for identifying ligands and other factors that stabilize specific proteins. Increased stability is indicated by an increase in the protein's melting temperature (Tm). In optimizing the assays for subsequent screening of compound libraries, it is important to minimize the variability of Tm measurements so as to maximize the assay's ability to detect potential ligands. The authors present an investigation of Tm variability in recombinant proteins from Plasmodium parasites. Ligands of Plasmodium proteins are particularly interesting as potential starting points for drugs for malaria, and new drugs are urgently needed. A single standard buffer (100 mM HEPES [pH 7.5], 150 mM NaCl) permitted estimation of Tm for 58 of 61 Plasmodium proteins tested. However, with several proteins, Tm could not be measured with a consistency suitable for high-throughput screening unless alternative protein-specific buffers were employed. The authors conclude that buffer optimization to minimize variability in Tm measurements increases the success of thermal melt screens involving proteins for which a standard buffer is suboptimal. PMID:19470714

  9. Development of a scintillation proximity assay for the Mycobacterium tuberculosis KasA and KasB enzymes involved in mycolic acid biosynthesis.

    PubMed

    Schaeffer, M L Merrill L; Carson, J D Jeffrey D; Kallender, Howard; Lonsdale, J T John T

    2004-01-01

    Tuberculosis remains a global health problem, and programs dedicated to discovery of novel compounds against Mycobacterium tuberculosis require robust assays for high-throughput screening of chemical and natural product libraries. Enzymes involved in the biosynthesis of mycolic acids, vital components of the mycobacterial cell wall, have received much attention as potential drug targets. KasA and KasB, examples of the beta-ketoacyl-acyl carrier protein synthase I/II (KASI/II) class of condensing enzymes of the M. tuberculosis fatty acid synthase II system have been the focus of several studies designed to biochemically characterize these enzymes. Whilst robust methods have been developed for FabH-like proteins, fast and sensitive assays for high-throughput screening of KASI/II enzymes have not been available. Here we report the development of a direct scintillation proximity assay (SPA) for the KASI/II enzymes, KasA and KasB. The SPA was more sensitive than existing assays, as shown by its ability to measure activity using less enzyme than other assay formats, and the SPA was validated using the known KAS inhibitor thiolactomycin. In addition, the KasA and KasB SPA was adapted for use with Staphylococcus aureus FabF to show the versatility of this assay format to KAS enzymes from other pathogenic organisms. PMID:15525558

  10. Assaying the kinetics of protein denaturation catalyzed by AAA+ unfolding machines and proteases.

    PubMed

    Baytshtok, Vladimir; Baker, Tania A; Sauer, Robert T

    2015-04-28

    ATP-dependent molecular machines of the AAA+ superfamily unfold or remodel proteins in all cells. For example, AAA+ ClpX and ClpA hexamers collaborate with the self-compartmentalized ClpP peptidase to unfold and degrade specific proteins in bacteria and some eukaryotic organelles. Although degradation assays are straightforward, robust methods to assay the kinetics of enzyme-catalyzed protein unfolding in the absence of proteolysis have been lacking. Here, we describe a FRET-based assay in which enzymatic unfolding converts a mixture of donor-labeled and acceptor-labeled homodimers into heterodimers. In this assay, ClpX is a more efficient protein-unfolding machine than ClpA both kinetically and in terms of ATP consumed. However, ClpP enhances the mechanical activities of ClpA substantially, and ClpAP degrades the dimeric substrate faster than ClpXP. When ClpXP or ClpAP engage the dimeric subunit, one subunit is actively unfolded and degraded, whereas the other subunit is passively unfolded by loss of its partner and released. This assay should be broadly applicable for studying the mechanisms of AAA+ proteases and remodeling chaperones. PMID:25870262

  11. Novel bioluminescent binding assays for interaction studies of protein/peptide hormones with their receptors.

    PubMed

    Liu, Ya-Li; Guo, Zhan-Yun

    2016-05-01

    Protein/peptide hormones are the largest group of endogenous signaling molecules and exert various biological functions by binding to specific cell membrane receptors. To study the interactions between these hormones and their receptors, quantitative ligand-receptor binding assays have been widely used for decades. However, the assays conventionally relied on the use of radioligands, which have some major drawbacks and can only be used in laboratories with a radioactive material license. We recently developed novel bioluminescent binding assays for several protein/peptide hormones using the brightest bioluminescent reporter known to date, nanoluciferase (NanoLuc). The NanoLuc reporter can be either chemically conjugated to an appropriate position, or genetically fused at one terminus, of protein/peptide hormones. Compared to conventional radioligands, these bioluminescent ligands have higher sensitivity, better safety, and longer shelf lives, and thus, represent a novel class of non-radioactive tracers for quantitative receptor binding assays. In the present review, we provide some general considerations and specific examples for setting up the bioluminescent binding assays. Such techniques can be applied to other protein/peptide hormones in future to facilitate their interaction studies with their receptors. PMID:27020777

  12. Validation of soluble amyloid-β precursor protein assays as diagnostic CSF biomarkers for neurodegenerative diseases.

    PubMed

    van Waalwijk van Doorn, Linda J C; Koel-Simmelink, Marleen J; Haußmann, Ute; Klafki, Hans; Struyfs, Hanne; Linning, Philipp; Knölker, Hans-Joachim; Twaalfhoven, Harry; Kuiperij, H Bea; Engelborghs, Sebastiaan; Scheltens, Philip; Verbeek, Marcel M; Vanmechelen, Eugeen; Wiltfang, Jens; Teunissen, Charlotte E

    2016-04-01

    Analytical validation of a biomarker assay is essential before implementation in clinical practice can occur. In this study, we analytically validated the performance of assays detecting soluble amyloid-β precursor protein (sAPP) α and β in CSF in two laboratories according to previously standard operating procedures serving this goal. sAPPα and sAPPβ ELISA assays from two vendors (IBL-international, Meso Scale Diagnostics) were validated. The performance parameters included precision, sensitivity, dilutional linearity, recovery, and parallelism. Inter-laboratory variation, biomarker comparison (sAPPα vs. sAPPβ) and clinical performance was determined in three laboratories using 60 samples of patients with subjective memory complaints, Alzheimer's disease, or frontotemporal dementia. All performance parameters of the assays were similar between labs and within predefined acceptance criteria. The only exceptions were minor out-of-range results for recovery at low concentrations and, despite being within predefined acceptance criteria, non-comparability of the results for evaluation of the dilutional linearity and hook-effect. Based on the inter-laboratory correlation between Lab #1 and Lab #2, the IBL-international assays were more robust (sAPPα: r(2) = 0.92, sAPPβ: r(2) = 0.94) than the Meso Scale Diagnostics (MSD) assay (sAPPα: r(2) = 0.70, sAPPβ: r(2) = 0.80). Specificity of assays was confirmed using assay-specific peptide competitors. Clinical validation showed consistent results across the clinical groups in the different laboratories for all assays. The validated sAPP assays appear to be of sufficient technical quality and perform well. Moreover, the study shows that the newly developed standard operating procedures provide highly useful tools for the validation of new biomarker assays. A recommendation was made for renewed instructions to evaluate the dilutional linearity and hook-effect. We analytically validated the performance of assays

  13. A simplified assay for the quantification of circulating activated protein C.

    PubMed

    Martos, Laura; Bonanad, Santiago; Ramón, Luis A; Cid, Ana-Rosa; Bonet, Elena; Corral, Javier; Miralles, Manuel; España, Francisco; Navarro, Silvia; Medina, Pilar

    2016-08-01

    Available assays for circulating levels of activated protein C (APC) are either time-consuming or difficult to use in a routine laboratory, or have a detection limit above normal levels. We have developed a simplified assay that measures both the in vivo free APC and the in vivo APC complexed to PC inhibitor (PCI). We measured APC levels, with both assays, in 339 plasma samples, 165 from patients with venous thromboembolism (VTE) and 174 from healthy individuals. The mean APC level in the 339 samples was 0.038±0.010 nM, using a previous assay that measures only the in vivo APC level, and 0.041±0.010 nM with the present new assay. The coefficient of correlation between assays was r=0.954 (P<0.001). The mean APC level in VTE patients was 0.034±0.009 nM (previous assay) and 0.037±0.009 nM (new assay), significantly lower than those in controls (P<0.001). In both groups there was a significant correlation between the levels obtained by the two assays (P<0.001). These results show that both assays are equivalent, and confirm that the APC level is lower in VTE patients than in healthy individuals. Therefore, the new simplified assay, which measures the sum of circulating free APC and APC complexed to PCI, may be used to estimate the level of circulating APC, and will allow its use in routine laboratories. PMID:27262823

  14. Integrated sample-to-detection chip for nucleic acid test assays.

    PubMed

    Prakash, R; Pabbaraju, K; Wong, S; Tellier, R; Kaler, K V I S

    2016-06-01

    Nucleic acid based diagnostic techniques are routinely used for the detection of infectious agents. Most of these assays rely on nucleic acid extraction platforms for the extraction and purification of nucleic acids and a separate real-time PCR platform for quantitative nucleic acid amplification tests (NATs). Several microfluidic lab on chip (LOC) technologies have been developed, where mechanical and chemical methods are used for the extraction and purification of nucleic acids. Microfluidic technologies have also been effectively utilized for chip based real-time PCR assays. However, there are few examples of microfluidic systems which have successfully integrated these two key processes. In this study, we have implemented an electro-actuation based LOC micro-device that leverages multi-frequency actuation of samples and reagents droplets for chip based nucleic acid extraction and real-time, reverse transcription (RT) PCR (qRT-PCR) amplification from clinical samples. Our prototype micro-device combines chemical lysis with electric field assisted isolation of nucleic acid in a four channel parallel processing scheme. Furthermore, a four channel parallel qRT-PCR amplification and detection assay is integrated to deliver the sample-to-detection NAT chip. The NAT chip combines dielectrophoresis and electrostatic/electrowetting actuation methods with resistive micro-heaters and temperature sensors to perform chip based integrated NATs. The two chip modules have been validated using different panels of clinical samples and their performance compared with standard platforms. This study has established that our integrated NAT chip system has a sensitivity and specificity comparable to that of the standard platforms while providing up to 10 fold reduction in sample/reagent volumes. PMID:27165104

  15. Multiplex Assay for Protein Profiling and Potency Measurement of German Cockroach Allergen Extracts

    PubMed Central

    Khurana, Taruna; Dobrovolskaia, Ekaterina; Shartouny, Jessica R.; Slater, Jay E.

    2015-01-01

    Background German cockroach (GCr) allergens induce IgE responses and may cause asthma. Commercial GCr allergen extracts are variable and existing assays may not be appropriate for determining extract composition and potency. Objective Our aim was to develop a multiplex antibody/bead-based assay for assessment of GCr allergen extracts. Methods Single chain fragment variable (scFv) antibodies against GCr were obtained by screening libraries derived from naïve human lymphocytes and hyperimmunized chicken splenocytes and bone marrow. Selected clones were sequenced and characterized by immunoblotting. Eighteen scFv antibodies (17 chicken, 1 human) coupled to polystyrene beads were used in this suspension assay; binding of targeted GCr allergens to antibody-coated beads was detected using rabbit antisera against GCr, and against specific allergens rBla g 1, rBla g 2, and rBla g 4. The assay was tested for specificity, accuracy, and precision. Extracts were also compared by IgE competition ELISA. Results Chicken scFv’s generated eight different binding patterns to GCr proteins from 14 to 150 kDa molecular weight. Human scFv’s recognized a 100 kDa GCr protein. The multiplex assay was found to be specific and reproducible with intra-assay coefficient of variation (CV) of 2.64% and inter-assay CV of 10.0%. Overall potencies of various GCr extracts were calculated using mean logEC50s for eight selected scFvs. Overall potency measures were also analyzed by assessing the contributions to potency of each target. Conclusions An scFv antibody-based multiplex assay has been developed capable of simultaneously measuring different proteins in a complex mixture, and to determine the potencies and compositions of allergen extracts. PMID:26444288

  16. Screening Oligosaccharide Libraries against Lectins Using the Proxy Protein Electrospray Ionization Mass Spectrometry Assay.

    PubMed

    Han, Ling; Shams-Ud-Doha, Km; Kitova, Elena N; Klassen, John S

    2016-08-16

    An electrospray ionization mass spectrometry (ESI-MS) assay for screening carbohydrate libraries against lectins is described. The assay is based on the proxy protein ESI-MS method, which combines direct ESI-MS protein-ligand binding measurements and competitive protein binding, to simultaneously detect and quantify protein-carbohydrate interactions. Specific interactions between components of the library and the target protein (PT) are identified from changes in the relative abundances (as measured by ESI-MS) of the carbohydrate complexes of a proxy protein (Pproxy), which binds to all components of the library with known affinity, upon addition of PT to the solution. The magnitude of the change in relative abundance of a given Pproxy-ligand complex provides a quantitative measure of the affinity of the corresponding PT-ligand interaction. A mathematical framework for the implementation of the method in the case of monovalent (single binding site) Pproxy and monovalent and multivalent (multiple equivalent and independent binding sites) PT is described. The application of the method to screen small libraries of oligosaccharides, on the basis of human histo-blood group antigens and milk oligosaccharides, against an N-terminal fragment of the family 51 carbohydrate-binding module, a fucose-binding lectin from Ralstonia solanacearum, and human norovirus VA387 P particle (24-mer of the protruding domain of the capsid protein), serves to demonstrate the reliability and versatility of the assay. PMID:27366913

  17. Protein stability regulators screening assay (Pro-SRSA): protein degradation meets the CRISPR-Cas9 library.

    PubMed

    Wu, Yuanzhong; Kang, Tiebang

    2016-01-01

    The regulation of protein stability is a fundamental issue for biophysical processes, but there has not previously been a convenient and unbiased method of identifying regulators of protein stability. However, as reported in the article entitled "A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A," recently published in Cell Discovery, our team developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome clustered regularly interspaced short palindromic repeats Cas9 (CRISPR-Cas9) library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Based on our findings, we are confident that this efficient and unbiased screening method at the genome scale will be used by researchers worldwide to identify regulators of protein stability. PMID:27357860

  18. Fluorometric functional assay for ion channel proteins in lipid nanovesicle membranes

    NASA Astrophysics Data System (ADS)

    Patti, J. T.; Montemagno, C. D.

    2007-08-01

    Voltage-gated membrane proteins function as biomolecular transistors, making them attractive components for biologically based nanodevices. A functional assay for purified channel proteins is described and demonstrated with sodium selective, voltage-gated NaChBac ion channels. Purified NaChBac proteins were incorporated into a nanovesicle system utilizing oxonol VI, a fluorescent indicator of trans-membrane voltage. The ionophore valinomycin was used to trigger a change in membrane potential, allowing the observation of sodium permeability using a fluorometer. This method is suitable for concurrently testing a large population of purified proteins prior to incorporation in nanodevices.

  19. Fluorinated amino acids: compatibility with native protein structures and effects on protein-protein interactions.

    PubMed

    Salwiczek, Mario; Nyakatura, Elisabeth K; Gerling, Ulla I M; Ye, Shijie; Koksch, Beate

    2012-03-21

    Fluorinated analogues of the canonical α-L-amino acids have gained widespread attention as building blocks that may endow peptides and proteins with advantageous biophysical, chemical and biological properties. This critical review covers the literature dealing with investigations of peptides and proteins containing fluorinated analogues of the canonical amino acids published over the course of the past decade including the late nineties. It focuses on side-chain fluorinated amino acids, the carbon backbone of which is identical to their natural analogues. Each class of amino acids--aliphatic, aromatic, charged and polar as well as proline--is presented in a separate section. General effects of fluorine on essential properties such as hydrophobicity, acidity/basicity and conformation of the specific side chains and the impact of these altered properties on stability, folding kinetics and activity of peptides and proteins are discussed (245 references). PMID:22130572

  20. Protein analytical assays for diagnosing, monitoring, and choosing treatment for cancer patients

    PubMed Central

    Powers, Alicia D.; Palecek, Sean P.

    2013-01-01

    Cancer treatment is often hindered by inadequate methods for diagnosing the disease or insufficient predictive capacity regarding therapeutic efficacy. Targeted cancer treatments, including Bcr-Abl and EGFR kinase inhibitors, have increased survival for some cancer patients but are ineffective in other patients. In addition, many patients who initially respond to targeted inhibitor therapy develop resistance during the course of treatment. Molecular analysis of cancer cells has emerged as a means to tailor treatment to particular patients. While DNA analysis can provide important diagnostic information, protein analysis is particularly valuable because proteins are more direct mediators of normal and diseased cellular processes. In this review article, we discuss current and emerging protein assays for improving cancer treatment, including trends toward assay miniaturization and measurement of protein activity. PMID:25147725

  1. Demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins

    PubMed Central

    Kennedy, Jacob J.; Abbatiello, Susan E.; Kim, Kyunggon; Yan, Ping; Whiteaker, Jeffrey R.; Lin, Chenwei; Kim, Jun Seok; Zhang, Yuzheng; Wang, Xianlong; Ivey, Richard G.; Zhao, Lei; Min, Hophil; Lee, Youngju; Yu, Myeong-Hee; Yang, Eun Gyeong; Lee, Cheolju; Wang, Pei; Rodriguez, Henry; Kim, Youngsoo; Carr, Steven A.; Paulovich, Amanda G.

    2014-01-01

    The successful application of MRM in biological specimens raises the exciting possibility that assays can be configured to measure all human proteins, resulting in an assay resource that would promote advances in biomedical research. We report the results of a pilot study designed to test the feasibility of a large-scale, international effort in MRM assay generation. We have configured, validated across three laboratories, and made publicly available as a resource to the community 645 novel MRM assays representing 319 proteins expressed in human breast cancer. Assays were multiplexed in groups of >150 peptides and deployed to quantify endogenous analyte in a panel of breast cancer-related cell lines. Median assay precision was 5.4%, with high inter-laboratory correlation (R2 >0.96). Peptide measurements in breast cancer cell lines were able to discriminate amongst molecular subtypes and identify genome-driven changes in the cancer proteome. These results establish the feasibility of a scaled, international effort. PMID:24317253

  2. Rapid Sensitive Assay for Interferons Based on the Inhibition of MM Virus Nucleic Acid Synthesis

    PubMed Central

    Allen, Patton T.; Giron, David J.

    1970-01-01

    A method for assaying mouse interferon based on the inhibition of viral ribonucleic acid (RNA) synthesis was devised. The amount of MM virus and RNA synthesized in interferon-treated L-cell cultures was determined by measuring the amount of 3H-uridine converted into a trichloroacetic acid-insoluble form after treatment of the infected cultures with 2.5 μg of actinomycin D per ml. The amount of RNA synthesized was inversely related to the concentration of interferon used for treatment. A linear dose-response regression curve was obtained by plotting the log of the amount of RNA made, expressed as a percentage of the control, versus the log of the reciprocal of the interferon dilution. A unit of interferon was defined as that concentration which inhibited nucleic acid synthesis by 50% (INAS50). The concentration of mouse interferon could be determined within 24 hr. This assay method, on the average, was approximately half as sensitive as the method which measured the 50% reduction of MM virus plaque number (PDD50-MM method), but was, on the average, almost 1.7 times as sensitive as the PDD50-VSV method. It averaged approximately 20 times the sensitivity of the methods which used as end points the 70% reduction in yield of MM virus or the complete inhibition of cytopathic effect by MM virus. The reproducibility of the INAS50 technique was tested in two ways. (i) Four independent assays of an interferon specimen were performed with replicate cultures. The standard deviation was 11.2% of the mean titer. (ii) On different dates, one interferon specimen was assayed seven times and another was assayed four times. The standard deviations were 21.5 and 26.6% of the respective mean titers. PMID:4320919

  3. Final Report Nucleic Acid System - PCR, Multiplex Assays and Sample Preparation Project

    SciTech Connect

    Koopman, R.P.; Langlois, R.G.; Nasarabadi, S.; Benett, W.J.; Richards, J.B.; Hadley, D.R.; Miles, R.R.; Brown, S.B.; Stratton, P.L.; Milanovich, F.P.

    2001-04-20

    The objective of this project was to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction). This entailed not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This project had two principal deliverables: (1) design, construct, test and deliver a 24 chamber, multiplex capable suitcase sized PCR instrument, and (2) develop and reduce to practice a multiplex assay for the detection of PCR product by flow cytometry. In addition, significant resources were allocated to test and evaluation of the Hand-held Advanced Nucleic Acid Analyzer (HANAA). This project helps provide the signature and intelligence gathering community the ability to perform, on-site or remote, rapid analysis of environmental or like samples for the presence of a suite of biological warfare pathogens.

  4. A helicase assay based on the displacement of fluorescent, nucleic acid-binding ligands.

    PubMed Central

    Eggleston, A K; Rahim, N A; Kowalczykowski, S C

    1996-01-01

    We have developed a new helicase assay that overcomes many limitations of other assays used to measure this activity. This continuous, kinetic assay is based on the displacement of fluorescent dyes from dsDNA upon DNA unwinding. These ligands exhibit significant fluorescence enhancement when bound to duplex nucleic acids and serve as the reporter molecules of DNA unwinding. We evaluated the potential of several dyes [acridine orange, ethidium bromide, ethidium homodimer, bis-benzimide (DAPI), Hoechst 33258 and thiazole orange] to function as suitable reporter molecules and demonstrate that the latter three dyes can be used to monitor the helicase activity of Escherichia coli RecBCD enzyme. Both the binding stoichiometry of RecBCD enzyme for the ends of duplex DNA and the apparent rate of unwinding are not significantly perturbed by two of these dyes. The effects of temperature and salt concentration on the rate of unwinding were also examined. We propose that this dye displacement assay can be readily adapted for use with other DNA helicases, with RNA helicases, and with other enzymes that act on nucleic acids. PMID:8614617

  5. A continuous fluorescence displacement assay for the measurement of phospholipase A2 and other lipases that release long-chain fatty acids.

    PubMed Central

    Wilton, D C

    1990-01-01

    1. A new continuous fluorescence assay for phospholipase A2 is described which involves the displacement of the highly fluorescent fatty-acid probe 11-(dansylamino)undecanoic acid from rat liver fatty-acid-binding protein by long-chain fatty acids released as a result of phospholipase A2-catalysed hydrolysis of phospholipids. The initial rate of decrease in fluorescence is linearly related to enzyme activity. 2. The assay will detect enzyme activity down to about 10 pmol/min per ml and gives a linear response up to about 10 nmol/min per ml. 3. The assay will work with all phospholipids that have been tested including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and phosphatidylglycerol. Substrates carrying a net negative charge showed the highest rates of hydrolysis. 4. The assay will work, in principle, with an enzyme catalysing the release of long-chain fatty acids from a fatty-acylated substrate. This has been confirmed with pancreatic lipase and cholesterol esterase. PMID:2317197

  6. In vitro and in vivo assays of protein kinase CK2 activity.

    PubMed

    Prudent, Renaud; Sautel, Céline F; Moucadel, Virginie; Laudet, Béatrice; Filhol, Odile; Cochet, Claude

    2010-01-01

    Protein kinase CK2 (formerly casein kinase 2) is recognized as a central component in the control of the cellular homeostasis; however, much remains unknown regarding its regulation and its implication in cellular transformation and carcinogenesis. Moreover, study of CK2 function and regulation in a cellular context is complicated by the dynamic multisubunit architecture of this protein kinase. Although a number of robust techniques are available to assay CK2 activity in vitro, there is a demand for sensitive and specific assays to evaluate its activity in living cells. We hereby provide a detailed description of several assays for monitoring the CK2 activity and its subunit interaction in living cells. The guidelines presented herein should enable researchers in the field to establish strategies for cellular screenings of CK2 inhibitors. PMID:21050938

  7. Bioanalytical method validation considerations for LC-MS/MS assays of therapeutic proteins.

    PubMed

    Duggan, Jeffrey X; Vazvaei, Faye; Jenkins, Rand

    2015-01-01

    This paper highlights the recommendations of a group of industry scientists in validating regulated bioanalytical LC-MS/MS methods for protein therapeutics in a 2015 AAPSJ White Paper. This group recommends that most of the same precision and accuracy validation criteria used for ligand-binding assays (LBAs) be applied to LC-MS/MS-based assays where proteins are quantified using the LC-MS/MS signal from a surrogate peptide after proteolytic digestion (PrD-LCMS methods). PrD-LCMS methods are generally more complex than small molecule LC-MS/MS assays and may often include LBA procedures, leading to the recommendation for a combination of chromatographic and LBA validation strategies and appropriate acceptance criteria. Several key aspects of this bioanalytical approach that are discussed in the White Paper are treated here in additional detail. These topics include selectivity/specificity, matrix effect, digestion efficiency, stability and critical reagent considerations. PMID:26110712

  8. Nucleic acids encoding human trithorax protein

    DOEpatents

    Evans, Glen A.; Djabali, Malek; Selleri, Licia; Parry, Pauline

    2001-01-01

    In accordance with the present invention, there is provided an isolated peptide having the characteristics of human trithorax protein (as well as DNA encoding same, antisense DNA derived therefrom and antagonists therefor). The invention peptide is characterized by having a DNA binding domain comprising multiple zinc fingers and at least 40% amino acid identity with respect to the DNA binding domain of Drosophila trithorax protein and at least 70% conserved sequence with respect to the DNA binding domain of Drosophila trithorax protein, and wherein said peptide is encoded by a gene located at chromosome 11 of the human genome at q23. Also provided are methods for the treatment of subject(s) suffering from immunodeficiency, developmental abnormality, inherited disease, or cancer by administering to said subject a therapeutically effective amount of one of the above-described agents (i.e., peptide, antagonist therefor, DNA encoding said peptide or antisense DNA derived therefrom). Also provided is a method for the diagnosis, in a subject, of immunodeficiency, developmental abnormality, inherited disease, or cancer associated with disruption of chromosome 11 at q23.

  9. In situ screening assay for cell viability using a dimeric cyanine nucleic acid stain.

    PubMed

    Becker, B; Clapper, J; Harkins, K R; Olson, J A

    1994-08-15

    A rapid and sensitive assay is described for the determination of cell viability of adherent and nonadherent cells that can be performed in situ in 96-well microtiter plates using fluorescence plate scanners. The assay, based on dye exclusion, utilizes a plasma membrane-impermeable, dimeric cyanine dye (YOYO-1). YOYO-1 fluoresces brightly only when bound to nucleic acids. Cells are incubated with YOYO-1, and fluorescence is measured before and after the addition of detergent, which allows the dye to enter the cells. The fluorescence before detergent treatment originates from nonviable cells that have membrane damage and take up YOYO-1. The fluorescence after detergent treatment originates from all cells in the sample. The ratio of the two fluorescence values is used as an indicator of cell viability. The cell viability results of this microplate assay closely resemble those of dye exclusion studies by flow cytometry and are similar but not identical to those of the thiazolyl blue assay, which uses a metabolic indicator of cell death. Because the assay can be performed in situ, without removing the medium, disintegrated cells, cell aggregates, and cells that stick to culture vessel walls are all included in the measurement. PMID:7527190

  10. Determination of boron in produced water using the carminic acid assay.

    PubMed

    Floquet, Cedric F A; Sieben, Vincent J; MacKay, Bruce A; Mostowfi, Farshid

    2016-04-01

    Using the carminic acid assay, we determined the concentration of boron in oilfield waters. We investigated the effect of high concentrations of salts and dissolved metals on the assay performance. The influence of temperature, development time, reagent concentration, and water volume was studied. Ten produced and flowback water samples of different origins were measured, and the method was successfully validated against ICP-MS measurements. In water-stressed regions, produced water is a potential source of fresh water for irrigation, industrial applications, or consumption. Therefore, boron concentration must be determined and controlled to match the envisaged waste water reuse. Fast, precise, and onsite measurements are needed to minimize errors introduced by sample transportation to laboratories. We found that the optimum conditions for our application were a 5:1 mixing volume ratio (reagent to sample), a 1 g L(-1) carminic acid concentration in 99.99% sulfuric acid, and a 30 min reaction time at ambient temperature (20 °C to 23 °C). Absorption values were best measured at 610 nm and 630 nm and baseline corrected at 865 nm. Under these conditions, the sensitivity of the assay to boron was maximized while its cross-sensitivity to dissolved titanium, iron, barium and zirconium was minimized, alleviating the need for masking agents and extraction methods. PMID:26838405

  11. DEVELOPMENT OF ENZYME-LINKED IMMUNOSORBENT ASSAYS FOR ISOCUPRESSIC ACID AND SERUM METABOLITES OF ISOCUPRESSIC ACID

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The consumption of ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), common juniper (Juniperus communis) and Monterey cypress (Cupressus macrocarpa) causes abortions in pregnant cattle. Recent studies have identified isocupressic acid as the primary abortifacient compound in these ...

  12. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2011-12-06

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  13. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2011-03-22

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  14. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2008-10-07

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  15. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2012-02-14

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  16. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2009-04-28

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  17. Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

    PubMed Central

    Hollender, Courtney A.; Liu, Zhongchi

    2010-01-01

    Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells. PMID:20567209

  18. Bimolecular fluorescence complementation (BiFC) assay for protein-protein interaction in onion cells using the helios gene gun.

    PubMed

    Hollender, Courtney A; Liu, Zhongchi

    2010-01-01

    Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay(1) allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)(2) and the visualization of their interactions mediated by BiFC in onion epidermal cells. PMID:20567209

  19. Development of a microplate-based, electrophoretic fluorescent protein kinase a assay: comparison with filter-binding and fluorescence polarization assay formats.

    PubMed

    Miick, Siobhan M; Jalali, Shila; Dwyer, Brian P; Havens, John; Thomas, Donald; Jimenez, Manuel A; Simpson, Mathew T; Zile, Betsy; Huss, Karen L; Campbell, Robert M

    2005-06-01

    A microplate-based electrophoretic assay has been developed for the serine/threonine kinase protein kinase A (PKA). The ElectroCapture PKA assay developed uses a positively charged, lissamine-rhodamine-labeled kemptide peptide substrate for the kinase reaction and Nanogen's ElectroCapture HTS Workstation and 384-well laminated membrane plates to electrophoretically separate the negatively charged phosphorylated peptide product from the kinase reaction mix. After the electrophoretic separation, the amount of rhodamine-labeled phosphopeptide product was quantified using a Tecan Ultra384 fluorescence reader. The ElectroCapture PKA assay was validated with both known PKA inhibitors and library compounds. The pK(iapp) results obtained in the ElectroCapture PKA assay were comparable to those generated with current radioactive filter-binding assay and antibody-based competitive fluorescence polarization PKA assay formats. PMID:15964934

  20. A high-throughput differential filtration assay to screen and select detergents for membrane proteins

    PubMed Central

    Vergis, James M.; Purdy, Michael D.; Wiener, Michael C.

    2015-01-01

    Structural studies on integral membrane proteins are routinely performed on protein–detergent complexes (PDCs) consisting of purified protein solubilized in a particular detergent. Of all the membrane protein crystal structures solved to date, a subset of only four detergents has been used in more than half of these structures. Unfortunately, many membrane proteins are not well behaved in these four detergents and/or fail to yield well-diffracting crystals. Identification of detergents that maintain the solubility and stability of a membrane protein is a critical step and can be a lengthy and “protein-expensive” process. We have developed an assay that characterizes the stability and size of membrane proteins exchanged into a panel of 94 commercially available and chemically diverse detergents. This differential filtration assay (DFA), using a set of filtered microplates, requires sub-milligram quantities of purified protein and small quantities of detergents and other reagents and is performed in its entirety in several hours. PMID:20667442

  1. A novel photoelectrochemical biosensor for protein kinase activity assay based on phosphorylated graphite-like carbon nitride.

    PubMed

    Li, Xue; Zhou, Yunlei; Xu, Yan; Xu, Huijie; Wang, Minghui; Yin, Huanshun; Ai, Shiyun

    2016-08-31

    Protein kinases are general and significant regulators in the cell signaling pathway, and it is still greatly desired to achieve simple and quick kinase detection. Herein, we develop a simple and sensitive photoelectrochemical strategy for the detection of protein kinase activity based on the bond between phosphorylated peptide and phosphorylated graphite-like carbon nitride (P-g-C3N4) conjugates triggered by Zr(4+) ion coordination. Under optimal conditions, the increased photocurrent is proportional to the protein kinase A (PKA) concentration ranging from 0.05 to 50 U/mL with a detection limit of 0.077 U/mL. Moreover, this photoelectrochemical assay can be also applied to quantitative analysis of kinase inhibition. The results indicated that the IC50 value (inhibitor concentration producing 50% inhibitor) for ellagic acid was 9.1 μM. Moreover, the developed method is further applied to detect PKA activity in real samples, which contains serum from healthy person and gastric cancer patients and breast tissue from healthy person and breast cancer patients. Therefore, the established protocol provides a new and simple tool for assay of kinase activity and its inhibitors with low cost and high sensitivity. PMID:27506341

  2. A fluorometric assay platform for caffeic acid detection based on the G-quadruplex/hemin DNAzyme.

    PubMed

    Cai, Nan; Li, Yan; Chen, Shufan; Su, Xingguang

    2016-07-21

    In this paper, a fluorometric assay platform for fluorescence detection of caffeic acid was designed based on the peroxidase-mimicking activities of G-quadruplex/hemin DNAzyme. Under the catalysis of the formed G-quadruplex/hemin complex, H2O2 could be decomposed into hydroxyl radicals with strong oxidation properties. Then caffeic acid would be oxidized by the released hydroxyl radicals, resulting in the product caffeic acid-quinone. Normally, caffeic acid has no influence on the fluorescence of graphene quantum dots. But when mixed with the G-quadruplex/hemin complex and H2O2, the fluorescence of graphene quantum dots was obviously quenched by the oxidized caffeic acid. Under the optimized experimental conditions, the quenched fluorescence intensity was linearly correlated with the concentration of caffeic acid, ranging from 2 μM to 350 μM with a detection limit of 200 nM. The proposed method was applied to the determination of caffeic acid in human serum samples with satisfactory results. PMID:27220084

  3. Final Report Nucleic Acid System - Hybrid PCR and Multiplex Assay Project Phase 2

    SciTech Connect

    Koopman, R P; Langlois, R G; Nasarabadi, S; Benett, W J; Colston, B W; Johnson, D C; Brown, S B; Stratton, P L; Milanovich, F P

    2002-04-17

    This report covers phase 2 (year 2) of the Nucleic Acid System--Hybrid PCR and Multiplex Assay project. The objective of the project is to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction) in a multiplex mode using flow cytometry. The Hybrid instrument consists of a flow-through PCR module capable of handling a multiplexed PCR assay, a hybridizing module capable of hybridizing multiplexed PCR amplicons and beads, and a flow cytometer module for bead-based identification, all controlled by a single computer. Multiplex immunoassay using bead-based Luminex flow cytometry is available, allowing rapid screening for many agents. PCR is highly specific and complements and verifies immunoassay. It can also be multiplexed and detection provided using the bead-based Luminex flow cytometer. This approach allows full access to the speed and 100-fold multiplex capability of flow cytometry for rapid screening as well as the accuracy and specificity of PCR. This project has two principal activities: (1) Design, build and test a prototype hybrid PCR/flow cytometer with the basic capabilities for rapid, broad spectrum detection and identification, and (2) Develop and evaluate multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products. This project requires not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This involves development and evaluation of multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products.

  4. A bioluminescent assay for monitoring conjugation of ubiquitin and ubiquitin-like proteins.

    PubMed

    Mondal, Subhanjan; Hsiao, Kevin; Goueli, Said A

    2016-10-01

    Post-translational modification of target proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins is a critical mechanism for regulating protein functions affecting diverse cellular processes. Ub/Ubl proteins are conjugated to lysine residues in substrate proteins through an adenosine triphosphate (ATP)-dependent enzymatic cascade involving enzyme 1 (E1)-activating enzyme, E2-conjugating enzyme, and E3 ligase. The amount of adenosine monophosphate (AMP) produced in the first step, involving E1-mediated Ub/Ubl activation, represents an accurate measure of Ub/Ubl transfer during the process. Here we describe a novel bioluminescent assay platform, AMP-Glo, to quantify Ub/Ubl conjugation by measuring the AMP generated. The AMP-Glo assay is performed in a two-step reaction. The first step terminates the ubiquitination reaction, depletes the remaining ATP, and converts the AMP generated in the ubiquitination reaction to adenosine diphosphate (ADP), and in the second step the ADP generated is converted to ATP, which is detected as a bioluminescent signal using luciferase/luciferin, proportional to the AMP concentration and correlated with the Ub/Ubl transfer activity. We demonstrate the use of the assay to study Ub/Ubl conjugation and screen for chemical modulators of enzymes involved in the process. Because there is a sequential enhancement in light output in the presence of E1, E2, and E3, the AMP-Glo system can be used to deconvolute inhibitor specificity. PMID:27325501

  5. Radiometric immunosorbent assay for the detection of anti-hormone-binding protein antibodies

    SciTech Connect

    Pierce, E.A.; Dame, M.C.; DeLuca, H.F.

    1986-02-15

    A radiometric immunosorbent assay (RISA) for the detection of monoclonal antibodies to hormone-binding proteins has been developed. The assay involves incubating hybridoma supernatants in microtiter wells that have been coated with goat anti-mouse IgG antibodies. Any mouse IgG in the test supernatant is thus specifically retained in the wells. Radioactive ligand-binding protein complexes are then incubated in the wells. The presence of anti-binding protein antibodies in the supernatant is indicated by specific retention of radioactive ligand-binding protein complexes in the wells. Crude antigen preparations, such as tissue homogenates, can be used to detect antibodies. The assay is capable of detecting antibody at concentrations 20 ng/ml (approx. 100 pM IgG). The RISA has been used successfully to screen for monoclonal antibodies to the intracellular receptor for 1,25-dihydroxyvitamin D/sub 3/ and should be useful for the detection of antibodies to ligand-binding proteins in general.

  6. An ultra-high performance liquid chromatography-tandem mass spectrometric assay for quantifying 3-ketocholanoic acid: Application to the human liver microsomal CYP3A-dependent lithocholic acid 3-oxidation assay.

    PubMed

    Bansal, Sumit; Chai, Swee Fen; Lau, Aik Jiang

    2016-06-15

    Lithocholic acid (LCA), a hepatotoxic and carcinogenic bile acid, is metabolized to 3-ketocholanoic acid (3-KCA) by cytochrome P450 3A (CYP3A). In the present study, the objectives were to develop and validate an ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify 3-KCA and apply it to the human liver microsomal CYP3A-dependent LCA 3-oxidation assay. Chromatographic separation was achieved on a Waters ACQUITY™ UPLC C18 column (50×2.1mm, 1.7μm) with a gradient system consisting of 0.1% v/v formic acid in water (solvent A) and 0.1% v/v formic acid in acetonitrile (solvent B). The retention time was 3.73min for 3-KCA and 2.73min for cortisol (internal standard). Positive electrospray ionization with multiple reaction monitoring (MRM) mode was used to quantify 3-KCA (m/z 375.4→135.2) and cortisol (m/z 363.5→121.0). The limit of detection of 3-KCA was 10μM, the lower limit of quantification was 33.3μM, and the calibration curve was linear from 0.05-10μM with r(2)>0.99. Intra-day and inter-day accuracy and precision were <13.7%. The quality control samples were stable when assessed after 4h at room temperature, 24h at 4°C, 14days at -20°C, and three freeze-thaw cycles. The liver microsomal matrix did not affect 3-KCA quantification. The amount of KCA formed in the human liver microsomal LCA 3-oxidation assay was linear with respect to the amount of microsomal protein (up to 40μg) and incubation time (5-30min). Enzyme kinetics experiment indicated that LCA 3-oxidation followed the Michaelis-Menten model with an apparent Km of 26±7μM and Vmax of 303±50pmol/min/mg protein. This novel UPLC-MS/MS method for quantifying 3-KCA offers a specific, sensitive, and fast approach to determine liver microsomal LCA 3-oxidation. PMID:27153105

  7. Assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay.

    PubMed

    Johnson, Christopher J; Carlson, Christina M; Morawski, Aaron R; Manthei, Alyson; Cashman, Neil R

    2015-01-01

    Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host's species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host's species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent. PMID:25867521

  8. Assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay

    USGS Publications Warehouse

    Johnson, Christopher J.; Carlson, Christina M.; Morawski, Aaron R.; Manthei, Alyson; Cashman, Neil R.

    2015-01-01

    Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitroprion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host’s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host’s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

  9. Photocleavable Peptide-Oligonucleotide Conjugates for Protein Kinase Assays by MALDI-TOF MS†

    PubMed Central

    Zhou, Guangchang; Khan, Faraz; Dai, Qing; Sylvester, Juliesta E.; Kron, Stephen J.

    2012-01-01

    Robust methods for highly parallel, quantitative analysis of cellular protein tyrosine kinase activities may provide tools critically needed to decipher oncogenic signaling, discover new targeted drugs, diagnose cancer and monitor patients. Here, we describe proof-of-principle for a novel protein kinase assay with potential to answer these challenges. MALDI-TOF mass spectrometry provides an ideal tool for label-free multiplexed analysis of peptide phosphorylation, but is poorly matched to homogeneous assays and complex samples. Thus, we conjugated a common oligonucleotide tag to multiple peptide substrates, offering efficient capture from solution-phase kinase reactions by annealing to the complementary sequence tethered to PEG-passivated superparamagnetic microparticles. To enable reversible conjugation, we developed a novel bifunctional cross-linker allowing simple and efficient preparation of photocleavable peptide-oligonucleotide conjugates. After washing away contaminants and photorelease, MALDI-TOF analysis yielded relative phosphorylation of each peptide with high sensitivity and specificity. Validating the hybridization-mediated multiplexed kinase assay, when three peptide substrate-oligonucleotide conjugates were mixed with the tyrosine kinase c-Abl and ATP, we readily observed their differential phosphorylation yet measured a common IC50 for the Abl kinase inhibitor imatinib. This new assay enables analysis of protein kinase activities in a multiplexed format amenable to screening inhibitors against multiple kinases in parallel, an important capability for drug discovery and predictive diagnostics. PMID:22772337

  10. Annotation of human cancers with EGFR signaling-associated protein complexes using proximity ligation assays

    PubMed Central

    Smith, Matthew A.; Hall, Richard; Fisher, Kate; Haake, Scott M.; Khalil, Farah; Schabath, Matthew B.; Vuaroqueaux, Vincent; Fiebig, Heinz-Herbert; Altiok, Soner; Chen, Y. Ann; Haura, Eric B.

    2015-01-01

    Strategies to measure functional signaling-associated protein complexes have the potential to augment current molecular biomarker assays, such as genotyping and expression profiling, used to annotate diseases. Aberrant activation of epidermal growth factor receptor (EGFR) signaling contributes to diverse cancers. Here, we used a proximity ligation assay (PLA) to detect EGFR in a complex with growth factor receptor-bound protein 2 (GRB2), the major signaling adaptor for EGFR. We used multiple lung cancer cell lines to develop and characterize EGFR:GRB2 PLA and correlated this assay with established biochemical measures of EGFR signaling. In a panel of patient-derived xenografts in mice, the intensity of EGFR:GRB2 PLA correlated with the reduction in tumor size in response to the EGFR inhibitor cetuximab. In tumor biopsies from three cohorts of lung cancer patients, positive EGFR:GRB2 PLA was observed in patients with and without EGFR mutations and the intensity of EGFR:GRB2 PLA was predictive of overall survival in an EGFR inhibitor-treated cohort. Thus, we established the feasibility of using PLA to measure EGFR signaling-associated protein complexes in patient-based materials, suggesting the potential for similar assays for a broader array of receptor tyrosine kinases and other key signaling molecules. PMID:25587191

  11. Development of Microcolumn-Based One-Site Immunometric Assays for Protein Biomarkers

    PubMed Central

    Pfaunmiller, Erika L.; Anguizola, Jeanethe A.; Milanuk, Mitchell L.; Papastavros, Efthimia; Carter, NaTasha; Matsuda, Ryan; Zheng, Xiwei; Hage, David S.

    2014-01-01

    One-site immunometric assays that utilize affinity microcolumns were developed and evaluated for the analysis of protein biomarkers. This approach used labeled antibodies that were monitored through on-line fluorescence or near-infrared (NIR) fluorescence detection. Human serum albumin (HSA) was utilized as a model target protein for this approach. In these assays, a fixed amount of labeled anti-HSA antibodies was mixed with samples or standards containing HSA, followed by the injection of this mixture onto an HSA microcolumn to remove excess antibodies and detect the non-retained labeled antibodies that were bound to HSA from the sample. The affinity microcolumns were 2.1 mm i.d. × 5 mm and contained 8-9 nmol of immobilized HSA. These microcolumns were used from 0.10-1.0 mL/min and gave results within 35 s to 2.8 min of sample injection. Limits of detection down to 0.10-0.28 ng/mL (1.5-4.2 pM) or 25-30 pg/mL (0.38-0.45 pM) were achieved when using fluorescein or a NIR fluorescent dye as the label, with an assay precision of ± 0.1-4.2%. Several parameters were examined during the optimization of these assays, and general guidelines and procedures were developed for the extension of this approach for use with other types of affinity microcolumns and protein biomarkers. PMID:25263063

  12. A CAPS-based binding assay provides semi-quantitative validation of protein-DNA interactions

    PubMed Central

    Xie, Yongyao; Zhang, Yaling; Zhao, Xiucai; Liu, Yao-Guang; Chen, Letian

    2016-01-01

    Investigation of protein-DNA interactions provides crucial information for understanding the mechanisms of gene regulation. Current methods for studying protein-DNA interactions, such as DNaseI footprinting or gel shift assays, involve labeling DNA with radioactive or fluorescent tags, making these methods costly, laborious, and potentially damaging to the environment. Here, we describe a novel cleaved amplified polymorphic sequence (CAPS)-based binding assay (CBA), which is a label-free method that can simplify the semi-quantitative validation of protein-DNA interactions. The CBA tests the interaction between a protein and its target DNA, based on the CAPS pattern produced due to differences in the accessibility of a restriction endonuclease site (intrinsic or artificial) in amplified DNA in the presence and absence of the protein of interest. Thus, the CBA can produce a semi-quantitative readout of the interaction strength based on the dose of the binding protein. We demonstrate the principle and feasibility of CBA using B3, MADS3 proteins and the corresponding RY or CArG-box containing DNAs. PMID:26877240

  13. Rapid turbidimetric assay for quantification of fusidic acid in a dermatological cream.

    PubMed

    Curbete, Mariane Machado; Salgado, Hérida Regina Nunes

    2016-06-01

    Fusidic acid is an antibiotic steroid widely used for the treatment of serious infections caused by methicillin-resistant Staphylococcus aureus (MRSA) strains. Microbiological methods are indispensable to determine the mean percentage of antimicrobial in medicaments during manufacturing and quality control processes. The aim of this study was to develop and validate a microbiological method for the quantification of fusidic acid in dermatological cream by turbidimetry, using Staphylococcus epidermidis (ATCC 12228) and casoy broth as the culture medium. The validation parameters were in accordance with ICH specifications and demonstrated accuracy, precision, selectivity, and robustness, with linear ranges from 0.25 to 2.25μgmL(-1). This method is an alternative to the diffusion agar assay currently employed to quantify fusidic acid in dermatological cream, since it is sensitive, fast, and more economical. PMID:27130089

  14. Degradation of caffeic acid in subcritical water and online HPLC-DPPH assay of degradation products.

    PubMed

    Khuwijitjaru, Pramote; Suaylam, Boonyanuch; Adachi, Shuji

    2014-02-26

    Caffeic acid was subjected to degradation under subcritical water conditions within 160-240 °C and at a constant pressure of 5 MPa in a continuous tubular reactor. Caffeic acid degraded quickly at these temperatures; the main products identified by liquid chromatography-diode array detection/mass spectrometry were hydroxytyrosol, protocatechuic aldehyde, and 4-vinylcatechol. The reaction rates for the degradation of caffeic acid and the formation of products were evaluated. Online high-performance liquid chromatography/2,2-diphenyl-1-picryhydrazyl assay was used to determine the antioxidant activity of each product in the solution. It was found that the overall antioxidant activity of the treated solution did not change during the degradation process. This study showed a potential of formation of antioxidants from natural phenolic compounds under these subcritical water conditions, and this may lead to a discovering of novel antioxidants compounds during the extraction by this technique. PMID:24483598

  15. Enzyme-linked immunosorbent assay (ELISA) for the anthropogenic marker isolithocholic acid in water.

    PubMed

    Baldofski, Stefanie; Hoffmann, Holger; Lehmann, Andreas; Breitfeld, Stefan; Garbe, Leif-Alexander; Schneider, Rudolf J

    2016-11-01

    Bile acids are promising chemical markers to assess the pollution of water samples with fecal material. This study describes the optimization and validation of a direct competitive enzyme-linked immunosorbent assay for the bile acid isolithocholic acid (ILA). The quantification range of the optimized assay was between 0.09 and 15 μg/L. The assay was applied to environmental water samples. Most studies until now were focused on bile acid fractions in the particulate phase of water samples. In order to avoid tedious sample preparation, we undertook to evaluate the dynamics and significance of ILA levels in the aqueous phase. Very low concentrations in tap and surface water samples made a pre-concentration step necessary for this matrix as well as for wastewater treatment plant (WWTP) effluent. Mean recoveries for spiked water samples were between 97% and 109% for tap water and WWTP influent samples and between 102% and 136% for WWTP effluent samples. 90th percentiles of intra-plate and inter-plate coefficients of variation were below 10% for influents and below 20% for effluents and surface water. ILA concentrations were quantified in the range of 33-72 μg/L in influent, 21-49 ng/L in effluent and 18-48 ng/L in surface water samples. During wastewater treatment the ILA levels were reduced by more than 99%. ILA concentrations of influents determined by ELISA and LC-MS/MS were in good agreement. However, findings in LC-ELISA experiments suggest that the true ILA levels in concentrated samples are lower due to interfering effects of matrix compounds and/or cross-reactants. Yet, the ELISA will be a valuable tool for the performance check and comparison of WWTPs and the localization of fecal matter input into surface waters. PMID:27544648

  16. Microspectrophotometric quantitation of nucleic acid and protein in irradiated epidermis.

    PubMed

    Conti, C J; Giménez, I B; Cabrini, R L

    1976-03-01

    Nucleic acid and proteins of newborn rat tail subjected to local X-irradiation were microspectrophotometrically studied. Feulgen, gallocyanine chrom-alum and naphthol yellow S methods were performed for demonstration of DNA, total nucleic acid and proteins respectively. The amount of proteins and total nucleic acid increases concomitantly with reactional acanthosis. However, the proteins and nucleic acid decrease as from day 3 post-irradiation. A tentative interpretation of the results would point to a giantization of the epidermic cells not only caused by aqueous imbition but also by an actual increase of the cellular protoplasm. PMID:1258094

  17. Relatedness of acyl carrier proteins shown by amino acid compositions.

    PubMed

    Walker, T A; Ernst-Fonberg, M L

    1982-01-01

    1. Relatedness among the following carrier proteins was assessed on the basis of amino acid compositions: eight acyl carrier proteins (ACP's) associated with fatty acid synthesis, ACP's associated with citrate lyase and citramalate lyase, a biotin carboxyl carrier protein and cytochrome 552. Two independent indices of amino acid composition were used. 2. The fatty acid synthesis-associated ACP's of many organisms and the lyase-associated ACP's show a high degree of relatedness among one another. 3. The ACP's show no relatedness to biotin carboxyl carrier protein or cytochrome 552. PMID:7128903

  18. The distinct C-terminal acidic domains of HMGB proteins are functionally relevant in Schistosoma mansoni.

    PubMed

    de Abreu da Silva, Isabel Caetano; Carneiro, Vitor Coutinho; Vicentino, Amanda Roberta Revoredo; Aguilera, Estefania Anahi; Mohana-Borges, Ronaldo; Thiengo, Silvana; Fernandez, Monica Ammon; Fantappié, Marcelo Rosado

    2016-04-01

    The Schistosoma mansoni High Mobility Group Box (HMGB) proteins SmHMGB1, SmHMGB2 and SmHMGB3 share highly conserved HMG box DNA binding domains but have significantly different C-terminal acidic tails. Here, we used three full-length and tailless forms of the S. mansoni HMGB proteins to examine the functional roles of their acidic tails. DNA binding assays revealed that the different lengths of the acidic tails among the three SmHMGB proteins significantly and distinctively influenced their DNA transactions. Spectroscopic analyses indicated that the longest acidic tail of SmHMGB3 contributes to the structural stabilisation of this protein. Using immunohistochemical analysis, we showed distinct patterns of SmHMGB1, SmHMGB2 and SmHMGB3 expression in different tissues of adult worms. RNA interference approaches indicated a role for SmHMGB2 and SmHMGB3 in the reproductive system of female worms, whereas for SmHMGB1 no clear phenotype was observed. Schistosome HMGB proteins can be phosphorylated, acetylated and methylated. Importantly, the acetylation and methylation of schistosome HMGBs were greatly enhanced upon removal of the acidic tail. These data support the notion that the C-terminal acidic tails dictate the differences in the structure, expression and function of schistosome HMGB proteins. PMID:26820302

  19. An electrochemical clamp assay for direct, rapid analysis of circulating nucleic acids in serum

    NASA Astrophysics Data System (ADS)

    Das, Jagotamoy; Ivanov, Ivaylo; Montermini, Laura; Rak, Janusz; Sargent, Edward H.; Kelley, Shana O.

    2015-07-01

    The analysis of cell-free nucleic acids (cfNAs), which are present at significant levels in the blood of cancer patients, can reveal the mutational spectrum of a tumour without the need for invasive sampling of the tissue. However, this requires differentiation between the nucleic acids that originate from healthy cells and the mutated sequences shed by tumour cells. Here we report an electrochemical clamp assay that directly detects mutated sequences in patient serum. This is the first successful detection of cfNAs without the need for enzymatic amplification, a step that normally requires extensive sample processing and is prone to interference. The new chip-based assay reads out the presence of mutations within 15 minutes using a collection of oligonucleotides that sequester closely related sequences in solution, and thus allow only the mutated sequence to bind to a chip-based sensor. We demonstrate excellent levels of sensitivity and specificity and show that the clamp assay accurately detects mutated sequences in a collection of samples taken from lung cancer and melanoma patients.

  20. Gamma aminobutyric acid radioreceptor-assay a possible biomarker for human exposure to certain agrochemicals.

    PubMed

    Saleh, M A; Abou Zied, M; el-Baroty, G; Abdel-Reheim, E; Abdel-Rahman, F; Wallace, C; el-Sebae, A H; Blancato, J N

    1993-12-01

    Cyclodiene insecticides, hexachlorocyclohexanes, pyrethroids, bicyclophosphates, the bicycloorthocarboxylate insecticides and some of their metabolites and environmental degradation products are central nervous system toxicants with high specific binding affinity to the chloride channel of the gamma-aminobutyric acid (GABA)A receptor-ionophore sites. [35S] tertiary-butylbicyclophosphorothionate (TBPS) with specific activity higher than 60 Ci/mmole has a high binding affinity to the same sites and is now commercially available and can be used to label the GABAA receptor for the development of a radioreceptor assay technique. The GABA receptor was prepared by ultra centrifugation and dialysis of brain homogenates of either cow, goat, rat or catfish. The receptor was then labeled with [35S] TBPS and the assay was conducted by measuring the displacement of radioactivity following incubation with samples containing the analytes. A radioreceptor assay protocol was developed to measure the amount of the alpha-endosulfan in blood samples. The assay was extremely sensitive, and can detect 0.2 nM of endosulfan at a level equivalent to 0.08 ppb or 8 x 10(-11) gm of endosulfan in each ml of the blood samples. PMID:8270763

  1. Modified bimolecular fluorescence complementation assay to study the inhibition of transcription complex formation by JAZ proteins.

    PubMed

    Qi, Tiancong; Song, Susheng; Xie, Daoxin

    2013-01-01

    The jasmonate (JA) ZIM-domain (JAZ) proteins of Arabidopsis thaliana repress JA signaling and negatively regulate the JA responses. Recently, JAZ proteins have been found to inhibit the transcriptional function of several transcription factors, among which the basic helix-loop-helix (bHLH) (GLABRA3 [GL3], ENHANCER OF GLABRA3 [EGL3], and TRANSPARENT TESTA8 [TT8]) and R2R3-MYB (GL1 and MYB75) that can interact with each other to form bHLH-MYB complexes and further control gene expression. The bimolecular fluorescence complementation (BiFC) assay is a widely used technique to study protein-protein interactions in living cells. Here we describe a modified BiFC experimental procedure to study the inhibition of the formation of the bHLH (GL3)-MYB (GL1) complex by JAZ proteins. PMID:23615997

  2. A repository of assays to quantify 10,000 human proteins by SWATH-MS

    PubMed Central

    Rosenberger, George; Koh, Ching Chiek; Guo, Tiannan; Röst, Hannes L.; Kouvonen, Petri; Collins, Ben C.; Heusel, Moritz; Liu, Yansheng; Caron, Etienne; Vichalkovski, Anton; Faini, Marco; Schubert, Olga T.; Faridi, Pouya; Ebhardt, H. Alexander; Matondo, Mariette; Lam, Henry; Bader, Samuel L.; Campbell, David S.; Deutsch, Eric W.; Moritz, Robert L.; Tate, Stephen; Aebersold, Ruedi

    2014-01-01

    Mass spectrometry is the method of choice for deep and reliable exploration of the (human) proteome. Targeted mass spectrometry reliably detects and quantifies pre-determined sets of proteins in a complex biological matrix and is used in studies that rely on the quantitatively accurate and reproducible measurement of proteins across multiple samples. It requires the one-time, a priori generation of a specific measurement assay for each targeted protein. SWATH-MS is a mass spectrometric method that combines data-independent acquisition (DIA) and targeted data analysis and vastly extends the throughput of proteins that can be targeted in a sample compared to selected reaction monitoring (SRM). Here we present a compendium of highly specific assays covering more than 10,000 human proteins and enabling their targeted analysis in SWATH-MS datasets acquired from research or clinical specimens. This resource supports the confident detection and quantification of 50.9% of all human proteins annotated by UniProtKB/Swiss-Prot and is therefore expected to find wide application in basic and clinical research. Data are available via ProteomeXchange (PXD000953-954) and SWATHAtlas (SAL00016-35). PMID:25977788

  3. GFP-complementation assay to detect functional CPP and protein delivery into living cells

    PubMed Central

    Milech, Nadia; Longville, Brooke AC; Cunningham, Paula T; Scobie, Marie N; Bogdawa, Heique M; Winslow, Scott; Anastasas, Mark; Connor, Theresa; Ong, Ferrer; Stone, Shane R; Kerfoot, Maria; Heinrich, Tatjana; Kroeger, Karen M; Tan, Yew-Foon; Hoffmann, Katrin; Thomas, Wayne R; Watt, Paul M; Hopkins, Richard M

    2015-01-01

    Efficient cargo uptake is essential for cell-penetrating peptide (CPP) therapeutics, which deliver widely diverse cargoes by exploiting natural cell processes to penetrate the cell’s membranes. Yet most current CPP activity assays are hampered by limitations in assessing uptake, including confounding effects of conjugated fluorophores or ligands, indirect read-outs requiring secondary processing, and difficulty in discriminating internalization from endosomally trapped cargo. Split-complementation Endosomal Escape (SEE) provides the first direct assay visualizing true cytoplasmic-delivery of proteins at biologically relevant concentrations. The SEE assay has minimal background, is amenable to high-throughput processes, and adaptable to different transient and stable cell lines. This split-GFP-based platform can be useful to study transduction mechanisms, cellular imaging, and characterizing novel CPPs as pharmaceutical delivery agents in the treatment of disease. PMID:26671759

  4. Strained cycloalkynes as new protein sulfenic acid traps.

    PubMed

    Poole, Thomas H; Reisz, Julie A; Zhao, Weiling; Poole, Leslie B; Furdui, Cristina M; King, S Bruce

    2014-04-30

    Protein sulfenic acids are formed by the reaction of biologically relevant reactive oxygen species with protein thiols. Sulfenic acid formation modulates the function of enzymes and transcription factors either directly or through the subsequent formation of protein disulfide bonds. Identifying the site, timing, and conditions of protein sulfenic acid formation remains crucial to understanding cellular redox regulation. Current methods for trapping and analyzing sulfenic acids involve the use of dimedone and other nucleophilic 1,3-dicarbonyl probes that form covalent adducts with cysteine-derived protein sulfenic acids. As a mechanistic alternative, the present study describes highly strained bicyclo[6.1.0]nonyne (BCN) derivatives as concerted traps of sulfenic acids. These strained cycloalkynes react efficiently with sulfenic acids in proteins and small molecules yielding stable alkenyl sulfoxide products at rates more than 100× greater than 1,3-dicarbonyl reagents enabling kinetic competition with physiological sulfur chemistry. Similar to the 1,3-dicarbonyl reagents, the BCN compounds distinguish the sulfenic acid oxoform from the thiol, disulfide, sulfinic acid, and S-nitrosated forms of cysteine while displaying an acceptable cell toxicity profile. The enhanced rates demonstrated by these strained alkynes identify them as new bioorthogonal probes that should facilitate the discovery of previously unknown sulfenic acid sites and their parent proteins. PMID:24724926

  5. A high-throughput, in-vitro assay for Bacillus thuringiensis insecticidal proteins.

    PubMed

    Izumi Willcoxon, Michi; Dennis, Jaclyn R; Lau, Sabina I; Xie, Weiping; You, You; Leng, Song; Fong, Ryan C; Yamamoto, Takashi

    2016-01-10

    A high-throughput, in-vitro assay for Bacillus thuringiensis (Bt) insecticidal proteins designated as Cry was developed and evaluated for screening a large number of Cry protein variants produced by DNA shuffling. This automation-amenable assay exploits an insect cell line expressing a single receptor of Bt Cry proteins. The Cry toxin used to develop this assay is a variant of the Cry1Ab protein called IP1-88, which was produced previously by DNA shuffling. Cell mortality caused by the activated Bt Cry toxin was determined by chemical cell viability assay in 96/384-well microtiter plates utilizing CellTiter 96(®) obtained from Promega. A widely-accepted mode-of-action theory of certain Bt Cry proteins suggests that the activated toxin binds to one or more receptors and forms a pore through the insect gut epithelial cell apical membrane. A number of insect proteins such as cadherin-like protein (Cad), aminopeptidase-N (APN), alkaline phosphatase (ALP) and ABC transporter (ABCC) have been identified as the receptors of Bt Cry toxins. In this study, Bt Cry toxin receptors Ostrinia nubilalis (European corn borer) cadherin-like protein (On-Cad) and aminopeptidase-N 1 and 3 (On-APN1, On-APN3) and Spodoptera frugiperda (fall armyworm) cadherin-like protein (Sf-Cad) were cloned in an insect cell line, Sf21, and a mammalian cell line, Expi293F. It was observed by ligand blotting and immunofluorescence microscopy that trypsin-activated IP1-88 bound to On-Cad and On-APN1, but not Sf-Cad or On-APN3. In contrast, IP1-88 bound only to APN1 in BBMV (Brush Border Membrane Vesicles) prepared from the third and fourth-instar O. nubilalis larval midgut. The sensitivity of the recombinant cells to the toxin was then tested. IP1-88 showed no toxicity to non-recombinant Sf21 and Expi293F. Toxicity was observed only when the On-Cad gene was cloned and expressed. Sf-Cad and On-APN1 were not able to make those cells sensitive to the toxin. Since the expression of On-Cad alone was

  6. Antioxidant assay-guided purification and LC determination of ellagic acid in pomegranate peel.

    PubMed

    Panichayupakarananta, Pharkphoom; Issuriya, Atcharaporn; Sirikatitham, Anusak; Wang, Wei

    2010-07-01

    On the basis of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay-guided purification, ellagic acid was isolated from the methanol extract of pomegranate fruit peel by liquid-liquid extraction and chromatographic techniques. A reversed-phase high-performance liquid chromatography was described for determination of ellagic acid in pomegranate fruit peel extract. The method involved the use of a TSK-gel ODS-80Tm column with a mixture of 2% aqueous acetic acid and methanol (gradient elution mode: 0-15 min, 40-60% v/v methanol and 15-20 min, 60% v/v methanol) as the mobile phase and detection at 254 nm. The parameters of linearity, repeatability, reproducibility, accuracy, and specificity of the method were evaluated. The recovery of the method was 98.5% and linearity (r(2) > 0.9995) was obtained for ellagic acid. A high degree of specificity as well as repeatability and reproducibility (relative standard deviation values less than 5%) were also achieved. The limits of detection and quantification were 1.00 and 2.50 microg/mL, respectively. The solvent for extraction of ellagic acid from pomegranate fruit peel was examined in order to maximize the ellagic acid content of the extract. A solution of 10% v/v water in methanol was capable of increasing the ellagic acid content in the extract up to 7.66% w/w. The ellagic acid content and antioxidant activity of the ethyl acetate fraction separated from the crude extract using water and ethyl acetate partition was higher than that of the crude extract. PMID:20822660

  7. Endogenous fatty acids in olfactory hairs influence pheromone binding protein structure and function in Lymantria dispar.

    PubMed

    Nardella, Jason; Terrado, Mailyn; Honson, Nicolette S; Plettner, Erika

    2015-08-01

    The gypsy moth utilizes a pheromone, (7R,8S)-2-methyl-7,8-epoxyoctadecane, for mate location. The pheromone is detected by sensory hairs (sensilla) on the antennae of adult males. Sensilla contain the dendrites of olfactory neurons bathed in lymph, which contains pheromone binding proteins (PBPs). We have extracted and identified free fatty acids from lymph of sensory hairs, and we demonstrate that these function as endogenous ligands for gypsy moth PBP1 and PBP2. Homology modeling of both PBPs, and docking of fatty acids reveal multiple binding sites: one internal, the others external. Pheromone binding assays suggest that these fatty acids increase PBP-pheromone binding affinity. We show that fatty acid binding causes an increase in α-helix content in the N-terminal domain, but not in the C-terminal peptide of both proteins. The C-terminal peptide was shown to form a α-helix in a hydrophobic, homogeneous environment, but not in the presence of fatty acid micelles. Through partition assays we show that the fatty acids prevent adsorption of the pheromone on hydrophobic surfaces and facilitate pheromone partition into an aqueous phase. We propose that lymph is an emulsion of fatty acids and PBP that influence each other and thereby control the partition equilibria of hydrophobic odorants. PMID:26032337

  8. A simple and cost-effective solid-phase protein nano-assay using polyacrylamide-coated glass plates.

    PubMed

    Krajewski, Wladyslaw A

    2015-02-01

    A new solid-phase protein nano-assay is suggested for simple and sensitive estimation of protein content in sample buffers (a 1-μl sample is sufficient for analysis). The assay is different from conventional "on-filter" assays in that it uses inexpensive fully transparent polyacrylamide gel (PAAG)-coated glass plates as solid support and, thus, combines the convenience of "on-membrane" staining with the sensitivity and ease of documentation of "in-gel" staining (and, therefore, is especially suited for standard lab gel documentation systems). The PAAG plates assay is compatible with all dyes for in-gel protein staining. Depending on the sensitivity of the staining protocol, the assay can be used in macro-, micro-, and nano-assay formats. We also describe a low-cost two-component colloidal Coomassie brilliant blue G-250 (CBB G-250) staining protocol for fast quantitative visualization of proteins spotted on a PAAG plate (the detection limit is up to 2 ng of proteins even when using a Nikon CoolPix digital camera and white light transilluminator instead of a gel scanner). The suggested colloidal CBB G-250 protocol could also be used for visualizing nano-amounts of proteins in polyacrylamide gels. The PAAG plate assay could be useful for proteomic applications and, in general, for all cases where a fast, sensitive, and easily documentable cost-effective solid-phase protein assay is required. PMID:25449300

  9. A Filter Binding Assay to Quantify the Association of Cyclic di-GMP to Proteins

    PubMed Central

    Srivastava, Disha; Waters, Christopher M.

    2016-01-01

    Cyclic di-GMP (c-di-GMP) is a ubiquitous second messenger that regulates many processes in bacteria including biofilm formation, motility, and virulence (Hengge, 2009). Analysis of c-di-GMP binding properties of bacterial proteins is an important step to characterize c-di-GMP signaling pathways. C-di-GMP binds numerous proteins such as transcription factors, enzymes, and multimeric protein complexes (Hickman and Harwood, 2008, Ryjenkov et al., 2006, Weinhouse et al., 1997). The c-di-GMP binding assay described here is a relatively simple and cost effective method to characterize c-di-GMP binding to a protein using [32P]-labeled c-di-GMP. Radiolabeled c-di-GMP is readily synthesized with a purified GGDEF enzyme [such as WspR from Pseudomonas aeruginosa (P. aeruginosa)] and [a-32P]-GTP (Srivastava et al., 2013). After incubation of the labeled c-di-GMP with the protein of interest in solution, the resulting mixture is filtered through a nitrocellulose protein binding membrane. The amount of labeled c-di-GMP that is retained on the membrane indicates the interaction between the signal and protein. The specificity of c-di-GMP binding can be tested by competing with unlabeled c-di-GMP or other nucleotides such as GTP in the reaction. By examining binding of a fixed protein concentration to increasing concentrations of c-di-GMP, this method is able to determine the dissociation constant of c-di-GMP-protein interaction.

  10. Ultrasensitive protein detection: a case for microfluidic magnetic bead-based assays.

    PubMed

    Tekin, H Cumhur; Gijs, Martin A M

    2013-12-21

    We review the use of magnetic micro- and nanoparticles ('magnetic beads') in microfluidic systems for ultrasensitive protein detection. During recent years magnetic beads have been used frequently in immunoassays, either as mobile substrates on which the target antigen is captured, as detection labels, or simultaneously as substrates and labels. The major part of the reviewed work has as application the detection of antibodies or disease biomarkers in serum or of biotoxins from food samples. Several of the most sensitive assays allow protein detection down to fg mL(-1) concentrations. We benchmark the performance of these microfluidic magnetic bead-based assays with the most promising earlier work and with alternative solutions. PMID:24145920

  11. Selection of direct transesterification as the preferred method for assay of fatty acid content of microalgae.

    PubMed

    Griffiths, M J; van Hille, R P; Harrison, S T L

    2010-11-01

    Assays for total lipid content in microalgae are usually based on the Folch or the Bligh and Dyer methods of solvent extraction followed by quantification either gravimetrically or by chromatography. Direct transesterification (DT) is a method of converting saponifiable lipids in situ directly to fatty acid methyl esters which can be quantified by gas chromatography (GC). This eliminates the extraction step and results in a rapid, one-step procedure applicable to small samples. This study compared the effectiveness of DT in quantifying the total fatty acid content in three species of microalgae to extraction using the Folch, the Bligh and Dyer and the Smedes and Askland methods, followed by transesterification and GC. The use of two catalysts in sequence, as well as the effect of reaction water content on the efficiency of DT were investigated. The Folch method was the most effective of the extraction methods tested, but comparison with DT illustrated that all extraction methods were incomplete. Higher levels of fatty acid in the cells were obtained with DT in comparison with the extraction-transesterification methods. A combination of acidic and basic transesterification catalysts was more effective than each individually when the sample contained water. The two-catalyst reaction was insensitive to water up to 10% of total reaction volume. DT proved a convenient and more accurate method than the extraction techniques for quantifying total fatty acid content in microalgae. PMID:20820931

  12. Nucleic acid sequence-based amplification assays for rapid detection of West Nile and St. Louis encephalitis viruses.

    PubMed

    Lanciotti, R S; Kerst, A J

    2001-12-01

    The development and application of nucleic acid sequence-based amplification (NASBA) assays for the detection of West Nile (WN) and St. Louis encephalitis (SLE) viruses are reported. Two unique detection formats were developed for the NASBA assays: a postamplification detection step with a virus-specific internal capture probe and electrochemiluminescence (NASBA-ECL assay) and a real-time assay with 6-carboxyfluorescein-labeled virus-specific molecular beacon probes (NASBA-beacon assay). The sensitivities and specificities of these NASBA assays were compared to those of a newly described standard reverse transcription (RT)-PCR and TaqMan assays for SLE virus and to a previously published TaqMan assay for WN virus. The NASBA assays demonstrated exceptional sensitivities and specificities compared to those of virus isolation, the TaqMan assays, and standard RT-PCR, with the NASBA-beacon assay yielding results in less than 1 h. These assays should be of utility in the diagnostic laboratory to complement existing diagnostic testing methodologies and as a tool in conducting flavivirus surveillance in the United States. PMID:11724870

  13. Selectivity analysis of single binder assays used in plasma protein profiling

    PubMed Central

    Neiman, Maja; Fredolini, Claudia; Johansson, Henrik; Lehtiö, Janne; Nygren, Per-Åke; Uhlén, Mathias; Nilsson, Peter; Schwenk, Jochen M

    2013-01-01

    The increasing availability of antibodies toward human proteins enables broad explorations of the proteomic landscape in cells, tissues, and body fluids. This includes assays with antibody suspension bead arrays that generate protein profiles of plasma samples by flow cytometer analysis. However, antibody selectivity is context dependent so it is necessary to corroborate on-target detection over off-target binding. To address this, we describe a concept to directly verify interactions from antibody-coupled beads by analysis of their eluates by Western blots and MS. We demonstrate selective antibody binding in complex samples with antibodies toward a set of chosen proteins with different abundance in plasma and serum, and illustrate the need to adjust sample and bead concentrations accordingly. The presented approach will serve as an important tool for resolving differential protein profiles from antibody arrays within plasma biomarker discoveries. PMID:24151238

  14. Development of an Assay for the Identification of Receptor Binding Proteins from Bacteriophages

    PubMed Central

    Simpson, David J.; Sacher, Jessica C.; Szymanski, Christine M.

    2016-01-01

    Recently, a large number of new technologies have been developed that exploit the unique properties of bacteriophage receptor binding proteins (RBPs). These include their use in diagnostic applications that selectively capture bacteria and as therapeutics that reduce bacterial colonization in vivo. RBPs exhibit comparable, and in many cases superior, stability, receptor specificity, and affinity to other carbohydrate binding proteins such as antibodies or lectins. In order to further exploit the use of RBPs, we have developed an assay for discovering RBPs using phage genome expression libraries and protein screens to identify binding partners that recognize the host bacterium. When phage P22 was screened using this assay, Gp9 was the only RBP discovered, confirming previous predictions that this is the sole RBP encoded by this phage. We then examined the Escherichia coli O157:H7 typing phage 1 in our assay and identified a previously undescribed RBP. This general approach has the potential to assist in the identification of RBPs from other bacteriophages. PMID:26761028

  15. Development of an Assay for the Identification of Receptor Binding Proteins from Bacteriophages.

    PubMed

    Simpson, David J; Sacher, Jessica C; Szymanski, Christine M

    2016-01-01

    Recently, a large number of new technologies have been developed that exploit the unique properties of bacteriophage receptor binding proteins (RBPs). These include their use in diagnostic applications that selectively capture bacteria and as therapeutics that reduce bacterial colonization in vivo. RBPs exhibit comparable, and in many cases superior, stability, receptor specificity, and affinity to other carbohydrate binding proteins such as antibodies or lectins. In order to further exploit the use of RBPs, we have developed an assay for discovering RBPs using phage genome expression libraries and protein screens to identify binding partners that recognize the host bacterium. When phage P22 was screened using this assay, Gp9 was the only RBP discovered, confirming previous predictions that this is the sole RBP encoded by this phage. We then examined the Escherichia coli O157:H7 typing phage 1 in our assay and identified a previously undescribed RBP. This general approach has the potential to assist in the identification of RBPs from other bacteriophages. PMID:26761028

  16. Novel Phakopsora pachyrhizi Extracellular Proteins Are Ideal Targets for Immunological Diagnostic Assays

    PubMed Central

    McMahon, Michael B.; Edwards, H. Herb; Boerma, Britney L.; Lewis Ivey, Melanie L.; Miller, Sally A.; Dorrance, Anne E.

    2012-01-01

    Phakopsora pachyrhizi, the causal agent of Asian soybean rust (ASR), continues to spread across the southeast and midsouth regions of the United States, necessitating the use of fungicides by producers. Our objective in this research was to identify ASR proteins expressed early during infection for the development of immunodiagnostic assays. We have identified and partially characterized a small gene family encoding extracellular proteins in the P. pachyrhizi urediniospore wall, termed PHEPs (for Phakopsora extracellular protein). Two highly expressed protein family members, PHEP 107 and PHEP 369, were selected as ideal immunodiagnostic targets for antibody development, after we detected PHEPs in plants as early as 3 days postinfection (dpi). Monoclonal antibodies (MAbs; 2E8E5-1 and 3G6H7-3) generated against recombinant PHEP 369 were tested for sensitivity against the recombinant protein and extracts from ASR-infected plants and for specificity against a set of common soybean pathogens. These antibodies should prove applicable in immunodiagnostic assays to detect infected soybeans and to identify ASR spores from sentinel surveillance plots. PMID:22447596

  17. Kinetic exclusion assay of monoclonal antibody affinity to the membrane protein Roundabout 1 displayed on baculovirus.

    PubMed

    Kusano-Arai, Osamu; Fukuda, Rie; Kamiya, Wakana; Iwanari, Hiroko; Hamakubo, Takao

    2016-07-01

    The reliable assessment of monoclonal antibody (mAb) affinity against membrane proteins in vivo is a major issue in the development of cancer therapeutics. We describe here a simple and highly sensitive method for the evaluation of mAbs against membrane proteins by means of a kinetic exclusion assay (KinExA) in combination with our previously developed membrane protein display system using budded baculovirus (BV). In our BV display system, the membrane proteins are displayed on the viral surface in their native form. The BVs on which the liver cancer antigen Roundabout 1 (Robo1) was displayed were adsorbed onto magnetic beads without fixative (BV beads). The dissociation constant (Kd, ∼10(-11) M) that was measured on the Robo1 expressed BV beads correlated well with the value from a whole cell assay (the coefficient of determination, R(2) = 0.998) but not with the value for the soluble extracellular domains of Robo1 (R(2) = 0.834). These results suggest that the BV-KinExA method described here provides a suitably accurate Kd evaluation of mAbs against proteins on the cell surface. PMID:27095060

  18. Tryptophan fluorescence quenching as a binding assay to monitor protein conformation changes in the membrane of intact mitochondria.

    PubMed

    Akbar, S Md; Sreeramulu, K; Sharma, Hari C

    2016-06-01

    Intrinsic protein fluorescence is due to aromatic amino acids, mainly tryptophan, which can be selectively measured by exciting at 295 nm. Changes in emission spectra of tryptophan are due to the protein conformational transitions, subunit association, ligand binding or denaturation, which affect the local environment surrounding the indole ring. In this study, tryptophan fluorescence was monitored in intact mitochondria at 333 nm following excitation at 295 nm in presence of insecticides using spectrofluorometer. Methyl-parathion, carbofuran, and endosulfan induced Trp fluorescence quenching and release of cytochrome c when incubated with the mitochondria, except fenvalarate. Mechanism of insecticide-induced mitochondrial toxicity for the tested insecticides has been discussed. Reduction in the intensity of tryptophan emission spectra of mitochondrial membrane proteins in presence of an increasing concentration of a ligand can be used to study the interaction of insecticides/drugs with the intact mitochondria. Furthermore, this assay can be readily adapted for studying protein-ligand interactions in intact mitochondria and in other cell organelles extending its implications for pesticide and pharma industry and in drug discovery. PMID:26905428

  19. Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family.

    PubMed

    Broussard, Tyler C; Miller, Darcie J; Jackson, Pamela; Nourse, Amanda; White, Stephen W; Rock, Charles O

    2016-03-18

    Fatty acid kinase (Fak) is a ubiquitous Gram-positive bacterial enzyme consisting of an ATP-binding protein (FakA) that phosphorylates the fatty acid bound to FakB. In Staphylococcus aureus, Fak is a global regulator of virulence factor transcription and is essential for the activation of exogenous fatty acids for incorporation into phospholipids. The 1.2-Å x-ray structure of S. aureus FakB2, activity assays, solution studies, site-directed mutagenesis, and in vivo complementation were used to define the functions of the five conserved residues that define the FakB protein family (Pfam02645). The fatty acid tail is buried within the protein, and the exposed carboxyl group is bound by a Ser-93-fatty acid carboxyl-Thr-61-His-266 hydrogen bond network. The guanidinium of the invariant Arg-170 is positioned to potentially interact with a bound acylphosphate. The reduced thermal denaturation temperatures of the T61A, S93A, and H266A FakB2 mutants illustrate the importance of the hydrogen bond network in protein stability. The FakB2 T61A, S93A, and H266A mutants are 1000-fold less active in the Fak assay, and the R170A mutant is completely inactive. All FakB2 mutants form FakA(FakB2)2 complexes except FakB2(R202A), which is deficient in FakA binding. Allelic replacement shows that strains expressing FakB2 mutants are defective in fatty acid incorporation into phospholipids and virulence gene transcription. These conserved residues are likely to perform the same critical functions in all bacterial fatty acid-binding proteins. PMID:26774272

  20. Assay of deoxyribonucleic acid homology using a single-strand-specific nuclease at 75 C.

    PubMed Central

    Barth, P T; Grinter, N J

    1975-01-01

    We investigated the conditions under which a crude preparation of endonuclease S1 gives maximal hydrolysis of denatured deoxyribonucleic acid (DNA) while giving minimal hydrolysis of native DNA. The hydrolysis was measured by filtering and determining the acid-insoluble reaction product using 3H-labeled substrates. We also investigated various parameters in making this measurement. Under appropriate conditions (in 1 mM ZnSO-4, 0.168 M NaCl at pH 4.8) denatured DNA is hydrolyzed within 3% of completion whereas native DNA is essentially unaffected. The reaction was applied to assay plasmid DNA homoand heteroduplexes for which the method proves to be simple, fast, and reproducible. PMID:234416

  1. Echinococcus granulosus fatty acid binding proteins subcellular localization.

    PubMed

    Alvite, Gabriela; Esteves, Adriana

    2016-05-01

    Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes. PMID:26873273

  2. Validation of a UV Spectrometric Method for the Assay of Tolfenamic Acid in Organic Solvents

    PubMed Central

    Ahmed, Sofia; Mustaan, Nafeesa; Sheraz, Muhammad Ali; Nabi, Syeda Ayesha Ahmed un; Ahmad, Iqbal

    2015-01-01

    The present study has been carried out to validate a UV spectrometric method for the assay of tolfenamic acid (TA) in organic solvents. TA is insoluble in water; therefore, a total of thirteen commonly used organic solvents have been selected in which the drug is soluble. Fresh stock solutions of TA in each solvent in a concentration of 1 × 10−4 M (2.62 mg%) were prepared for the assay. The method has been validated according to the guideline of International Conference on Harmonization and parameters like linearity, range, accuracy, precision, sensitivity, and robustness have been studied. Although the method was found to be efficient for the determination of TA in all solvents on the basis of statistical data 1-octanol, followed by ethanol and methanol, was found to be comparatively better than the other studied solvents. No change in the stock solution stability of TA has been observed in each solvent for 24 hours stored either at room (25 ± 1°C) or at refrigerated temperature (2–8°C). A shift in the absorption maxima has been observed for TA in various solvents indicating drug-solvent interactions. The studied method is simple, rapid, economical, accurate, and precise for the assay of TA in different organic solvents. PMID:26783497

  3. Development and utilization of activated STAT3 detection assays for screening a library of secreted proteins.

    PubMed

    Fursov, Natalie; Gates, Irina V; Panavas, Tadas; Giles-Komar, Jill; Powers, Gordon

    2011-08-01

    Interleukin-6 (IL-6) family of cytokines are multifunctional proteins that play an important role in host defenses, acute phase reactions, immune responses, hematopoiesis, and tumorigenesis. The cytokines are produced by various lymphoid and nonlymphoid cells and mediate their biological activity through initial low-affinity binding to cell surface receptors, which are specific for their respective ligands. Ligand-specific receptor binding results in the receptor heterodimerization with ubiquitously expressed signal-transducing transmembrane component gp130 followed by activation of the gp130-associated Janus kinase, which, in turn, phosphorylates signal transducer and activator of transcription 3 (STAT3). Phosphorylated STAT3 (pSTAT3) dimerizes and translocates to the nucleus, where it activates gene transcription. Activation of STAT3 is essential to IL-6 family-associated physiological effects. Therefore, the ability to assess STAT3 phosphorylation is important for drug discovery efforts targeting IL-6 family cytokines. Various reagents and technologies are available to detect the effect of IL-6 type cytokines in treated cells. The present study describes the development of two pSTAT3 detection assays: the high-throughput screening assay based on Meso-Scale Discovery technology, which utilizes electrochemoluminescent signal measurements for the detection of pSTAT3 in treated cell extracts, and the secondary characterization assay based on fluorescent imaging analysis, which monitors pSTAT3 nuclear translocation in cells after activation. We have successfully utilized these assays to screen a small library of secreted proteins and identified inducers of STAT3 phosphorylation. The results obtained in this study demonstrate that both assays are robust, reliable, and amenable to high-throughput screening applications. PMID:21294636

  4. Development of FRET assay into quantitative and high-throughput screening technology platforms for protein-protein interactions.

    PubMed

    Song, Yang; Madahar, Vipul; Liao, Jiayu

    2011-04-01

    Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research and is a very powerful tool in elucidating protein interactions in many cellular processes. Ubiquitination and SUMOylation are multi-step cascade reactions, involving multiple enzymes and protein-protein interactions. Here we report the development of dissociation constant (K (d)) determination for protein-protein interaction and cell-based high-throughput screening (HTS) assay in SUMOylation cascade using FRET technology. These developments are based on steady state and high efficiency of fluorescent energy transfer between CyPet and YPet fused with SUMO1 and Ubc9, respectively. The developments in theoretical and experimental procedures for protein interaction K (d) determination and cell-based HTS provide novel tools in affinity measurement and protein interaction inhibitor screening. The K (d) determined by FRET between SUMO1 and Ubc9 is compatible with those determined with other traditional approaches, such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). The FRET-based HTS is pioneer in cell-based HTS. Both K (d) determination and cell-based HTS, carried out in 384-well plate format, provide powerful tools for large-scale and high-throughput applications. PMID:21174150

  5. Photoaffinity labeling of retinoic acid-binding proteins.

    PubMed Central

    Bernstein, P S; Choi, S Y; Ho, Y C; Rando, R R

    1995-01-01

    Retinoid-binding proteins are essential mediators of vitamin A function in vertebrate organisms. They solubilize and stabilize retinoids, and they direct the intercellular and intracellular trafficking, transport, and metabolic function of vitamin A compounds in vision and in growth and development. Although many soluble retinoid-binding proteins and receptors have been purified and extensively characterized, relatively few membrane-associated enzymes and other proteins that interact with retinoids have been isolated and studied, due primarily to their inherent instabilities during purification. In an effort to identify and purify previously uncharacterized retinoid-binding proteins, it is shown that radioactively labeled all-trans-retinoic acid can be used as a photoaffinity labeling reagent to specifically tag two known retinoic acid-binding proteins, cellular retinoic acid-binding protein and albumin, in complex mixtures of cytosolic proteins. Additionally, a number of other soluble and membrane-associated proteins that bind all-trans-[11,12-3H]retinoic acid with high specificity are labeled utilizing the same photoaffinity techniques. Most of these labeled proteins have molecular weights that do not correspond to any known retinoid-binding proteins. Thus, photoaffinity labeling with all-trans-retinoic acid and related photoactivatable retinoids is a method that should prove extremely useful in the identification and purification of novel soluble and membrane-associated retinoid-binding proteins from ocular and nonocular tissues. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7846032

  6. A Substrate Mimic Allows High-Throughput Assay of the FabA Protein and Consequently the Identification of a Novel Inhibitor of Pseudomonas aeruginosa FabA

    PubMed Central

    Moynié, Lucile; Hope, Anthony G.; Finzel, Kara; Schmidberger, Jason; Leckie, Stuart M.; Schneider, Gunter; Burkart, Michael D.; Smith, Andrew D.; Gray, David W.; Naismith, James H.

    2016-01-01

    Eukaryotes and prokaryotes possess fatty acid synthase (FAS) biosynthetic pathways that comprise iterative chain elongation, reduction, and dehydration reactions. The bacterial FASII pathway differs significantly from human FAS pathways and is a long-standing target for antibiotic development against Gram-negative bacteria due to differences from the human FAS, and several existing antibacterial agents are known to inhibit FASII enzymes. N-Acetylcysteamine (NAC) fatty acid thioesters have been used as mimics of the natural acyl carrier protein pathway intermediates to assay FASII enzymes, and we now report an assay of FabV from Pseudomonas aeruginosa using (E)-2-decenoyl-NAC. In addition, we have converted an existing UV absorbance assay for FabA, the bifunctional dehydration/epimerization enzyme and key target in the FASII pathway, into a high-throughput enzyme coupled fluorescence assay that has been employed to screen a library of diverse small molecules. With this approach, N-(4-chlorobenzyl)-3-(2-furyl)-1H-1,2,4-triazol-5-amine (N42FTA) was found to competitively inhibit (pIC50 = 5.7 ± 0.2) the processing of 3-hydroxydecanoyl-NAC by P. aeruginosa FabA. N42FTA was shown to be potent in blocking crosslinking of Escherichia coli acyl carrier protein and FabA, a direct mimic of the biological process. The co-complex structure of N42FTA with P. aeruginosa FabA protein rationalises affinity and suggests future design opportunities. Employing NAC fatty acid mimics to develop further high-throughput assays for individual enzymes in the FASII pathway should aid in the discovery of new antimicrobials. PMID:26562505

  7. A Substrate Mimic Allows High-Throughput Assay of the FabA Protein and Consequently the Identification of a Novel Inhibitor of Pseudomonas aeruginosa FabA.

    PubMed

    Moynié, Lucile; Hope, Anthony G; Finzel, Kara; Schmidberger, Jason; Leckie, Stuart M; Schneider, Gunter; Burkart, Michael D; Smith, Andrew D; Gray, David W; Naismith, James H

    2016-01-16

    Eukaryotes and prokaryotes possess fatty acid synthase (FAS) biosynthetic pathways that comprise iterative chain elongation, reduction, and dehydration reactions. The bacterial FASII pathway differs significantly from human FAS pathways and is a long-standing target for antibiotic development against Gram-negative bacteria due to differences from the human FAS, and several existing antibacterial agents are known to inhibit FASII enzymes. N-Acetylcysteamine (NAC) fatty acid thioesters have been used as mimics of the natural acyl carrier protein pathway intermediates to assay FASII enzymes, and we now report an assay of FabV from Pseudomonas aeruginosa using (E)-2-decenoyl-NAC. In addition, we have converted an existing UV absorbance assay for FabA, the bifunctional dehydration/epimerization enzyme and key target in the FASII pathway, into a high-throughput enzyme coupled fluorescence assay that has been employed to screen a library of diverse small molecules. With this approach, N-(4-chlorobenzyl)-3-(2-furyl)-1H-1,2,4-triazol-5-amine (N42FTA) was found to competitively inhibit (pIC50=5.7±0.2) the processing of 3-hydroxydecanoyl-NAC by P. aeruginosa FabA. N42FTA was shown to be potent in blocking crosslinking of Escherichia coli acyl carrier protein and FabA, a direct mimic of the biological process. The co-complex structure of N42FTA with P. aeruginosa FabA protein rationalises affinity and suggests future design opportunities. Employing NAC fatty acid mimics to develop further high-throughput assays for individual enzymes in the FASII pathway should aid in the discovery of new antimicrobials. PMID:26562505

  8. Properties of kojic acid and curcumin: Assay on cell B16-F1

    NASA Astrophysics Data System (ADS)

    Sugiharto, Ariff, Arbakariya; Ahmad, Syahida; Hamid, Muhajir

    2016-03-01

    Ultra violet (UV) exposure and oxidative stress are casually linked to skin disorders. They can increase melanin synthesis, proliferation of melanocytes, and hyperpigmentation. It is possible that antioxidants or inhibitors may have a beneficial effect on skin health to reduce hyperpigmentation. In the last few years, a huge number of natural herbal extracts have been tested to reduce hyperpigmentation. The objective of this study was to determine and to compare of kojic acid and curcumin properties to viability cell B16-F1. In this study, our data showed that the viability of cell B16-F1 was 63.91% for kojic acid and 64.12% for curcumin at concentration 100 µg/ml. Further investigation assay of antioxidant activities, indicated that IC50 for kojic acid is 63.8 µg/ml and curcumin is 16.05 µg/ml. Based on the data, kojic acid and curcumin have potential antioxidant properties to reduce hyperpigmentation with low toxicity effect in cell B16-F1.

  9. An enzymic assay for uric acid in serum and urine compared with HPLC.

    PubMed

    Dubois, H; Delvoux, B; Ehrhardt, V; Greiling, H

    1989-03-01

    We evaluated a colorimetric method for the assay of uric acid in serum or urine, which utilises a Trinder chromogenic system modified by the inclusion of 2,4,6-tribromo-3-hydroxybenzoic acid for oxidative coupling to p-aminophenazone. Colour development (Amax: 512 nm) is complete within five minutes. Measurement of a sample blank is not needed. The procedure involves pre-incubation with ascorbic acid oxidase and detergent to eliminate interference by ascorbic acid and to abolish turbidity due to lipaemia; this pretreatment was effective up to 1.14 mmol/l ascorbate and up to at least 25 mmol/l triacylglycerol. Interference by icteric sera was insignificant up to about 170 mumol/l bilirubin. The method is linear up to at least 1428 mumol/l. In human serum and urine the procedure correlates well with HPLC and the uricase p-aminophenazone method on the SMAC analyser. Within-run and between-run imprecisions of the enzymic test were higher than for HPLC, but did not exceed 1.2% (CV) and 2.5% (CV), respectively. PMID:2708944

  10. Serological assays based on recombinant viral proteins for the diagnosis of arenavirus hemorrhagic fevers.

    PubMed

    Fukushi, Shuetsu; Tani, Hideki; Yoshikawa, Tomoki; Saijo, Masayuki; Morikawa, Shigeru

    2012-10-01

    The family Arenaviridae, genus Arenavirus, consists of two phylogenetically independent groups: Old World (OW) and New World (NW) complexes. The Lassa and Lujo viruses in the OW complex and the Guanarito, Junin, Machupo, Sabia, and Chapare viruses in the NW complex cause viral hemorrhagic fever (VHF) in humans, leading to serious public health concerns. These viruses are also considered potential bioterrorism agents. Therefore, it is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of arenavirus outbreaks. However, these arenaviruses are classified as BSL-4 pathogens, thus making it difficult to develop diagnostic techniques for these virus infections in institutes without BSL-4 facilities. To overcome these difficulties, antibody detection systems in the form of an enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence assay were developed using recombinant nucleoproteins (rNPs) derived from these viruses. Furthermore, several antigen-detection assays were developed. For example, novel monoclonal antibodies (mAbs) to the rNPs of Lassa and Junin viruses were generated. Sandwich antigen-capture (Ag-capture) ELISAs using these mAbs as capture antibodies were developed and confirmed to be sensitive and specific for detecting the respective arenavirus NPs. These rNP-based assays were proposed to be useful not only for an etiological diagnosis of VHFs, but also for seroepidemiological studies on VHFs. We recently developed arenavirus neutralization assays using vesicular stomatitis virus (VSV)-based pseudotypes bearing arenavirus recombinant glycoproteins. The goal of this article is to review the recent advances in developing laboratory diagnostic assays based on recombinant viral proteins for the diagnosis of VHFs and epidemiological studies on the VHFs caused by arenaviruses. PMID:23202455

  11. A protein chip membrane-capture assay for botulinum neurotoxin activity

    SciTech Connect

    Marconi, Severine; Ferracci, Geraldine; Berthomieu, Maelys; Kozaki, Shunji; Miquelis, Raymond; Boucraut, Jose; Seagar, Michael

    2008-12-15

    Botulinum neurotoxins A and B (BoNT/A and B) are neuromuscular blocking agents which inhibit neurotransmission by cleaving the intra-cellular presynaptic SNARE proteins SNAP-25 and VAMP2, localized respectively in plasma membrane and synaptic vesicles. These neurotoxins are both dangerous pathogens and powerful therapeutic agents with numerous clinical and cosmetic applications. Consequently there is a need for in vitro assays of their biological activity to screen for potential inhibitors and to replace the widely used in vivo mouse assay. Surface plasmon resonance (SPR) was used to measure membrane vesicle capture by antibodies against SNAP-25 and VAMP2. Substrate cleavage by BoNTs modified capture providing a method to assay toxin activity. Firstly using synaptic vesicles as a substrate, a comparison of the EC{sub 50}s for BoNT/B obtained by SPR, ELISA or flow cytometry indicated similar sensitivity although SPR assays were more rapid. Sonication of brain or neuronal cultures generated plasma membrane fragments with accessible intra-cellular epitopes adapted to measurement of BoNT/A activity. SPR responses were proportional to antigen concentration permitting detection of as little as 4 pM SNAP-25 in crude lysates. BoNT/A activity was assayed using monoclonal antibodies that specifically recognize a SNAP-25 epitope generated by the proteolytic action of the toxin. Incubation of intact primary cultured neurons with BoNT/A yielded an EC{sub 50} of 0.5 pM. The SPR biosensor method was sensitive enough to monitor BoNT/A and B activity in cells cultured in a 96-well format providing an alternative to experimental animals for toxicological assays.

  12. Development of Lentivirus-Based Reference Materials for Ebola Virus Nucleic Acid Amplification Technology-Based Assays

    PubMed Central

    Mattiuzzo, Giada; Ashall, James; Doris, Kathryn S.; MacLellan-Gibson, Kirsty; Nicolson, Carolyn; Wilkinson, Dianna E.; Harvey, Ruth; Almond, Neil; Anderson, Robert; Efstathiou, Stacey; Minor, Philip D.; Page, Mark

    2015-01-01

    The 2013-present Ebola virus outbreak in Western Africa has prompted the production of many diagnostic assays, mostly based on nucleic acid amplification technologies (NAT). The calibration and performance assessment of established assays and those under evaluation requires reference materials that can be used in parallel with the clinical sample to standardise or control for every step of the procedure, from extraction to the final qualitative/quantitative result. We have developed safe and stable Ebola virus RNA reference materials by encapsidating anti sense viral RNA into HIV-1-like particles. The lentiviral particles are replication-deficient and non-infectious due to the lack of HIV-1 genes and Envelope protein. Ebola virus genes were subcloned for encapsidation into two lentiviral preparations, one containing NP-VP35-GP and the other VP40 and L RNA. Each reference material was formulated as a high-titre standard for use as a calibrator for secondary or internal standards, and a 10,000-fold lower titre preparation to serve as an in-run control. The preparations have been freeze-dried to maximise stability. These HIV-Ebola virus RNA reference materials were suitable for use with in-house and commercial quantitative RT-PCR assays and with digital RT-PCR. The HIV-Ebola virus RNA reference materials are stable at up to 37°C for two weeks, allowing the shipment of the material worldwide at ambient temperature. These results support further evaluation of the HIV-Ebola virus RNA reference materials as part of an International collaborative study for the establishment of the 1st International Standard for Ebola virus RNA. PMID:26562415

  13. New quantitative total protein S-assay system for diagnosing protein S type II deficiency: clinical application of the screening system for protein S type II deficiency.

    PubMed

    Tsuda, Tomohide; Jin, Xiuri; Tsuda, Hiroko; Ieko, Masahiro; Morishita, Eriko; Adachi, Tomoko; Hamasaki, Naotaka

    2012-01-01

    Venous thromboembolism (VTE) incidence is rising rapidly in Japan with lifestyle westernization and aging. Deficiency of protein S, an important blood coagulation regulator, is a risk factor for VTE. Protein S deficiency prevalence in Asians is approximately 10 times that in Caucasians and that of protein S type II deficiency, associated with the protein S Tokushima mutation (K155E), is quite high in Japan. However, currently available methods for measuring protein S are not precise enough for detection of this deficiency. We developed a novel assay system for precise simultaneous determinations of total protein S activity and total protein S antigen level, using a general-purpose automated analyzer, allowing protein S-specific activity (ratio of total protein S activity to total protein S antigen level) to be calculated. Mean specific activity was 0.99 for samples from healthy individuals but 0.69 or less (mean-3SD) in protein S type II-deficient and warfarin-treated samples, but was 1.0 in an estrogen-treated sample with significantly decreased protein S antigen. Protein S gene analyses in healthy individuals with specific activity 0.69 or less revealed the K155E mutation in all three. These results show our new assay system to be an effective screening tool for protein S type II deficiency. This system can also be used in an automated analyzer, facilitating numerous sample measurements, and is, thus, applicable to regular medical checkups and diagnosing VTE. Such applications would potentially contribute to early detection of protein S type II deficiency, and, thereby, to thrombosis prevention. PMID:22157257

  14. Continuous colorimetric screening assays for the detection of specific L- or D-α-amino acid transaminases in enzyme libraries.

    PubMed

    Heuson, Egon; Petit, Jean-Louis; Debard, Adrien; Job, Aurélie; Charmantray, Franck; de Berardinis, Véronique; Gefflaut, Thierry

    2016-01-01

    In the course of a project devoted to the stereoselective synthesis of non-proteinogenic α-amino acids using α-transaminases (α-TA), we report the design and optimization of generic high-throughput continuous assays for the screening of α-TA libraries. These assays are based on the use of L- or D-cysteine sulfinic acid (CSA) as irreversible amino donor and subsequent sulfite titration by colorimetry. The assays' quality was assessed under screening conditions. Hit selection thresholds were accurately determined for every couple of substrates and a library of 232 putative transaminases expressed in Escherichia coli host cells was screened. The reported high throughput screening assays proved very sensitive allowing the detection with high confidence of activities as low as 10 μU (i.e., 0.01 nmol substrate converted per min). The assays were also evidenced to be stereochemically discriminant since L-CSA and D-CSA allowed the exclusive detection of L-TA and D-TA, respectively. These generic assays thus allow testing the stereoselective conversion of a wide range of α-keto acids into α-amino acids of interest. As a proof of principle, the use of 2-oxo-4-phenylbutyric acid as acceptor substrate led to the identification of 54 new α-TA offering an access to valuable L- or D-homophenylalanine. PMID:26452497

  15. A Low-Dose Electron Diffraction Assay for Protection of Protein Structure against Damage from Drying

    NASA Astrophysics Data System (ADS)

    Massover, William H.

    2004-04-01

    A new assay using low-dose electron diffraction to measure the protection of protein structure against damage from drying is described. When thin single crystals of catalase are dried within water alone, low-dose electron diffraction yields no Bragg spots. Drying within an experimental aqueous solution that permits detection of diffraction spots thereby indicates a positive result, and the extent of these Bragg reflections into the high angle range gives a quantitative measure of the degree of protection. Bragg spots out to 3.7 3.9 [Angstrom capital A, ring] are recorded for drying within 100 mM solutions of the known structure-preserving sugars, sucrose, tannin, and trehalose. The ability of trehalose to maintain native protein structure during drying starts between 10 and 25 mM, and changes only slightly at concentrations above this threshold; with drying in 150-mM trehalose, catalase crystals yield diffraction spots out to 3.7 [Angstrom capital A, ring]. Drying within the organic nonsugar polymer polyvinylpyrrolidone gives Bragg spots to 4.0 [Angstrom capital A, ring]. This new assay should be useful to measure the unexamined structure-preserving capabilities of modified sugars, other nonsugars, and mixtures to identify which protective matrix maintains native protein structure to the greatest extent during drying; electron crystallography using that optimal matrix should yield protein structure at improved levels of high resolution.

  16. A novel protease activity assay using a protease-responsive chaperone protein

    SciTech Connect

    Sao, Kentaro; Murata, Masaharu; Fujisaki, Yuri; Umezaki, Kaori; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki; Hashizume, Makoto

    2009-06-05

    Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

  17. A Force-Based, Parallel Assay for the Quantification of Protein-DNA Interactions

    PubMed Central

    Limmer, Katja; Pippig, Diana A.; Aschenbrenner, Daniela; Gaub, Hermann E.

    2014-01-01

    Analysis of transcription factor binding to DNA sequences is of utmost importance to understand the intricate regulatory mechanisms that underlie gene expression. Several techniques exist that quantify DNA-protein affinity, but they are either very time-consuming or suffer from possible misinterpretation due to complicated algorithms or approximations like many high-throughput techniques. We present a more direct method to quantify DNA-protein interaction in a force-based assay. In contrast to single-molecule force spectroscopy, our technique, the Molecular Force Assay (MFA), parallelizes force measurements so that it can test one or multiple proteins against several DNA sequences in a single experiment. The interaction strength is quantified by comparison to the well-defined rupture stability of different DNA duplexes. As a proof-of-principle, we measured the interaction of the zinc finger construct Zif268/NRE against six different DNA constructs. We could show the specificity of our approach and quantify the strength of the protein-DNA interaction. PMID:24586920

  18. Uric acid photo-oxidation assay: in vitro comparison of sunscreening agents.

    PubMed

    Dunlap, W C; Yamamoto, Y; Inoue, M; Kashiba-Iwatsuki, M; Yamaguchi, M; Tomita, K

    1998-02-01

    We present a new method to evaluate the photo-oxidative activity of sunscreening agents based on the photodynamic oxidation of uric acid. Uric acid was selected as the oxidant probe for its high reactivity to singlet oxygen and oxygen radicals, high sensitivity of detection using electrochemical (EC) techniques, low light absorptivity and high photochemical stability in the UVA/B region of interest, and stability to autoxidation. The method is demonstrated by the photodynamic oxidation of uric acid on co-irradiation with Rose Bengal, a highly efficient photosensitizing dye for the production of singlet oxygen (1O2). Using this assay we found that the relative photodynamic oxidation rates of UVB-absorbing sunscreens in 80% methanol on irradiation with >290 nm light decreased in the order 2-ethylhexyl 4-dimethylaminobenzoate (DMABA-2EH) > 2-ethylhexyl 4-methoxycinnamate (MCA-2EH) and the experimental sunscreens, 1-(1,1-dimethylethyl)-3-octanoyl-4,4-dimethyl- 1,4,5,6,-tetrahydropyridine (ICI-319) and 1-(2-methylpropyl)-3-propionyl-4,4-dimethyl-1,4,5,6-tetrahydropyridine (ICI-855). The relative photodynamic oxidation rates of UVA-absorbing sunscreens decreased in the order 4-tert-butyl-4'-methoxydibenzoylmethane (BMDBM) and 4-(2-propyl)benzophenone (PB) > 2-hydroxy-4'-methoxy-benzophenone (HMB) and 2,2'-dihydroxy-4-methoxybenzophenone (DHMB). We have confirmed the photodynamic activity of DMABA-2EH for the production of singlet oxygen (1O2) using electron paramagnetic resonance (EPR) spectroscopy and the reagent 2,2,6,6-tetramethyl-4-piperidone (4-oxo-TEMP). We failed to detect the photodynamic production of the oxyradicals, superoxide (O2.-) and hydroxyl radical (HO.) using N-tert-butyl-a-phenylnitrone (PBN) and 5,5-dimethyl-1-pyrrolidine-1-oxide (DMPO) as a result of photochemical interference caused by these spin-trapping reagents. The uric acid photo-oxidation assay was also used to compare the photodynamic reactivity of light-reflective, microfine oxides TiO2, Zn

  19. Evaluation of Multiplexed Foot-and-Mouth Disease Nonstructural Protein Antibody Assay Against Standardized Bovine Serum Panel

    SciTech Connect

    Perkins, J; Parida, S; Clavijo, A

    2007-05-14

    Liquid array technology has previously been used to show proof-of-principle of a multiplexed non structural protein serological assay to differentiate foot-and-mouth infected and vaccinated animals. The current multiplexed assay consists of synthetically produced peptide signatures 3A, 3B and 3D and recombinant protein signature 3ABC in combination with four controls. To determine diagnostic specificity of each signature in the multiplex, the assay was evaluated against a naive population (n = 104) and a vaccinated population (n = 94). Subsequently, the multiplexed assay was assessed using a panel of bovine sera generated by the World Reference Laboratory for foot-and-mouth disease in Pirbright, UK. This sera panel has been used to assess the performance of other singleplex ELISA-based non-structural protein antibody assays. The 3ABC signature in the multiplexed assay showed comparative performance to a commercially available non-structural protein 3ABC ELISA (Cedi test{reg_sign}) and additional information pertaining to the relative diagnostic sensitivity of each signature in the multiplex is acquired in one experiment. The encouraging results of the evaluation of the multiplexed assay against a panel of diagnostically relevant samples promotes further assay development and optimization to generate an assay for routine use in foot-and-mouth disease surveillance.

  20. On-chip microtubule gliding assay for parallel measurement of tau protein species.

    PubMed

    Subramaniyan Parimalam, Subhathirai; Tarhan, Mehmet C; Karsten, Stanislav L; Fujita, Hiroyuki; Shintaku, Hirofumi; Kotera, Hidetoshi; Yokokawa, Ryuji

    2016-04-26

    Tau protein is a well-established biomarker for a group of neurodegenerative diseases collectively called tauopathies. So far, clinically relevant detection of tau species in cerebrospinal fluid (CSF) cannot be achieved without immunological methods. Recently, it was shown that different tau isoforms including the ones carrying various types of mutations affect microtubule (MT)-kinesin binding and velocity in an isoform specific manner. Here, based on these observations, we developed a microfluidic device to analyze tau mutations, isoforms and their ratios. The assay device consists of three regions: a MT reservoir which captures MTs from a solution to a kinesin-coated surface, a microchannel which guides gliding MTs, and an arrowhead-shaped collector which concentrates MTs. Tau-bound fluorescently labeled MTs (tau-MTs) were assayed, and the increase in fluorescence intensity (FI) corresponding to the total number of MTs accumulated was measured at the collector. We show that our device is capable of differentiating 3R and 4R tau isoform ratios and effects of point mutations within 5 minutes. Furthermore, radially oriented collector regions enable simultaneous FI measurements for six independent assays. Performing parallel assays in the proposed device with minimal image processing provides a cost-efficient, easy-to-use and fast tau detection platform. PMID:27056640

  1. Optimized DPPH assay in a detergent-based buffer system for measuring antioxidant activity of proteins

    PubMed Central

    Nicklisch, Sascha C.T.; Waite, J. Herbert

    2014-01-01

    The free radical method using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) is a well established assay for the in vitro determination of antioxidant activity in food and biological extracts. The standard DPPH assay uses methanol or ethanol as solvents, or buffered alcoholic solutions in a ratio of 40%/60% (buffer/alcohol, v/v) to keep the hydrophobic hydrazyl radical and phenolic test compounds soluble while offering sufficient buffering capacity at different pHs tested. Following this protocol, we were unable to keep proteinaceous antioxidants soluble at different pHs to test for their antioxidant activity. Thus, the assay protocol was modified as follows to improve its utility:•Non-ionic detergents were added to keep the DPPH radical soluble and to provide a mild and non-denaturing environment for the antioxidant protein.•Maximal concentration of DPPH was limited to 100 μM to stay within the sensitivity range of the detector at the given wavelength (515 nm) and to increase the dynamic range of the assay.•0.1 M citrate phosphate buffer was introduced to prevent experimental artifacts due to changing buffer compositions at different pHs. PMID:25530949

  2. Amino acid metabolism and protein synthesis in malarial parasites*

    PubMed Central

    Sherman, I. W.

    1977-01-01

    Malaria-infected red cells and free parasites have limited capabilities for the biosynthesis of amino acids. Therefore, the principal amino acid sources for parasite protein synthesis are the plasma free amino acids and host cell haemoglobin. Infected cells and plasmodia incorporate exogenously supplied amino acids into protein. However, the hypothesis that amino acid utilization (from an external source) is related to availability of that amino acid in haemoglobin is without universal support: it is true for isoleucine and for Plasmodium knowlesi and P. falciparum, but not for methionine, cysteine, and other amino acids, and it does not apply to P. lophurae. More by default than by direct evidence, haemoglobin is believed to be the main amino acid reservoir available to the intraerythrocytic plasmodium. Haemoglobin, ingested via the cytostome, is held in food vacuoles where auto-oxidation takes place. As a consequence, haem is released and accumulates in the vacuole as particulate haemozoin (= malaria pigment). Current evidence favours the view that haemozoin is mainly haematin. Acid and alkaline proteases (identified in crude extracts from mammalian and avian malarias) are presumably secreted directly into the food vacuole. They then digest the denatured globin and the resulting amino acids are incorporated into parasite protein. Cell-free protein synthesizing systems have been developed using P. knowlesi and P. lophurae ribosomes. In the main these systems are typically eukaryotic. Studies of amino acid metabolism are exceedingly limited. Arginine, lysine, methionine, and proline are incorporated into protein, whereas glutamic acid is metabolized via an NADP-specific glutamic dehydrogenase. Glutamate oxidation generates NADPH and auxiliary energy (in the form of α-ketoglutarate). The role of red cell glutathione in the economy of the parasite remains obscure. Important goals for future research should be: quantitative assessment of the relative importance of

  3. In vitro Assay for Cytidine Deaminase Activity of APOBEC3 Protein

    PubMed Central

    Nair, Smita; Rein, Alan

    2016-01-01

    Cytidine deaminases are enzymes that catalyze the removal of an amino group from cytidine, forming uridine. APOBEC3 (ApolipoproteinB mRNA editing enzyme, catalytic polypeptide like) proteins are cytidine deaminases that deaminate cytidines in polynucleotides (RNA/DNA), resulting in editing of their target substrates. Mammalian APOBEC3 proteins are an important element in cellular defenses against retrovirus replication, and this “restriction” of retroviral infections is partially due to the cytidine deaminase activity of the APOBEC3. The present protocol (Nair et al., 2014) describes the assay to detect the deaminase activity of mouse APOBEC3 protein, which targets cytidines present in TCC or TTC motifs in a single-stranded DNA substrate. In brief, the protein preparation to be assayed is incubated with a fluorophore-labeled oligodeoxynucleotide containing the deamination target motif (radiolabeled oligonucleotide substrates have also been successfully used by other groups). Cytidines in the oligonucleotide are deaminated to uridines; the addition of Uracil DNA Glycosylase (UDG) catalyzes the hydrolysis of the N-glycosylic bond between uracil and sugar, generating an abasic (AB) site in the oligonucleotide. Mild alkali treatment cleaves the substrate oligonucleotide at the AB site; cleaved products are resolved from uncleaved substrate by denaturing polyacrylamide gel electrophoresis and visualized on a fluorescence scanner. The protocol described here is mainly adapted from that described by Iwatani et al. (2006) with modifications. The assay can, of course, be used to detect the activity of other APOBEC3 deaminases targeting DNA substrates, using oligonucleotides containing the cytidine-containing target sequence for the deaminase.

  4. Pantothenic acid quantification by a stable isotope dilution assay based on liquid chromatography-tandem mass spectrometry.

    PubMed

    Rychlik, Michael

    2003-07-01

    A stable isotope dilution assay for the quantification of free and total pantothenic acid has been developed by using [13C3,15N]-pantothenic acid as the internal standard. The three-dimensional specificity of liquid chromatography-tandem mass spectrometry enabled unequivocal determination of the vitamin. Due to the very simple extraction and clean-up procedure, free pantothenic acid could be analysed within 2 h, which is much faster than by microbiological or gas chromatographic assays. For quantification of total pantothenic acid, the vitamin was liberated from its conjugates by an overnight incubation with pigeon liver pantetheinase and alkaline phosphatase. In analyses of corn flour, the intra-assay coefficient of variation was 8.5% (n = 5) and 15.3% (n = 4) for free and total pantothenic acid, respectively. When pantothenic acid was added to corn starch at a level of 6 mg kg(-1), a recovery of 97.5% was found. Application of the stable isotope dilution assay to whole egg powder, hazel nuts and corn revealed similar data compared to those listed in nutrition data bases, whereas the content in mushrooms and porcine liver determined by the newly developed assay appeared to be lower and that of cocoa higher than reported in the literature. PMID:12894818

  5. Phosphate acceptor amino acid residues in structural proteins of rhabdoviruses.

    PubMed

    Sokol, F; Tan, K B; McFalls, M L; Madore, P

    1974-07-01

    Partial acid hydrolysates of the [(32)P]phosphate- or [(3)H]serine-labeled proteins of purified vesicular stomatitis, rabies, Lagos bat, Mokola, or spring viremia of carp virions and of purified intracellular nucleocapsids of these viruses have been analyzed by paper electrophoresis for the presence of phosphorylated amino acids. Both phosphoserine and phosphothreonine, with the former predominant, were present in virion and nucleocapsid preparations that contained phosphoproteins. An exception was the fish rhabdovirus, which contained only phosphoserine. When vesicular stomatitis or rabies virus proteins were phosphorylated in a cell-free system by the virion-associated protein kinase and analyzed for the presence of phosphorylated amino acid residues, phosphoserine was again found to be more abundant than phosphothreonine. After in vitro protein phosphorylation, another phospho-compound, possibly a third phosphoamino acid, was detected in the partial acid hydrolysates of these viruses. PMID:4365328

  6. Protein and amino acid metabolism and requirements

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells of the body. Enzymes, membrane carriers, blood transport molecules, intracellular matrix, and even hair and fingernails are proteins, as are many hormones. Proteins also constitute a major portion of all membranes, and the cons...

  7. Interaction of milk whey protein with common phenolic acids

    NASA Astrophysics Data System (ADS)

    Zhang, Hao; Yu, Dandan; Sun, Jing; Guo, Huiyuan; Ding, Qingbo; Liu, Ruihai; Ren, Fazheng

    2014-01-01

    Phenolics-rich foods such as fruit juices and coffee are often consumed with milk. In this study, the interactions of α-lactalbumin and β-lactoglobulin with the phenolic acids (chlorogenic acid, caffeic acid, ferulic acid, and coumalic acid) were examined. Fluorescence, CD, and FTIR spectroscopies were used to analyze the binding modes, binding constants, and the effects of complexation on the conformation of whey protein. The results showed that binding constants of each whey protein-phenolic acid interaction ranged from 4 × 105 to 7 × 106 M-n and the number of binding sites n ranged from 1.28 ± 0.13 to 1.54 ± 0.34. Because of these interactions, the conformation of whey protein was altered, with a significant reduction in the amount of α-helix and an increase in the amounts of β-sheet and turn structures.

  8. Silver nanoclusters-based fluorescence assay of protein kinase activity and inhibition.

    PubMed

    Shen, Congcong; Xia, Xiaodong; Hu, Shengqiang; Yang, Minghui; Wang, Jianxiu

    2015-01-01

    A simple and sensitive fluorescence method for monitoring the activity and inhibition of protein kinase (PKA) has been developed using polycytosine oligonucleotide (dC12)-templated silver nanoclusters (Ag NCs). Adenosine-5'-triphosphate (ATP) was found to enhance the fluorescence of Ag NCs, while the hydrolysis of ATP to adenosine diphosphate (ADP) by PKA decreased the fluorescence of Ag NCs. Compared to the existing methods for kinase activity assay, the developed method does not involve phosphorylation of the substrate peptides, which significantly simplifies the detection procedures. The method exhibits high sensitivity, good selectivity, and wide linear range toward PKA detection. The inhibition effect of kinase inhibitor H-89 on the activity of PKA was also studied. The sensing protocol was also applied to the assay of drug-stimulated activation of PKA in HeLa cell lysates. PMID:25517425

  9. A Spectrophotometric Assay Optimizing Conditions for Pepsin Activity.

    ERIC Educational Resources Information Center

    Harding, Ethelynda E.; Kimsey, R. Scott

    1998-01-01

    Describes a laboratory protocol optimizing the conditions for the assay of pepsin activity using the Coomasie Blue dye binding assay of protein concentration. The dye bonds through strong, noncovalent interactions to basic and aromatic amino acid residues. (DDR)

  10. A Review of Cry Protein Detection with Enzyme-Linked Immunosorbent Assays.

    PubMed

    Albright, Vurtice C; Hellmich, Richard L; Coats, Joel R

    2016-03-23

    The widespread use of Cry proteins in insecticide formulations and transgenic crops for insect control has led to an increased interest in the environmental fate of these proteins. Although several detection methods are available to monitor the fate of Cry proteins in the environment, enzyme-linked immunosorbent assays (ELISAs) have emerged as the preferred detection method, due to their cost-effectiveness, ease of use, and rapid results. Validation of ELISAs is necessary to ensure accurate measurements of Cry protein concentrations in the environment. Validation methodology has been extensively researched and published for the areas of sensitivity, specificity, accuracy, and precision; however, cross validation of ELISA results has been studied to a lesser extent. This review discusses the use of ELISAs for detection of Cry proteins in environmental samples and validation of ELISAs and introduces cross validation. The state of Cry protein environmental fate research is considered through a critical review of published literature to identify areas where the use of validation protocols can be improved. PMID:26949828

  11. Chloroplast envelope protein targeting fidelity is independent of cytosolic components in dual organelle assays

    PubMed Central

    Kriechbaumer, Verena; Abell, Ben M.

    2012-01-01

    The general mechanisms of intracellular protein targeting are well established, and depend on a targeting sequence in the protein, which is recognized by a targeting factor. Once a membrane protein is delivered to the correct organelle its targeting sequence can be recognized by receptors and a translocase, leading to membrane insertion. However, the relative contribution of each step for generating fidelity and efficiency of the overall process has not been systematically addressed. Here, we use tail-anchored (TA) membrane proteins in cell-free competitive targeting assays to chloroplasts to show that targeting can occur efficiently and with high fidelity in the absence of all cytosolic components, suggesting that chloroplast envelope protein targeting is primarily dependent on events at the outer envelope. Efficiency of targeting was increased by the addition of complete cytosol, and by Hsp70 or Hsp90, depending on the protein, but none of these cytosolic components influenced the fidelity of targeting. Our results suggest that the main role of targeting factors in chloroplast localization is to increase targeting efficiency by maintaining recognition competency at the outer envelope. PMID:22783268

  12. A simple purification and activity assay of the coagulant protein from Moringa oleifera seed.

    PubMed

    Ghebremichael, Kebreab A; Gunaratna, K R; Henriksson, Hongbin; Brumer, Harry; Dalhammar, Gunnel

    2005-06-01

    Use of extracts from Moringa oleifera (MO) is of great interest for low-cost water treatment. This paper discusses water and salt extraction of a coagulant protein from the seed, purification using ion exchange, its chemical characteristics, coagulation and antimicrobial properties. The coagulant from both extracts is a cationic protein with pI greater than 9.6 and molecular mass less than 6.5 kDa. Mass spectrometric analysis of the purified water extract indicated that it contained at least four homologous proteins, based on MS/MS peptide sequence data. The protein is thermoresistant and remained active after 5h heat treatment at 95 degrees C. The coagulant protein showed both flocculating and antibacterial effects of 1.1--4 log reduction. With samples of high turbidity, the MO extract showed similar coagulation activity as alum. Cecropin A and MO extract were found to have similar flocculation effects for clay and microorganisms. Simple methods for both the purification and assay of MO coagulating proteins are presented, which are necessary for large-scale water treatment applications. PMID:15921719

  13. [Protein assay by the modified Dumas method applied to preparations of plasma proteins].

    PubMed

    Blondel, P; Vian, L

    1993-01-01

    Quantify protein according Pharmacopoeia method, based on Kjeldahl method, needs a long time to do. The development of an automaton which used the modified Dumas method divide the analysis time by 15 (6 minutes versus over 90 minutes). The results show no statistical differences between official method and this one. PMID:8154798

  14. Genotoxic effects of boric acid and borax in zebrafish, Danio rerio using alkaline comet assay.

    PubMed

    Gülsoy, Nagihan; Yavas, Cüneyd; Mutlu, Özal

    2015-01-01

    The present study is conducted to determine the potential mechanisms of Boron compounds, boric acid (BA) and borax (BX), on genotoxicity of zebrafish Danio rerio for 24, 48, 72 and 96-hours acute exposure (level:1, 4, 16, 64 mg/l BA and BX) in semi-static bioassay experiment. For that purpose, peripheral erythrocytes were drawn from caudal vein and Comet assay was applied to assess genotoxicity. Acute (96 hours) exposure and high concentrations of boric acid and borax increases % tail DNA and Olive tail moment. Genotoxicity was found for BA as concentration-dependent and BX as concentration and time dependent manner. In general, significant effects (P < 0,05) on both concentrations and exposure times were observed in experimental groups. DNA damage was highest at 96 h and 24 h for all BX and BA concentrations, respectively in peripheral blood of D. rerio. For the first time, our study demonstrates the effect of waterborne BA and BX exposure on genotoxicity at the molecular level, which may contribute to understanding the mechanism of boric acid and borax-induced genotoxicity in fish. PMID:26862320

  15. Urinary Excretion of Phenolic Acids by Infants and Children: A Randomised Double-Blind Clinical Assay

    PubMed Central

    Uberos, J.; Fernández-Puentes, V.; Molina-Oya, M.; Rodríguez-Belmonte, R.; Ruíz-López, A.; Tortosa-Pinto, P.; Molina-Carballo, A.; Muñoz-Hoyos, A.

    2012-01-01

    Objectives: The present study, which is part of the ISRCTN16968287 clinical assay, is aimed at determining the effects of cranberry syrup or trimethoprim treatment for UTI. Methods: This Phase III randomised clinical trial was conducted at the San Cecilio Clinical Hospital (Granada, Spain) with a study population of 192 patients, aged between 1 month and 13 years. Criteria for inclusion were a background of recurrent UTI, associated or otherwise with vesico-ureteral reflux of any degree, or renal pelvic dilatation associated with urinary infection. Each child was randomly given 0.2 mL/Kg/day of either cranberry syrup or trimethoprim (8 mg/mL). The primary and secondary objectives, respectively, were to determine the risk of UTI and the levels of phenolic acids in urine associated with each intervention. Results: With respect to UTI, the cranberry treatment was non-inferior to trimethoprim. Increased urinary excretion of ferulic acid was associated with a greater risk of UTI developing in infants aged under 1 year (RR 1.06; CI 95% 1.024–1.1; P = 0.001). Conclusions: The results obtained show the excretion of ferulic acid is higher in infants aged under 1 year, giving rise to an increased risk of UTI, for both treatment options. PMID:23641168

  16. Genotoxic effects of boric acid and borax in zebrafish, Danio rerio using alkaline comet assay

    PubMed Central

    Gülsoy, Nagihan; Yavas, Cüneyd; Mutlu, Özal

    2015-01-01

    The present study is conducted to determine the potential mechanisms of Boron compounds, boric acid (BA) and borax (BX), on genotoxicity of zebrafish Danio rerio for 24, 48, 72 and 96-hours acute exposure (level:1, 4, 16, 64 mg/l BA and BX) in semi-static bioassay experiment. For that purpose, peripheral erythrocytes were drawn from caudal vein and Comet assay was applied to assess genotoxicity. Acute (96 hours) exposure and high concentrations of boric acid and borax increases % tail DNA and Olive tail moment. Genotoxicity was found for BA as concentration-dependent and BX as concentration and time dependent manner. In general, significant effects (P < 0,05) on both concentrations and exposure times were observed in experimental groups. DNA damage was highest at 96 h and 24 h for all BX and BA concentrations, respectively in peripheral blood of D. rerio. For the first time, our study demonstrates the effect of waterborne BA and BX exposure on genotoxicity at the molecular level, which may contribute to understanding the mechanism of boric acid and borax-induced genotoxicity in fish. PMID:26862320

  17. Immunochemical Assays and Nucleic-Acid Detection Techniques for Clinical Diagnosis of Prostate Cancer

    PubMed Central

    Kanyong, Prosper; Rawlinson, Sean; Davis, James

    2016-01-01

    Prostate cancer (PCa) is a significant cause of morbidity and mortality and the most common cancer in men in Europe, North America, and some parts of Africa. The established methods for detecting PCa are normally based on tests using Prostate Specific Antigen (PSA) in blood, Prostate cancer antigen 3 (PCA3) in urine and tissue Alpha-methylacyl-CoA racemase (AMACR) as tumour markers in patient samples. Prior to the introduction of PSA in clinics, prostatic acid phosphatase (PAP) was the most widely used biomarker. An early diagnosis of PCa through the detection of these biomarkers requires the availability of simple, reliable, cost-effective and robust techniques. Immunoassays and nucleic acid detection techniques have experienced unprecedented growth in recent years and seem to be the most promising analytical tools. This growth has been driven in part by the surge in demand for near-patient-testing systems in clinical diagnosis. This article reviews immunochemical assays, and nucleic-acid detection techniques that have been used to clinically diagnose PCa. PMID:26958088

  18. Measuring protein-protein and protein-nucleic Acid interactions by biolayer interferometry.

    PubMed

    Sultana, Azmiri; Lee, Jeffrey E

    2015-01-01

    Biolayer interferometry (BLI) is a simple, optical dip-and-read system useful for measuring interactions between proteins, peptides, nucleic acids, small molecules, and/or lipids in real time. In BLI, a biomolecular bait is immobilized on a matrix at the tip of a fiber-optic sensor. The binding between the immobilized ligand and another molecule in an analyte solution produces a change in optical thickness at the tip and results in a wavelength shift proportional to binding. BLI provides direct binding affinities and rates of association and dissociation. This unit describes an efficient approach using streptavidin-based BLI to analyze DNA-protein and protein-protein interactions. A quantitative set of equilibrium binding affinities (K(d)) and rates of association and dissociation (k(a)/k(d)) can be measured in minutes using nanomole quantities of sample. PMID:25640894

  19. Interaction of perfluoroalkyl acids with human liver fatty acid-binding protein.

    PubMed

    Sheng, Nan; Li, Juan; Liu, Hui; Zhang, Aiqian; Dai, Jiayin

    2016-01-01

    Perfluoroalkyl acids (PFAAs) are highly persistent and bioaccumulative, resulting in their broad distribution in humans and the environment. The liver is an important target for PFAAs, but the mechanisms behind PFAAs interaction with hepatocyte proteins remain poorly understood. We characterized the binding of PFAAs to human liver fatty acid-binding protein (hL-FABP) and identified critical structural features in their interaction. The binding interaction of PFAAs with hL-FABP was determined by fluorescence displacement and isothermal titration calorimetry (ITC) assay. Molecular simulation was conducted to define interactions at the binding sites. ITC measurement revealed that PFOA/PFNA displayed a moderate affinity for hL-FABP at a 1:1 molar ratio, a weak binding affinity for PFHxS and no binding for PFHxA. Moreover, the interaction was mainly mediated by electrostatic attraction and hydrogen bonding. Substitution of Asn111 with Asp caused loss of binding affinity to PFAA, indicating its crucial role for the initial PFAA binding to the outer binding site. Substitution of Arg122 with Gly caused only one molecule of PFAA to bind to hL-FABP. Molecular simulation showed that substitution of Arg122 increased the volume of the outer binding pocket, making it impossible to form intensive hydrophobic stacking and hydrogen bonds with PFOA, and highlighting its crucial role in the binding process. The binding affinity of PFAAs increased significantly with their carbon number. Arg122 and Asn111 played a pivotal role in these interactions. Our findings may help understand the distribution pattern, bioaccumulation, elimination, and toxicity of PFAAs in humans. PMID:25370009

  20. Quantitative mass spectrometry-based assay development and validation: from small molecules to proteins.

    PubMed

    Božović, Andrea; Kulasingam, Vathany

    2013-04-01

    Mass spectrometry (MS) has emerged as a powerful analytical tool for the identification, characterization and quantification of various biomolecules (small molecules, drug metabolites and proteins) in biological specimens. The use of mass spectrometers in the clinical diagnostic laboratories have gained popularity due to its ease of development of new assays, ability to measure multiple analytes in a single analytical run, low volume requirements and low reagent costs. Novel technological advancements in ionization sources, instrumentation and software have increased the popularity of these platforms. Consequently, a number of home-brew assays, utilizing the power of MS, are being developed and validated for clinical diagnostic use. In this review, we will discuss the two phases that precede method implementation: method development and validation for both small molecule analysis and protein quantification using liquid chromatography tandem mass spectrometry (LC-MS/MS). Some of the challenges facing protein quantification will be highlighted and an outlook for the future of laboratory medicine and MS will be provided. PMID:23041077

  1. Simultaneous Measurement of Multiple Mouse Ear Proteins with Multiplex ELISA Assays

    PubMed Central

    Trune, Dennis R.; Larrain, Barbara E.; Hausman, Frances A.; Kempton, J. Beth; MacArthur, Carol J.

    2011-01-01

    A recent advancement in enzyme-linked immunosorbent assay (ELISA) technology is the multiplex antibody array that measures multiple proteins simultaneously within a single sample. This allows reduction in sample volume, time, labor, and material costs, while increasing sensitivity over single ELISA. Current multiplex platforms include planar-based systems using microplates or slides, or bead-based suspension assay with microspheres. To determine the applicability of this technology for ear research, we used 3 different multiplex ELISA-based immunoassay arrays from 4 different companies to measure cytokine levels in mouse middle and inner ear tissue lysate extracts 24 hours following transtympanic Haemophilus influenzae inoculation. Middle and inner ear tissue lysates were analyzed using testing services from Quansys BioSciences, Aushon Biosystems SearchLight (both microplate-based), Milliplex MAP Sample (bead-based), and a RayBiotech, Inc. (slide-based) kit. Samples were assayed in duplicate or triplicate. Results were compared to determine their relative sensitivity and reliability for measures of cytokine related to inflammation. The cytokine pg/ml amounts varied among the multiplex assays, so a comparison also was made of the mean fold increase in cytokines from untreated controls. Several cytokines and chemokines were elevated, the extent dependent upon the assay sensitivity. Those most significantly elevated were IL-1α, IL-1β, IL-6, TNFα, VEGF, IL-8/MIP-2. The results of the multiplex systems were compared with single ELISA kits (IL-1β, IL-6) to assess sensitivity over the traditional method. Overall, the Quansys Biosciences and SearchLight arrays showed the greatest sensitivity, both employing the same multiplex methodology of a spotted array within a microplate well with chemiluminescent detection. They also were more sensitive than the traditional single ELISA performed with commercial kits and matched gene expression changes determined by quantitative

  2. Development of a multi-product leached protein A assay for bioprocess samples containing recombinant human monoclonal antibodies.

    PubMed

    Ren, Diya; Darlucio, Maria R; Chou, Judy H

    2011-03-01

    The detection of low level of protein A leached from monoclonal antibody downstream purification process is often interfered by the presence of excess amount of product antibody. In order to prevent this interference, we developed a new multi-product leached protein A assay that used acidification to completely dissociate the IgG-ProteinA complex, followed by neutralization under selected condition to prevent re-formation of the IgG-ProteinA complex. The amount of protein A was then determined by a sandwich immunoassay using Meso Scale Discovery technology. The assay takes approximately 3h to complete for one 96-well plate of samples, and this has been successfully applied to samples containing different monoclonal antibody products examined so far. The data demonstrates that this assay is robust and efficient in determining leached protein A contamination during purification of recombinant monoclonal antibodies. PMID:21315722

  3. Complementary Spectroscopic Assays for Investigating Protein-Ligand Binding Activity: A Project for the Advanced Chemistry Laboratory

    ERIC Educational Resources Information Center

    Mascotti, David P.; Waner, Mark J.

    2010-01-01

    A protein-ligand binding, guided-inquiry laboratory project with potential application across the advanced undergraduate curriculum is described. At the heart of the project are fluorescence and spectrophotometric assays utilizing biotin-4-fluorescein and streptavidin. The use of the same stock solutions for an assay that may be examined by two…

  4. A Cell-Based Fluorescent Assay to Detect the Activity of Shiga Toxin and Other Toxins That Inhibit Protein Synthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli O157:H7, a major cause of food-borne illness, produces Shiga toxins that block protein synthesis by inactivating the ribosome. In this chapter we describe a simple cell-based fluorescent assay to detect Shiga toxins and inhibitors of toxin activity. The assay can also be used to d...

  5. Investigations into the Use of a Protein Sensor Assay for Metabolite Analysis.

    PubMed

    Hamerly, Timothy; Bothner, Brian

    2016-01-01

    Rapid and definitive classification of biological samples has application in industrial, agricultural, and clinical settings. Considerable effort has been given to analytical methods to address such applications over the past 50 years, with the majority of successful solutions focusing on a single molecular target. However, in many cases, a single or even a few features are insufficient for accurate characterization or classification. Serum albumin (SA) proteins are a class of cargo-carrying proteins in blood that have evolved to transport a wide variety of metabolites and peptides in mammals. These proteins have up to seven binding sites which communicate allosterically to orchestrate a complex pick-up and delivery system involving a large number of different molecules at any time. The ability of SA proteins to bind multiple molecular species in a sophisticated manner inspired the development of assays to differentiate complex biological solutions. The combination of SA and high-resolution liquid chromatography mass spectrometry (LC-MS) is showing exciting promise as a protein sensor assay (PSA) for classification of complex biological samples. In this study, the PSA has been applied to cells undergoing and recovering from mild oxidative stress. Analysis using traditional LC-MS-based metabolomics failed to differentiate samples into treatment or temporal groups, whereas samples first treated with the PSA were cleanly classified into both correct treatment and temporal groups. The success of the PSA could be attributed to selective binding of metabolites, leading to a reduction in sample complexity and a general reduction in chemical noise. Metabolites important to successful sample classification were often enriched by 100-fold or more yet displayed a wide range of affinities for SA. The end result of PSA treatment is better classification of samples with a reduction in the number of features seen overall. Together, these results demonstrate how the use of a

  6. Promising Nucleic Acid Lateral Flow Assay Plus PCR for Shiga Toxin-Producing Escherichia coli.

    PubMed

    Terao, Yoshitaka; Takeshita, Kana; Nishiyama, Yasutaka; Morishita, Naoki; Matsumoto, Takashi; Morimatsu, Fumiki

    2015-08-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) is a frequent cause of foodborne infections, and methods for rapid and reliable detection of STEC are needed. A nucleic acid lateral flow assay (NALFA) plus PCR was evaluated for detecting STEC after enrichment. When cell suspensions of 45 STEC strains, 14 non-STEC strains, and 13 non-E. coli strains were tested with the NALFA plus PCR, all of the STEC strains yielded positive results, and all of the non-STEC and non-E. coli strains yielded negative results. The lower detection limit for the STEC strains ranged from 0.1 to 1 pg of genomic DNA (about 20 to 200 CFU) per test, and the NALFA plus PCR was able to detect Stx1- and Stx2-producing E. coli strains with similar sensitivities. The ability of the NALFA plus PCR to detect STEC in enrichment cultures of radish sprouts, tomato, raw ground beef, and beef liver inoculated with 10-fold serially diluted STEC cultures was comparable to that of a real-time PCR assay (at a level of 100 to 100,000 CFU/ml in enrichment culture). The bacterial inoculation test in raw ground beef revealed that the lower detection limit of the NALFA plus PCR was also comparable to that obtained with a real-time PCR assay that followed the U.S. Department of Agriculture guidelines. Although further evaluation is required, these results suggest that the NALFA plus PCR is a specific and sensitive method for detecting STEC in a food manufacturing plant. PMID:26219371

  7. Established and emerging fluorescence-based assays for G-protein function: Ras-superfamily GTPases.

    PubMed

    Rojas, Rafael J; Kimple, Randall J; Rossman, Kent L; Siderovski, David P; Sondek, John

    2003-06-01

    Ras and Rho GTPases are signaling proteins that regulate a variety of physiological events and are intimately linked to the progression of cancer. Recently, a variety of fluorescence-based assays have been refined to monitor activation of these GTPases. This review summarizes current fluorescence-based techniques for studying Ras superfamily GTPases with an emphasis on practical examples and high-throughput applications. These techniques are not only useful for biochemical characterization of Ras superfamily members, but will also facilitate the discovery of small molecule therapeutics designed to inhibit signal transduction mediated by GTPases. PMID:12769685

  8. Light and heavy dansyl reporter groups in food chemistry: amino acid assay in beverages.

    PubMed

    Mazzotti, Fabio; Benabdelkamel, Hicham; Di Donna, Leonardo; Athanassopoulos, Constantinos M; Napoli, Anna; Sindona, Giovanni

    2012-07-01

    5-Dimethylamino-1-sulfonyl naphthalene (DNS, commonly referred as dansyl) is a functionality, bearing well-established properties in directing the fragmentation, by mass spectrometry (MS), of the corresponding ionized sulfonylated derivatives. This property is shared also by its labeled analogs. The use of d(0)/d(6) DNS derivatives is now exploited in the application of the well-established isotope dilution mass spectrometric approach in the assay of complex mixtures. A new method for the quantitation of amino acids (AAs) in beverages is therefore presented, which relies on liquid chromatographic separation of their N-dansylated derivatives followed by comparative electrospray tandem MS/MS of the d(0)/d(6) isobaric mixtures. Labeled and unlabeled DNS derivatives of the selected AAs are readily available by microwave-assisted synthetic protocols. The novelty of the method is represented by the use of heavy and light DNS-isotopologue providing suitable reporter groups. Multiple-reaction monitoring has been applied in the assay of AAs in wine, pineapple juice and bergamot juice with good-to-excellent results as proved by both relative standard deviation, lower than 15%, and by the accuracy values in the range 90-110%. PMID:22791261

  9. A bacterial type III secretion assay for delivery of fungal effector proteins into wheat.

    PubMed

    Upadhyaya, Narayana M; Mago, Rohit; Staskawicz, Brian J; Ayliffe, Michael A; Ellis, Jeffrey G; Dodds, Peter N

    2014-03-01

    Large numbers of candidate effectors from fungal pathogens are being identified through whole-genome sequencing and in planta expression studies. Although Agrobacterium-mediated transient expression has enabled high-throughput functional analysis of effectors in dicot plants, this assay is not effective in cereal leaves. Here, we show that a nonpathogenic Pseudomonas fluorescens engineered to express the type III secretion system (T3SS) of P. syringae and the wheat pathogen Xanthomonas translucens can deliver fusion proteins containing T3SS signals from P. syringae (AvrRpm1) and X. campestris (AvrBs2) avirulence (Avr) proteins, respectively, into wheat leaf cells. A calmodulin-dependent adenylate cyclase reporter protein was delivered effectively into wheat and barley by both bacteria. Absence of any disease symptoms with P. fluorescens makes it more suitable than X. translucens for detecting a hypersensitive response (HR) induced by an effector protein with avirulence activity. We further modified the delivery system by removal of the myristoylation site from the AvrRpm1 fusion to prevent its localization to the plasma membrane which could inhibit recognition of an Avr protein. Delivery of the flax rust AvrM protein by the modified delivery system into transgenic tobacco leaves expressing the corresponding M resistance protein induced a strong HR, indicating that the system is capable of delivering a functional rust Avr protein. In a preliminary screen of effectors from the stem rust fungus Puccinia graminis f. sp. tritici, we identified one effector that induced a host genotype-specific HR in wheat. Thus, the modified AvrRpm1:effector-Pseudomonas fluorescens system is an effective tool for large-scale screening of pathogen effectors for recognition in wheat. PMID:24156769

  10. Unsaturated fatty acids show clear elicitation responses in a modified local lymph node assay with an elicitation phase, and test positive in the direct peptide reactivity assay.

    PubMed

    Yamashita, Kunihiko; Shinoda, Shinsuke; Hagiwara, Saori; Miyazaki, Hiroshi; Itagaki, Hiroshi

    2015-12-01

    The Organisation for Economic Co-operation and Development (OECD) Test Guidelines (TG) adopted the murine local lymph node assay (LLNA) and guinea pig maximization test (GPMT) as stand-alone skin sensitization test methods. However, unsaturated carbon-carbon double-bond and/or lipid acids afforded false-positive results more frequently in the LLNA compared to those in the GPMT and/or in human subjects. In the current study, oleic, linoleic, linolenic, undecylenic, fumaric, maleic, and succinic acid and squalene were tested in a modified LLNA with an elicitation phase (LLNA:DAE), and in a direct peptide reactivity assay (DPRA) to evaluate their skin-sensitizing potential. Oleic, linoleic, linolenic, undecylenic and maleic acid were positive in the LLNA:DAE, of which three, linoleic, linolenic, and maleic acid were positive in the DPRA. Furthermore, the results of the cross-sensitizing tests using four LLNA:DAE-positive chemicals were negative, indicating a chemical-specific elicitation response. In a previous report, the estimated concentration needed to produce a stimulation index of 3 (EC3) of linolenic acid, squalene, and maleic acid in the LLNA was < 10%. Therefore, these chemicals were classified as moderate skin sensitizers in the LLNA. However, the skin-sensitizing potential of all LLNA:DAE-positive chemicals was estimated as weak. These results suggested that oleic, linoleic, linolenic, undecylenic, and maleic acid had skin-sensitizing potential, and that the LLNA overestimated the skin-sensitizing potential compared to that estimated by the LLNA:DAE. PMID:26558466

  11. Heat shock protein 70-dependent protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells.

    PubMed

    Qin, Ying; Naito, Yuji; Handa, Osamu; Hayashi, Natsuko; Kuki, Aiko; Mizushima, Katsura; Omatsu, Tatsushi; Tanimura, Yuko; Morita, Mayuko; Adachi, Satoko; Fukui, Akifumi; Hirata, Ikuhiro; Kishimoto, Etsuko; Nishikawa, Taichiro; Uchiyama, Kazuhiko; Ishikawa, Takeshi; Takagi, Tomohisa; Yagi, Nobuaki; Kokura, Satoshi; Yoshikawa, Toshikazu

    2011-11-01

    Protection of the small intestine from mucosal injury induced by nonsteroidal anti-inflammatory drugs including acetylsalicylic acid is a critical issue in the field of gastroenterology. Polaprezinc an anti-ulcer drug, consisting of zinc and L-carnosine, provides gastric mucosal protection against various irritants. In this study, we investigated the protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of the RIE1 rat intestinal epithelial cell line. Confluent rat intestinal epithelial cells were incubated with 70 µM polaprezinc for 24 h, and then stimulated with or without 15 mM acetylsalicylic acid for a further 15 h. Subsequent cellular viability was quantified by fluorometric assay based on cell lysis and staining. Acetylsalicylic acid-induced cell death was also qualified by fluorescent microscopy of Hoechst33342 and propidium iodide. Heat shock proteins 70 protein expression after adding polaprezinc or acetylsalicylic acid was assessed by western blotting. To investigate the role of Heat shock protein 70, Heat shock protein 70-specific small interfering RNA was applied. Cell viability was quantified by fluorometric assay based on cell lysis and staining and apoptosis was analyzed by fluorescence-activated cell sorting. We found that acetylsalicylic acid significantly induced apoptosis of rat intestinal epithelial cells in a dose- and time-dependent manner. Polaprezinc significantly suppressed acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells at its late phase. At the same time, polaprezinc increased Heat shock protein 70 expressions of rat intestinal epithelial cells in a time-dependent manner. However, in Heat shock protein 70-silenced rat intestinal epithelial cells, polaprezinc could not suppress acetylsalicylic acid -induced apoptosis at its late phase. We conclude that polaprezinc-increased Heat shock protein 70 expression might be an important mechanism by which polaprezinc suppresses acetylsalicylic

  12. Determination of Cry9C protein in corn-based foods by enzyme-linked immunosorbent assay: interlaboratory study.

    PubMed

    Trucksess, N W

    2001-01-01

    The performance of a commercially available enzyme-linked immunosorbent assay kit (Enviro-Logix) was assessed for the determination of Cry9C protein, which is produced by the genetically modified corn StarLink, in 8 types of corn-based foods (starch, refined oil, soft tortillas, tortilla chips, corn flakes, corn puffs, corn muffins, and corn bread) in an interlaboratory study involving 7 laboratories in the United States. The assay kit is a double antibody sandwich and is based on the specific interaction between antibody and antigen. The Cry9C protein analyte is sandwiched between 2 antibodies, one to capture the analyte and the other is conjugated to the enzyme, horseradish peroxidase. The enzyme uses tetramethylbenzidine/peroxide for color development. A strong acid stopping reagent is then used to change the color from blue to a stable yellow. The intensity of the color is proportional to the concentration of the Cry9C protein. In this study blind duplicates of control samples (blank material prepared from non- StarLink corn), spiked samples (blank material with the addition of Cry9C protein), and samples containing incurred analyte (products prepared with StarLink corn) were analyzed. Cry9C protein from 2 different sources was used to spike the food products. Cry9C protein produced and purified from a bacterial host was used to prepare spiked test samples at 2.72 and 6.8 ng/g. Cry9C protein from StarLink corn flour was used to prepare spiked samples at 1.97 ng/g. Average recoveries for samples spiked with corn flour Cry9C protein at 1.97 ng/g ranged from 73 to 122%, within-laboratory relative standard deviations (RSDr) ranged from 6 to 22%, and between-laboratories relative standard deviations (RSDR) ranged from 16 to 56%. Average recoveries for samples spiked with bacterial Cry9C protein at 2.72 and 6.8 ng/g ranged from 27 to 96% and from 32 to 113%, respectively; RSDr values ranged from 10 to 35% and from 7 to 38%, respectively; and the RSDR ranged from 28

  13. Enhanced Photoelectrochemical Proximity Assay for Highly Selective Protein Detection in Biological Matrixes.

    PubMed

    Wen, Guangming; Ju, Huangxian

    2016-08-16

    This work proposes the first photoelectrochemical proximity assay (PECPA) method via the sensitization of CdTe quantum dots (QDs) on photoelectrochemical response of ITO/TiO2/CdS electrode for highly selective and sensitive detection of proteins. This detection was performed on a sensing interface formed via the hybridization of capture DNA immobilized on ITO/TiO2/CdS electrode with labeled antibody-DNA (DNA-Ab1). Upon the recognition of Ab1 to target protein, the immunocomplex of DNA-Ab1, target, and the detection antibody-DNA (DNA-Ab2) was formed, which led to the proximity hybridization of the DNA in DNA-Ab2, capture DNA, and signal DNA-CdTe QDs, and brought CdTe QDs to the ITO/TiO2/CdS electrode to produce a sensitized photocurrent. The photocurrent intensity increased with the increasing concentration of the specific target protein. Using insulin as a target, this sensitized method showed a detectable range of 10 fM to 10 nM and a detection limit of 3.0 fM without the need of a washing step. It possessed high selectivity and good accuracy for detection of proteins in biological matrixes. This method is extremely flexible and can be extended to varieties of protein targets. PMID:27464227

  14. Aptamer-mediated turn-on fluorescence assay for prion protein based on guanine quenched fluophor.

    PubMed

    Xiao, Sai Jin; Hu, Ping Ping; Li, Yuan Fang; Huang, Cheng Zhi; Huang, Tao; Xiao, Geng Fu

    2009-10-15

    An aptamer-participated haprin structure was designed by employing cellular prion protein (PrP(C)) as a model protein, and thus an aptamer-mediated turn-on fluorescence assay for proteins was developed in this contribution. The designed aptamer-participated haprin structure consists of three segments. Namely, an aptamer sequence located in the loop, three guanine bases at 3'-terminal, and a fluophor modified at 5'-terminal. It was found that the guanine bases at the 3'-terminal could quench the fluorescence of the fluophor such as tetramethyl-6-carboxyrhodamine (TAMRA) at the 5'-terminal about 76.6% via electron transfer if the guanine bases are close enough to the fluophor, and the quenched fluorescence could get restored when the target protein is present since the interaction, which could be confirmed by measuring fluorescence lifetime, between TAMRA-aptamer and the target protein forces the guanines away from TAMRA so that TAMRA-modified aptamer changes into turn-on state. A linear relationship was then constructed between the turn-on fluorescence intensity and the concentration of PrP(C) in the range from 1.1 to 44.7 microg/mL with a limit of detection of 0.3 microg/mL (3sigma). PMID:19635360

  15. Assay of adenosine 3',5' cyclic monophosphate by stimulation of protein kinase: a method not involving radioactivity

    SciTech Connect

    Handa, A.K.; Bressan, R.A.

    1980-03-01

    In order to meet a need for a cAMP assay which is not subject to interference by compounds in plant extracts, and which is suitable for use on occasions separated by many /sup 32/P half-lives, an assay based on cAMP-dependent protein kinase has been developed which does not require the use of (..gamma..-/sup 32/P)ATP. Instead of measuring the cAMP-stimulated increase in the rate of transfer of (..gamma..-/sup 32/P) phosphate from (..gamma..-/sup 32/P)ATP to protein, the rate of loss of ATP from the reaction mixture is determined. The ATP remaining after the protein kinase reaction is assayed by ATP-dependent chemiluminescence of the firefly luciferin-luciferase system. Under conditions of the protein kinase reaction in which a readily measurable decrease in ATP concentration occurs, the logarithm of the concentration of ATP decreases in proportion to the cAMP concentration, i.e., the reaction can be described by the equation: (ATP) = (ATP)/sub 0/ e/sup -(cAMP)kt/. The assay based on this relationship can detect less than 1 pmol of cAMP. The levels of cAMP found with this assay after partial purification of the cAMP from rat tissue, algal cells, and the media in which the cells were grown agreed with measurements made by the cAMP binding-competition assay of Gilman, and the potein kinase stimulation assay based on transfer of (/sup 32/P) phosphate from (..gamma..-/sup 32/P)ATP to protein. All of the enzymes and chemicals required for the assay of cAMP by protein kinase catalyzed loss of ATP can be stored frozen for months, making the assay suitable for occasional use.

  16. Ultrasensitive Detection of Low-Abundance Protein Biomarkers by Mass Spectrometry Signal Amplification Assay.

    PubMed

    Du, Ruijun; Zhu, Lina; Gan, Jinrui; Wang, Yuning; Qiao, Liang; Liu, Baohong

    2016-07-01

    A mass spectrometry signal amplification method is developed for the ultrasensitive and selective detection of low-abundance protein biomarkers by utilizing tag molecules on gold nanoparticles (AuNPs). EpCAM and thrombin as model targets are captured by specific aptamers immobilized on the AuNPs. With laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS), the mass tag molecules are detected to represent the protein biomarkers. Benefiting from the MS signal amplification, the assay can achieve a limit of detection of 100 aM. The method is further applied to detect thrombin in fetal bovine serum and EpCAM in cell lysates to demonstrate its selectivity and feasibility in complex biological samples. With the high sensitivity and specificity, the protocol shows great promise for providing a new route to single-cell analysis and early disease diagnosis. PMID:27253396

  17. Non-protein amino acids and neurodegeneration: the enemy within.

    PubMed

    Rodgers, Kenneth J

    2014-03-01

    Animals, in common with plants and microorganisms, synthesise proteins from a pool of 20 protein amino acids (plus selenocysteine and pyrolysine) (Hendrickson et al., 2004). This represents a small proportion (~2%) of the total number of amino acids known to exist in nature (Bell, 2003). Many 'non-protein' amino acids are synthesised by plants, and in some cases constitute part of their chemical armoury against pathogens, predators or other species competing for the same resources (Fowden et al., 1967). Microorganisms can also use selectively toxic amino acids to gain advantage over competing organisms (Nunn et al., 2010). Since non-protein amino acids (and imino acids) are present in legumes, fruits, seeds and nuts, they are ubiquitous in the diets of human populations around the world. Toxicity to humans is unlikely to have been the selective force for their evolution, but they have the clear potential to adversely affect human health. In this review we explore the links between exposure to non-protein amino acids and neurodegenerative disorders in humans. Environmental factors play a major role in these complex disorders which are predominantly sporadic (Coppede et al., 2006). The discovery of new genes associated with neurodegenerative diseases, many of which code for aggregation-prone proteins, continues at a spectacular pace but little progress is being made in identifying the environmental factors that impact on these disorders. We make the case that insidious entry of non-protein amino acids into the human food chain and their incorporation into protein might be contributing significantly to neurodegenerative damage. PMID:24374297

  18. Heat capacities of amino acids, peptides and proteins.

    PubMed

    Makhatadze, G I

    1998-04-20

    The heat capacity is one of the fundamental parameters describing thermodynamic properties of a system. It has wide applications in a number of areas such as polymer chemistry, protein folding and DNA stability. To aid the scientific community in the analysis of such data, I have compiled a database on the experimentally measured heat capacities of amino acids, polyamino acids, peptides, and proteins in solid state and in aqueous solutions. PMID:9648205

  19. A rapid fluorogenic palmitoyl-protein thioesterase assay: pre- and postnatal diagnosis of INCL.

    PubMed

    van Diggelen, O P; Keulemans, J L; Winchester, B; Hofman, I L; Vanhanen, S L; Santavuori, P; Voznyi, Y V

    1999-04-01

    A deficiency of palmitoyl-protein thioesterase (PPT) was recently shown to be the primary defect in infantile neuronal ceroid lipofuscinosis (INCL). The available enzyme assays are complicated and impractical for diagnostic use. We have recently developed a new, fluorometric assay for PPT based on the sensitive fluorochrome 4-methylumbelliferone, requiring an overnight incubation to measure PPT. Now we have synthesized an analogue of this substrate which allows PPT determinations in 1 h. This improved PPT assay is simple, sensitive, and robust and will facilitate the definition of the full clinical spectrum associated with a deficiency of PPT. PPT activity was readily detectable in fibroblasts, leukocytes, amniotic fluid cells, chorionic villi, plasma, and cerebrospinal fluid from controls. PPT activity was profoundly deficient in these tissues and fluids from INCL patients. Similarly, a deficiency of PPT activity was demonstrated in patients with the variant juvenile NCL with GROD. These results show the feasibility of rapid pre- and postnatal diagnosis of INCL and its variants. PMID:10191108

  20. An Affinity-Based Fluorescence Polarization Assay for Protein Tyrosine Phosphatases

    PubMed Central

    Zhang, Sheng; Chen, Lan; Kumar, Sanjai; Wu, Li; Lawrence, David S.; Zhang, Zhong-Yin

    2007-01-01

    Protein tyrosine phosphatases (PTPs) are important signaling enzymes that control such fundamental processes as proliferation, differentiation, survival/apoptosis, as well as adhesion and motility. Potent and selective PTP inhibitors serve not only as powerful research tools, but also as potential therapeutics against a variety illness including cancer and diabetes. PTP activity-based assays are widely used in high throughput screening (HTS) campaigns for PTP inhibitor discovery. These assays suffer from a major weakness, in that the reactivity of the active site Cys can cause serious problems as highly reactive oxidizing and alkylating agents may surface as hits. We describe the development of a fluorescence polarization (FP)-based displacement assay that makes the use of an active site Cys to Ser mutant PTP (e.g., PTP1B/C215S) that retains the wild type binding affinity. The potency of library compounds is assessed by their ability to compete with the fluorescently labeled active site ligand for binding to the Cys to Ser PTP mutant. Finally, the substitution of the active site Cys by a Ser renders the mutant PTP insensitive to oxidation and alkylation and thus will likely eliminate “false” positives due to modification of the active site Cys that destroy the phosphatase activity. PMID:17532513

  1. Multipurpose high-throughput filtering microarrays (HiFi) for DNA and protein assays.

    PubMed

    Le Goff, Gaelle C; Desmet, Cloé; Brès, Jean-Charles; Rigal, Dominique; Blum, Loïc J; Marquette, Christophe A

    2010-12-15

    We are reporting here a low cost colorimetric device for high-throughput multiplexed blood group genotyping and allergy diagnosis, displayed as an automated 96-well microtiter plate format. A porous polymeric membrane sealed at the bottom of each well accounts for the sensor support. For each sensing unit, a 6×6 matrix of specific probes is spotted on the external surface of the membrane resulting in 5 mm(2) microarrays. Thanks to the membrane porosity, reagents dispensed into the well can be eliminated through vacuum soaking. This unusual design drastically reduces the assay background signal. The system was first validated on robust models composed of either two complementary oligonucleotide sequences or one allergen/specific rabbit IgG pair. The quality of both oligonucleotide and protein immobilisation on the membrane substrate was then demonstrated together with the capacity to use the arrayed biomolecules as probes for the quantitative detection of specific targets (respectively complementary oligonucleotide and specific antibody). On the basis of these good results, two multiplex assays were developed for crude biological samples testing, focussing on two human in vitro diagnosis applications: a hybridisation assay for multiplex blood group genotyping and a multiparametric immunoassay for allergy diagnosis. In both cases, the transfer to crude biological samples testing was successful i.e. high signal to noise ratio of the stained membranes, reproducibility and good correlation with results obtained using routine testing procedures. PMID:20663657

  2. Assay of Protein and Peptide Adducts of Cholesterol Ozonolysis Products by Hydrophobic and Click Enrichment Methods

    PubMed Central

    2015-01-01

    Cholesterol undergoes ozonolysis to afford a variety of oxysterol products, including cholesterol-5,6-epoxide (CholEp) and the isomeric aldehydes secosterol A (seco A) and secosterol B (seco B). These oxysterols display numerous important biological activities, including protein adduction; however, much remains to be learned about the identity of the reactive species and the range of proteins modified by these oxysterols. Here, we synthesized alkynyl derivatives of cholesterol-derived oxysterols and employed a straightforward detection method to establish secosterols A and B as the most protein-reactive of the oxysterols tested. Model adduction studies with an amino acid, peptides, and proteins provide evidence for the potential role of secosterol dehydration products in protein adduction. Hydrophobic separation methods—Folch extraction and solid phase extraction (SPE)—were successfully applied to enrich oxysterol-adducted peptide species, and LC-MS/MS analysis of a model peptide–seco adduct revealed a unique fragmentation pattern (neutral loss of 390 Da) for that species. Coupling a hydrophobic enrichment method with proteomic analysis utilizing characteristic fragmentation patterns facilitates the identification of secosterol-modified peptides and proteins in an adducted protein. More broadly, these improved enrichment methods may give insight into the role of oxysterols and ozone exposure in the pathogenesis of a variety of diseases, including atherosclerosis, Alzheimer’s disease, Parkinson’s disease, and asthma. PMID:25185119

  3. Direct Assay of δ-Aminolevulinic Acid Dehydratase in Heme Biosynthesis for the Detection of Porphyrias by Tandem Mass Spectrometry

    PubMed Central

    Choiniere, John R.; Scott, C. Ronald; Gelb, Michael H.; Tureček, František

    2010-01-01

    We report a new assay of human δ-aminolevulinic acid dehydratase (ALAD), an enzyme converting δ-aminolevulinic acid (ALA) into porphobilinogen. The assay is developed for use in the clinical diagnosis of δ-aminolevulinic acid dehydratase-deficient porphyria, a rare enzymatic deficiency of the heme biosynthetic pathway. The assay involves the incubation of erythrocyte lysate with the natural substrate, ALA, followed by quantitative in situ conversion of porphobilinogen to its butyramide, and liquid-liquid extraction into a mass spectrometer-friendly solvent. Quantitation of the butyrylated porphobilinogen is done by electrospray ionization tandem mass spectrometry, using a deuterium labeled internal standard. The assay stays well within the range wherein ALAD activity is linear with time. The Km of ALAD for ALA was measured as 333 μM, and the Vmax was 19.3 μM/hr. Average enzyme activity among a random sample of 36 anonymous individuals was 277 μmol/L erythrocyte lysate/hour with a standard deviation of 90 μmol/L erythrocyte lysate/hour. The tandem mass spectrometric assay should easily detect the enzyme deficiency, which causes a reduction of activity by 95–99%. The assay shows good reproducibility, low background, requires a simple workup, and uses a commercially available substrate. PMID:20583792

  4. Phthalic acid chemical probes synthesized for protein-protein interaction analysis.

    PubMed

    Liang, Shih-Shin; Liao, Wei-Ting; Kuo, Chao-Jen; Chou, Chi-Hsien; Wu, Chin-Jen; Wang, Hui-Min

    2013-01-01

    Plasticizers are additives that are used to increase the flexibility of plastic during manufacturing. However, in injection molding processes, plasticizers cannot be generated with monomers because they can peel off from the plastics into the surrounding environment, water, or food, or become attached to skin. Among the various plasticizers that are used, 1,2-benzenedicarboxylic acid (phthalic acid) is a typical precursor to generate phthalates. In addition, phthalic acid is a metabolite of diethylhexyl phthalate (DEHP). According to Gene_Ontology gene/protein database, phthalates can cause genital diseases, cardiotoxicity, hepatotoxicity, nephrotoxicity, etc. In this study, a silanized linker (3-aminopropyl triethoxyslane, APTES) was deposited on silicon dioxides (SiO2) particles and phthalate chemical probes were manufactured from phthalic acid and APTES-SiO2. These probes could be used for detecting proteins that targeted phthalic acid and for protein-protein interactions. The phthalic acid chemical probes we produced were incubated with epithelioid cell lysates of normal rat kidney (NRK-52E cells) to detect the interactions between phthalic acid and NRK-52E extracted proteins. These chemical probes interacted with a number of chaperones such as protein disulfide-isomerase A6, heat shock proteins, and Serpin H1. Ingenuity Pathways Analysis (IPA) software showed that these chemical probes were a practical technique for protein-protein interaction analysis. PMID:23797655

  5. Library screening by means of mass spectrometry (MS) binding assays-exemplarily demonstrated for a pseudostatic library addressing γ-aminobutyric acid (GABA) transporter 1 (GAT1).

    PubMed

    Sindelar, Miriam; Wanner, Klaus T

    2012-09-01

    In the present study, the application of mass spectrometry (MS) binding assays as a tool for library screening is reported. For library generation, dynamic combinatorial chemistry (DCC) was used. These libraries can be screened by means of MS binding assays when appropriate measures are taken to render the libraries pseudostatic. That way, the efficiency of MS binding assays to determine ligand binding in compound screening with the ease of library generation by DCC is combined. The feasibility of this approach is shown for γ-aminobutyric acid (GABA) transporter 1 (GAT1) as a target, representing the most important subtype of the GABA transporters. For the screening, hydrazone libraries were employed that were generated in the presence of the target by reacting various sets of aldehydes with a hydrazine derivative that is delineated from piperidine-3-carboxylic acid (nipecotic acid), a common fragment of known GAT1 inhibitors. To ensure that the library generated is pseudostatic, a large excess of the nipecotic acid derivative is employed. As the library is generated in a buffer system suitable for binding and the target is already present, the mixtures can be directly analyzed by MS binding assays-the process of library generation and screening thus becoming simple to perform. The binding affinities of the hits identified by deconvolution were confirmed in conventional competitive MS binding assays performed with single compounds obtained by separate synthesis. In this way, two nipecotic acid derivatives exhibiting a biaryl moiety, 1-{2-[2'-(1,1'-biphenyl-2-ylmethylidene)hydrazine]ethyl}piperidine-3-carboxylic acid and 1-(2-{2'-[1-(2-thiophenylphenyl)methylidene]hydrazine}ethyl)piperidine-3-carboxylic acid, were found to be potent GAT1 ligands exhibiting pK(i) values of 6.186 ± 0.028 and 6.229 ± 0.039, respectively. This method enables screening of libraries, whether generated by conventional chemistry or DCC, and is applicable to all kinds of targets including

  6. [Development of direct competitive enzyme-linked immunosorbent assay for the determination of domoic acid].

    PubMed

    Wang, Qian; Cheng, Jin-Ping; Gao, Li-Li; Dong, Yu; Xi, Lei

    2012-02-01

    To develop a direct competitive enzyme-linked immunosorbent assay (ELISA) for rapid detection of domoic acid concentrations, HRP (horse radish peroxidase) was successfully linked to DA using EDC. The concentration of DA was quantitatively analyzed on the basic of the specific immune responses between the DA- HRP and the monoclonal antibodies made in advance. Calibration curve were established after the optimization of reaction conditions such as the type of blocking solution, the blocking time and the incubation temperature. The results show that, the best reaction condition of the direct competitive ELISA is 1% gelatin, blocking 1 h at 37 degrees C, incubating 1 h at 37 degrees C after the monoclonal antibodies added. The detect limit is 3.58 ng x mL(-1), the coefficient of variation between the holes is below 15%, and the recovery is 80% - 120%. The whole analysis process could be completed within 1.5 h. It meets the requirements of rapid and batch detection of domoic acid. The method will have broad development prospects. PMID:22509610

  7. HALOACETIC ACIDS PERTURB PROTEIN PHOSPHORYLATION IN MOUSE EMBRYOS IN VITRO

    EPA Science Inventory

    HALOACETIC ACIDS PERTURB PROTEIN PHOSPHORYLATION IN MOUSE EMBRYOS IN VITRO. MR Blanton and ES Hunter. Reproductive Toxicology Division, NHEERL, ORD, US EPA, RTP, NC, USA.
    Sponsor: JM Rogers.
    Haloacetic Acids (HAAs) formed during the disinfection process are present in drin...

  8. Conformational Entropy of Intrinsically Disordered Proteins from Amino Acid Triads

    PubMed Central

    Baruah, Anupaul; Rani, Pooja; Biswas, Parbati

    2015-01-01

    This work quantitatively characterizes intrinsic disorder in proteins in terms of sequence composition and backbone conformational entropy. Analysis of the normalized relative composition of the amino acid triads highlights a distinct boundary between globular and disordered proteins. The conformational entropy is calculated from the dihedral angles of the middle amino acid in the amino acid triad for the conformational ensemble of the globular, partially and completely disordered proteins relative to the non-redundant database. Both Monte Carlo (MC) and Molecular Dynamics (MD) simulations are used to characterize the conformational ensemble of the representative proteins of each group. The results show that the globular proteins span approximately half of the allowed conformational states in the Ramachandran space, while the amino acid triads in disordered proteins sample the entire range of the allowed dihedral angle space following Flory’s isolated-pair hypothesis. Therefore, only the sequence information in terms of the relative amino acid triad composition may be sufficient to predict protein disorder and the backbone conformational entropy, even in the absence of well-defined structure. The predicted entropies are found to agree with those calculated using mutual information expansion and the histogram method. PMID:26138206

  9. Rapid and quantitative detection of C-reactive protein based on quantum dots and immunofiltration assay

    PubMed Central

    Zhang, Pengfei; Bao, Yan; Draz, Mohamed Shehata; Lu, Huiqi; Liu, Chang; Han, Huanxing

    2015-01-01

    Convenient and rapid immunofiltration assays (IFAs) enable on-site “yes” or “no” determination of disease markers. However, traditional IFAs are commonly qualitative or semi-quantitative and are very limited for the efficient testing of samples in field diagnostics. Here, we overcome these limitations by developing a quantum dots (QDs)-based fluorescent IFA for the quantitative detection of C-reactive proteins (CRP). CRP, the well-known diagnostic marker for acute viral and bacterial infections, was used as a model analyte to demonstrate performance and sensitivity of our developed QDs-based IFA. QDs capped with both polyethylene glycol (PEG) and glutathione were used as fluorescent labels for our IFAs. The presence of the surface PEG layer, which reduced the non-specific protein interactions, in conjunction with the inherent optical properties of QDs, resulted in lower background signal, increased sensitivity, and ability to detect CRP down to 0.79 mg/L with only 5 µL serum sample. In addition, the developed assay is simple, fast and can quantitatively detect CRP with a detection limit up to 200 mg/L. Clinical test results of our QD-based IFA are well correlated with the traditional latex enhance immune-agglutination aggregation. The proposed QD-based fluorescent IFA is very promising, and potentially will be adopted for multiplexed immunoassay and in field point-of-care test. PMID:26491289

  10. A homogeneous fluorescence polarization assay adaptable for a range of protein serine/threonine and tyrosine kinases.

    PubMed

    Gaudet, Elizabeth A; Huang, Kuo-Sen; Zhang, Yan; Huang, Wei; Mark, David; Sportsman, J Richard

    2003-04-01

    Recently, a new technology for high-throughput screening has been developed, called IMAP(patent pending). IMAP technology has previously been implemented in an assay for cyclic nucleotide phosphodiesterases (PDE). The authors describe the development of a homogeneous, non-antibody-based fluorescence polarization (FP) assay for a variety of protein kinases. In this assay, fluorescently labeled peptide substrate phosphorylated by the kinase is captured on modified nanoparticles through interactions with immobilized metal (M(III)) coordination complexes, resulting in a change from low to high polarization values. This assay is applicable to protein kinases that phosphorylate serine, threonine, or tyrosine residues. The IMAP platform is very compatible with high-throughput robotics and can be applied to the 1536-well format. As there are hundreds of different kinases coded for in the human genome, the assay platform described in this report is a valuable new tool in drug discovery. PMID:12844437

  11. Free 25-Hydroxyvitamin D: Impact of Vitamin D Binding Protein Assays on Racial-Genotypic Associations

    PubMed Central

    Nielson, Carrie M.; Jones, Kerry S.; Chun, Rene F.; Jacobs, Jon M.; Wang, Ying; Hewison, Martin; Adams, John S.; Swanson, Christine M.; Lee, Christine G.; Vanderschueren, Dirk; Pauwels, Steven; Prentice, Ann; Smith, Richard D.; Shi, Tujin; Gao, Yuqian; Schepmoes, Athena A.; Zmuda, Joseph M.; Lapidus, Jodi; Cauley, Jane A.; Schoenmakers, Inez; Orwoll, Eric S.

    2016-01-01

    Context: Total 25-hydroxyvitamin D (25OHD) is a marker of vitamin D status and is lower in African Americans than in whites. Whether this difference holds for free 25OHOD (f25OHD) is unclear, considering reported genetic-racial differences in vitamin D binding protein (DBP) used to calculate f25OHD. Objectives: Our objective was to assess racial-geographic differences in f25OHD and to understand inconsistencies in racial associations with DBP and calculated f25OHD. Design: This study used a cross-sectional design. Setting: The general community in the United States, United Kingdom, and The Gambia were included in this study. Participants: Men in Osteoporotic Fractures in Men and Medical Research Council studies (N = 1057) were included. Exposures: Total 25OHD concentration, race, and DBP (GC) genotype exposures were included. Outcome Measures: Directly measured f25OHD, DBP assessed by proteomics, monoclonal and polyclonal immunoassays, and calculated f25OHD were the outcome measures. Results: Total 25OHD correlated strongly with directly measured f25OHD (Spearman r = 0.84). Measured by monoclonal assay, mean DBP in African-ancestry subjects was approximately 50% lower than in whites, whereas DBP measured by polyclonal DBP antibodies or proteomic methods was not lower in African-ancestry. Calculated f25OHD (using polyclonal DBP assays) correlated strongly with directly measured f25OHD (r = 0.80–0.83). Free 25OHD, measured or calculated from polyclonal DBP assays, reflected total 25OHD concentration irrespective of race and was lower in African Americans than in US whites. Conclusions: Previously reported racial differences in DBP concentration are likely from monoclonal assay bias, as there was no racial difference in DBP concentration by other methods. This confirms the poor vitamin D status of many African-Americans and the utility of total 25OHD in assessing vitamin D in the general population. PMID:27007693

  12. Biological Variations of Lupus Anticoagulant, Antithrombin, Protein C, Protein S, and von Willebrand Factor Assays.

    PubMed

    Shou, Weiling; Chen, Qian; Wu, Wei; Cui, Wei

    2016-02-01

    The results of lupus anticoagulant (LA), antithrombin (AT), protein C (PC), and protein S (PS) testing, and the values of von Willebrand factor antigen (VWF:Ag) are important in diagnosis and therapeutic monitoring of thrombosis and hemostasis diseases. Till now, no published study has focused on the biological variations in LA testing, and only a few studies have examined the biological variations of AT, PC, PS, and VWF:Ag. With the latest fully automated instruments and improved reagents, the analytical, within-subject, and between-subject biological variations were estimated for these five coagulant parameters in a cohort of 25 apparently healthy subjects. Blood specimens were collected at 8:00 am, 12:00 pm, and 4:00 pm on days 1, 3, and 5. The analytical biological variation (CV(A)) values of all the parameters were less than 3%. The within-subject biological variation (CV(W)) and between-subject biological variation (CV(G)) values of the LA normalized ratio were 4.64 and 6.83%, respectively. No significant differences were observed in the intraday and interday biological variations of LA tests, or in AT, PC, PS, and VWF:Ag values. Additionally, the utility of the conventional population-based reference intervals of the five coagulation parameters was evaluated by the index of individuality, and data on CV(W) and CV(A) were used to calculate the reference change value to identify the significance of changes in serial results from the same individual. PMID:26516946

  13. An in vivo imaging-based assay for detecting protein interactions over a wide range of binding affinities

    SciTech Connect

    Fowlkes, Jason Davidson; Owens, Elizabeth T; Standaert, Robert F; Pelletier, Dale A; Hurst, Gregory {Greg} B; Doktycz, Mitchel John; Morrell-Falvey, Jennifer L; Billings, Amanda N

    2009-01-01

    Identifying and characterizing protein interactions are fundamental steps towards understanding and modeling biological networks. Methods that detect protein interactions in intact cells rather than buffered solutions are likely more relevant to natural systems since molecular crowding events in the cytosol can influence the diffusion and reactivity of individual proteins. One in vivo, imaging-based method relies on the co-localization of two proteins of interest fused to DivIVA, a cell division protein from Bacillus subtilis, and green fluorescent protein (GFP). We have modified this imaging-based assay to facilitate rapid cloning by constructing new vectors encoding N- and C-terminal DivIVA or GFP molecular tag fusions based on site-specific recombination technology. The sensitivity of the assay was defined using a well-characterized protein interaction system involving the eukaryotic nuclear import receptor subunit, Importin (Imp ) and variant nuclear localization signals (NLS) representing a range of binding affinities. These data demonstrate that the modified co-localization assay is sensitive enough to detect protein interactions with Kd values that span over four orders of magnitude (1nM to 15 M). Lastly, this assay was used to confirm numerous protein interactions identified from mass spectrometry-based analyses of affinity isolates as part of an interactome mapping project in Rhodopseudomonas palustris

  14. Medical devices; immunology and microbiology devices; classification of quality control material for cystic fibrosis nucleic acid assays. Final rule.

    PubMed

    2007-01-10

    The Food and Drug Administration (FDA) is classifying quality control material for cystic fibrosis nucleic acid assays into class II (special controls). The special control that will apply to the device is the guidance document entitled "Class II Special Controls Guidance Document: Quality Control Material for Cystic Fibrosis Nucleic Acid Assays." The agency is classifying the device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of the device. Elsewhere in this issue of the Federal Register, FDA is announcing the availability of the guidance document that will serve as the special control for this device. PMID:17294552

  15. A Soluble, Folded Protein without Charged Amino Acid Residues.

    PubMed

    Højgaard, Casper; Kofoed, Christian; Espersen, Roall; Johansson, Kristoffer Enøe; Villa, Mara; Willemoës, Martin; Lindorff-Larsen, Kresten; Teilum, Kaare; Winther, Jakob R

    2016-07-19

    Charges are considered an integral part of protein structure and function, enhancing solubility and providing specificity in molecular interactions. We wished to investigate whether charged amino acids are indeed required for protein biogenesis and whether a protein completely free of titratable side chains can maintain solubility, stability, and function. As a model, we used a cellulose-binding domain from Cellulomonas fimi, which, among proteins of more than 100 amino acids, presently is the least charged in the Protein Data Bank, with a total of only four titratable residues. We find that the protein shows a surprising resilience toward extremes of pH, demonstrating stability and function (cellulose binding) in the pH range from 2 to 11. To ask whether the four charged residues present were required for these properties of this protein, we altered them to nontitratable ones. Remarkably, this chargeless protein is produced reasonably well in Escherichia coli, retains its stable three-dimensional structure, and is still capable of strong cellulose binding. To further deprive this protein of charges, we removed the N-terminal charge by acetylation and studied the protein at pH 2, where the C-terminus is effectively protonated. Under these conditions, the protein retains its function and proved to be both soluble and have a reversible folding-unfolding transition. To the best of our knowledge, this is the first time a soluble, functional protein with no titratable side chains has been produced. PMID:27307139

  16. Optimization of pH values to formulate the bireagent kit for serum uric acid assay.

    PubMed

    Huang, Ya; Chen, Yuanxiang; Yang, Xiaolan; Zhao, Hua; Hu, Xiaolei; Pu, Jun; Liao, Juan; Long, Gaobo; Liao, Fei

    2015-01-01

    A new formulation of the bireagent kit for serum uric acid assay was developed based on the effects of pH on enzyme stability. At 4 °C, half-lives of uricases from Bacillus fastidious and Arthrobacter globiforms were longer than 15 months at pH 9.2, but became shorter at pH below 8.0; half-lives of ascorbate oxidase and peroxidase were comparable at pH 6.5 and 7.0, but became much shorter at pH higher than 7.4. In the new formulation of the bireagent kit, Reagent A contained peroxidase, 4-aminoantipyrine, and ascorbate oxidase in 50 mM phosphate buffer at pH 6.5; Reagent B contained B. fastidious or A. globiforms uricase in 50 mM sodium borate buffer at pH 9.2; Reagents A and B were mixed at 4:1 to produce a final pH from 7.2 to 7.6 for developing a stable color. The new bireagent kit consumed smaller quantities of three enzymes for the same shelf life. With the new bireagent kit, there were linear responses of absorbance at 546 nm to uric acid up to 34 mM in reaction mixtures and a good correlation of uric acid levels in clinical sera with those by a commercial kit, but stronger resistance to ascorbate. Therefore, the new formulation was advantageous. PMID:24673428

  17. Predicting protein concentrations with ELISA microarray assays, monotonic splines and Monte Carlo simulation

    SciTech Connect

    Daly, Don S.; Anderson, Kevin K.; White, Amanda M.; Gonzalez, Rachel M.; Varnum, Susan M.; Zangar, Richard C.

    2008-07-14

    Background: A microarray of enzyme-linked immunosorbent assays, or ELISA microarray, predicts simultaneously the concentrations of numerous proteins in a small sample. These predictions, however, are uncertain due to processing error and biological variability. Making sound biological inferences as well as improving the ELISA microarray process require require both concentration predictions and creditable estimates of their errors. Methods: We present a statistical method based on monotonic spline statistical models, penalized constrained least squares fitting (PCLS) and Monte Carlo simulation (MC) to predict concentrations and estimate prediction errors in ELISA microarray. PCLS restrains the flexible spline to a fit of assay intensity that is a monotone function of protein concentration. With MC, both modeling and measurement errors are combined to estimate prediction error. The spline/PCLS/MC method is compared to a common method using simulated and real ELISA microarray data sets. Results: In contrast to the rigid logistic model, the flexible spline model gave credible fits in almost all test cases including troublesome cases with left and/or right censoring, or other asymmetries. For the real data sets, 61% of the spline predictions were more accurate than their comparable logistic predictions; especially the spline predictions at the extremes of the prediction curve. The relative errors of 50% of comparable spline and logistic predictions differed by less than 20%. Monte Carlo simulation rendered acceptable asymmetric prediction intervals for both spline and logistic models while propagation of error produced symmetric intervals that diverged unrealistically as the standard curves approached horizontal asymptotes. Conclusions: The spline/PCLS/MC method is a flexible, robust alternative to a logistic/NLS/propagation-of-error method to reliably predict protein concentrations and estimate their errors. The spline method simplifies model selection and fitting

  18. Using constitutive activity to define appropriate high-throughput screening assays for orphan g protein-coupled receptors.

    PubMed

    Ngo, Tony; Coleman, James L J; Smith, Nicola J

    2015-01-01

    Orphan G protein-coupled receptors represent an underexploited resource for drug discovery but pose a considerable challenge for assay development because their cognate G protein signaling pathways are often unknown. In this methodological chapter, we describe the use of constitutive activity, that is, the inherent ability of receptors to couple to their cognate G proteins in the absence of ligand, to inform the development of high-throughput screening assays for a particular orphan receptor. We specifically focus on a two-step process, whereby constitutive G protein coupling is first determined using yeast Gpa1/human G protein chimeras linked to growth and β-galactosidase generation. Coupling selectivity is then confirmed in mammalian cells expressing endogenous G proteins and driving accumulation of transcription factor-fused luciferase reporters specific to each of the classes of G protein. Based on these findings, high-throughput screening campaigns can be performed on the already miniaturized mammalian reporter system. PMID:25563179

  19. Improved Proteomic Analysis Following Trichloroacetic Acid Extraction of Bacillus anthracis Spore Proteins

    SciTech Connect

    Kaiser, Brooke LD; Wunschel, David S.; Sydor, Michael A.; Warner, Marvin G.; Wahl, Karen L.; Hutchison, Janine R.

    2015-08-07

    Proteomic analysis of bacterial samples provides valuable information about cellular responses and functions under different environmental pressures. Proteomic analysis is dependent upon efficient extraction of proteins from bacterial samples without introducing bias toward extraction of particular protein classes. While no single method can recover 100% of the bacterial proteins, selected protocols can improve overall protein isolation, peptide recovery, or enrich for certain classes of proteins. The method presented here is technically simple and does not require specialized equipment such as a mechanical disrupter. Our data reveal that for particularly challenging samples, such as B. anthracis Sterne spores, trichloroacetic acid extraction improved the number of proteins identified within a sample compared to bead beating (714 vs 660, respectively). Further, TCA extraction enriched for 103 known spore specific proteins whereas bead beating resulted in 49 unique proteins. Analysis of C. botulinum samples grown to 5 days, composed of vegetative biomass and spores, showed a similar trend with improved protein yields and identification using our method compared to bead beating. Interestingly, easily lysed samples, such as B. anthracis vegetative cells, were equally as effectively processed via TCA and bead beating, but TCA extraction remains the easiest and most cost effective option. As with all assays, supplemental methods such as implementation of an alternative preparation method may provide additional insight to the protein biology of the bacteria being studied.

  20. Isotope-dilution assay for urinary methylmalonic acid in the diagnosis of vitamin B12 deficiency. A prospective clinical evaluation

    SciTech Connect

    Matchar, D.B.; Feussner, J.R.; Millington, D.S.; Wilkinson, R.H. Jr.; Watson, D.J.; Gale, D.

    1987-05-01

    Vitamin B12 deficiency is a frequently considered diagnosis for which there is no single, commonly available and accurate test. A urinary methylmalonic acid assay using gas chromatography-mass spectrometry has been proposed as the preferred test. We reviewed vitamin B12 assays on 1599 consecutive patients and prospectively studied all patients with low serum B12 levels (n = 75) and a random sample of patients with normal levels (n = 68). Of 96 evaluable patients, 7 had clinical deficiency. All 7 deficient patients had urinary methylmalonic acid levels greater than 5 micrograms/mg creatine (sensitivity, 100%; confidence interval, 65% to 100%). Of the 89 patients who were not clinically deficient, 88 had urinary methylmalonic acid levels less than or equal to 5 micrograms/mg creatinine (specificity, 99%). The overall test accuracy in this population was 99%. If the high sensitivity and specificity of the gas chromatography-mass spectrometry assay for urinary methylmalonic acid is supported by other clinical studies, the methylmalonic acid assay may become the reference standard for the diagnosis of vitamin B12 deficiency.

  1. Manipulating Fatty Acid Biosynthesis in Microalgae for Biofuel through Protein-Protein Interactions

    PubMed Central

    Blatti, Jillian L.; Beld, Joris; Behnke, Craig A.; Mendez, Michael; Mayfield, Stephen P.; Burkart, Michael D.

    2012-01-01

    Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes. PMID:23028438

  2. Predicting protein disorder by analyzing amino acid sequence

    PubMed Central

    Yang, Jack Y; Yang, Mary Qu

    2008-01-01

    Background Many protein regions and some entire proteins have no definite tertiary structure, presenting instead as dynamic, disorder ensembles under different physiochemical circumstances. These proteins and regions are known as Intrinsically Unstructured Proteins (IUP). IUP have been associated with a wide range of protein functions, along with roles in diseases characterized by protein misfolding and aggregation. Results Identifying IUP is important task in structural and functional genomics. We exact useful features from sequences and develop machine learning algorithms for the above task. We compare our IUP predictor with PONDRs (mainly neural-network-based predictors), disEMBL (also based on neural networks) and Globplot (based on disorder propensity). Conclusion We find that augmenting features derived from physiochemical properties of amino acids (such as hydrophobicity, complexity etc.) and using ensemble method proved beneficial. The IUP predictor is a viable alternative software tool for identifying IUP protein regions and proteins. PMID:18831799

  3. IR-UV photochemistry of protein-nucleic acid systems

    SciTech Connect

    Kozub, J.; Edwards, G.

    1995-12-31

    UV light has often been used to induce the formation of covalent bonds between DNA (or RNA) and tightly-bound protein molecules. However, the internal photoreactions of nucleic acids and proteins limit the yield and complicate the analysis of intermolecular crosslinks. In an ongoing search for improved reaction specificity or new photoreactions in these systems, we have employed UV photons from a Nd:YAG-pumped dye laser and mid-IR photons from the Vanderbilt FEL. Having crosslinked several protein-nucleic acid systems with nanosecond UV laser pulses, we are currently studying the effect of various IR wavelengths on a model system (gene 32 protein and poly[dT]). We have found that irradiation with sufficiently intense FEL macropulses creates an altered form of gene 32 protein which was not observed with UV-only irradiation. The electrophoretic nobility of the product is consistent with the formation of a specific protein-protein crosslink. No evidence of the non-specific protein damage typically induced by UV light is found. The yield of the new photoproduct is apparently enhanced by exposure to FEL macropulses which are synchronized with UV laser pulses. With ideal exposure parameters, the two-color reaction effectively competes with UV-only reactions. Experiments designed to determine the reaction mechanism and to demonstrate FEL-induced reactions in other protein-nucleic acid systems are currently underway.

  4. Modifications of proteins by polyunsaturated fatty acid peroxidation products

    NASA Astrophysics Data System (ADS)

    Refsgaard, Hanne H. F.; Tsai, Lin; Stadtman, Earl R.

    2000-01-01

    The ability of unsaturated fatty acid methyl esters to modify amino acid residues in bovine serum albumin (BSA), glutamine synthetase, and insulin in the presence of a metal-catalyzed oxidation system [ascorbate/Fe(III)/O2] depends on the degree of unsaturation of the fatty acid. The fatty acid-dependent generation of carbonyl groups and loss of lysine residues increased in the order methyl linoleate < methyl linolenate < methyl arachidonate. The amounts of alkyl hydroperoxides, malondialdehyde, and a number of other aldehydes that accumulated when polyunsaturated fatty acids were oxidized in the presence of BSA were significantly lower than that observed in the absence of BSA. Direct treatment of proteins with various lipid hydroperoxides led to a slight increase in the formation of protein carbonyl derivatives, whereas treatment with the hydroperoxides together with Fe(II) led to a substantial increase in the formation of protein carbonyls. These results are consistent with the proposition that metal-catalyzed oxidation of polyunsaturated fatty acids can contribute to the generation of protein carbonyls by direct interaction of lipid oxidation products (α,β-unsaturated aldehydes) with lysine residues (Michael addition reactions) and also by interactions with alkoxyl radicals obtained by Fe(II) cleavage of lipid hydroperoxides that are formed. In addition, saturated aldehydes derived from the polyunsaturated fatty acids likely react with lysine residues to form Schiff base adducts.

  5. Modifications of proteins by polyunsaturated fatty acid peroxidation products

    PubMed Central

    Refsgaard, Hanne H. F.; Tsai, Lin; Stadtman, Earl R.

    2000-01-01

    The ability of unsaturated fatty acid methyl esters to modify amino acid residues in bovine serum albumin (BSA), glutamine synthetase, and insulin in the presence of a metal-catalyzed oxidation system [ascorbate/Fe(III)/O2] depends on the degree of unsaturation of the fatty acid. The fatty acid-dependent generation of carbonyl groups and loss of lysine residues increased in the order methyl linoleate < methyl linolenate < methyl arachidonate. The amounts of alkyl hydroperoxides, malondialdehyde, and a number of other aldehydes that accumulated when polyunsaturated fatty acids were oxidized in the presence of BSA were significantly lower than that observed in the absence of BSA. Direct treatment of proteins with various lipid hydroperoxides led to a slight increase in the formation of protein carbonyl derivatives, whereas treatment with the hydroperoxides together with Fe(II) led to a substantial increase in the formation of protein carbonyls. These results are consistent with the proposition that metal-catalyzed oxidation of polyunsaturated fatty acids can contribute to the generation of protein carbonyls by direct interaction of lipid oxidation products (α,β-unsaturated aldehydes) with lysine residues (Michael addition reactions) and also by interactions with alkoxyl radicals obtained by Fe(II) cleavage of lipid hydroperoxides that are formed. In addition, saturated aldehydes derived from the polyunsaturated fatty acids likely react with lysine residues to form Schiff base adducts. PMID:10639127

  6. Use of submitochondrial particle (SMP) assays for assessing wetlands constructed for sequestering acid mine runoff

    SciTech Connect

    Bettermann, A.D.; Haahr, J.E.; Lazorchak, J.M.

    1995-12-31

    Use of constructed wetlands to sequester metals from acid mine drainage is part of a USEPA SITE demonstration at Burleigh Tunnel near Silverplume, Colorado. Samples are collected on a seasonal basis for toxicity evaluation of two different pilot treatment systems. Water samples were obtained from the outflow of two experimental wetland cells utilizing either upflow and downflow treatment, as well as upstream and downstream of the discharge of Burleigh Tunnel to Clear Creek. Submitochondrial Particle (SMP), Ceriodaphnia dubia and Pimephales promelas acute bioassays were used to evaluate the water quality. The SMP bioassay is based on the electron transfer complex derived from mitochondria. Toxic responses result from subcellular perturbations of various subsets of enzyme systems contained in the complex. In prior work, a 0.79 r{sup 2} was reported between the SMP bioassay and P. promelas for 11 inorganics on the EPA Priority Pollutant list. The SMP bioassay provided data consistent with the whole organism results. The two most toxic samples: the Burleigh outflow, and the Clear Creek Upstream sample, gave C. dubia LC50s of 1.01% and 8.41%, respectively. The Burleigh outflow P. promelas LC50 was 1.55%. SMP EC50s for the Burleigh outflow and the Clear Creek Upstream sample were 0.63% and 1.63%, respectively. As the SMP bioassay requires 1 hour to run and costs approximately 1/10th of whole organism assays, it was feasible to determine EC50 values for 7 samples vs. the two sample LC50s determined using whole organism assays. The SMP bioassays can provide sufficient sampling density, at low cost, allowing effective delineation of wetland performance over time.

  7. Affinity regression predicts the recognition code of nucleic acid binding proteins

    PubMed Central

    Pelossof, Raphael; Singh, Irtisha; Yang, Julie L.; Weirauch, Matthew T.; Hughes, Timothy R.; Leslie, Christina S.

    2016-01-01

    Predicting the affinity profiles of nucleic acid-binding proteins directly from the protein sequence is a major unsolved problem. We present a statistical approach for learning the recognition code of a family of transcription factors (TFs) or RNA-binding proteins (RBPs) from high-throughput binding assays. Our method, called affinity regression, trains on protein binding microarray (PBM) or RNA compete experiments to learn an interaction model between proteins and nucleic acids, using only protein domain and probe sequences as inputs. By training on mouse homeodomain PBM profiles, our model correctly identifies residues that confer DNA-binding specificity and accurately predicts binding motifs for an independent set of divergent homeodomains. Similarly, learning from RNA compete profiles for diverse RBPs, our model can predict the binding affinities of held-out proteins and identify key RNA-binding residues. More broadly, we envision applying our method to model and predict biological interactions in any setting where there is a high-throughput ‘affinity’ readout. PMID:26571099

  8. A label-free impedance-based whole cell assay revealed a new G protein-coupled receptor ligand for mouse microglial cell migration.

    PubMed

    Fukano, Yasufumi; Okino, Nozomu; Furuya, Shigeki; Ito, Makoto

    2016-09-16

    We report the usefulness of an impedance-based label-free whole cell assay to identify new ligands for G protein-coupled receptors (GPCRs) involved in microglial cell migration. Authentic GPCR ligands were subjected to the impedance-based cell assay in order to examine the responses of ligands for MG5 mouse microglial cells. Complement component 5 (C5a), adenosine 5'-diphosphate (ADP), uridine 5'-triphosphate (UTP), lysophosphatidic acid (LPA), and lysophosphatidylserine (LysoPS) were found to elicit different cellular impedance patterns, i.e. C5a, ADP, and UTP caused a transient increase in cellular impedance, while LPA and LysoPS decreased it. The responses for C5a and ADP were abolished by pertussis toxin (PTX), but not rho-associated protein kinase inhibitor, Y-27632, indicating that C5a and ADP elicited responses through the Gαi pathway. On the other hand, the response for UTP, LPA or LysoPS was not cancelled by PTX or Y-27632. In a modified Boyden chamber assay, C5a and ADP, but not UTP, LPA, or LysoPS, induced the migration of MG5 cells. These results suggest that PTX-sensitive increase in cellular impedance with the assay is characteristic for ligands of GPCRs involved in microglial cell migration. We found using this assay that 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) is a new chemoattractant inducing microglial cell migration through the activation of Gαi. PMID:27480930

  9. Magnetic levitation as a platform for competitive protein-ligand binding assays.

    PubMed

    Shapiro, Nathan D; Soh, Siowling; Mirica, Katherine A; Whitesides, George M

    2012-07-17

    This paper describes a method based on magnetic levitation (MagLev) that is capable of indirectly measuring the binding of unlabeled ligands to unlabeled protein. We demonstrate this method by measuring the affinity of unlabeled bovine carbonic anhydrase (BCA) for a variety of ligands (most of which are benzene sulfonamide derivatives). This method utilizes porous gel beads that are functionalized with a common aryl sulfonamide ligand. The beads are incubated with BCA and allowed to reach an equilibrium state in which the majority of the immobilized ligands are bound to BCA. Since the beads are less dense than the protein, protein binding to the bead increases the overall density of the bead. This change in density can be monitored using MagLev. Transferring the beads to a solution containing no protein creates a situation where net protein efflux from the bead is thermodynamically favorable. The rate at which protein leaves the bead for the solution can be calculated from the rate at which the levitation height of the bead changes. If another small molecule ligand of BCA is dissolved in the solution, the rate of protein efflux is accelerated significantly. This paper develops a reaction-diffusion (RD) model to explain both this observation, and the physical-organic chemistry that underlies it. Using this model, we calculate the dissociation constants of several unlabeled ligands from BCA, using plots of levitation height versus time. Notably, although this method requires no electricity, and only a single piece of inexpensive equipment, it can measure accurately the binding of unlabeled proteins to small molecules over a wide range of dissociation constants (K(d) values within the range from ~10 nM to 100 μM are measured easily). Assays performed using this method generally can be completed within a relatively short time period (20 min-2 h). A deficiency of this system is that it is not, in its present form, applicable to proteins with molecular weight greater

  10. Magnetic Levitation as a Platform for Competitive Protein-Ligand Binding Assays

    PubMed Central

    Shapiro, Nathan D.; Soh, Siowling; Mirica, Katherine A.; Whitesides, George M.

    2012-01-01

    This paper describes a method based on magnetic levitation (MagLev) that is capable of indirectly measuring the binding of unlabeled ligands to unlabeled protein. We demonstrate this method by measuring the affinity of unlabeled bovine carbonic anhydrase (BCA) for a variety of ligands (most of which are benzene sulfonamide derivatives). This method utilizes porous gel beads that are functionalized with a common aryl sulfonamide ligand. The beads are incubated with BCA and allowed to reach an equilibrium state in which the majority of the immobilized ligands are bound to BCA. Since the beads are less dense than the protein, protein binding to the bead increases the overall density of the bead. This change in density can be monitored using MagLev. Transferring the beads to a solution containing no protein creates a situation where net protein efflux from the bead is thermodynamically favorable. The rate at which protein leaves the bead for the solution can be calculated from the rate at which the levitation height of the bead changes. If another small molecule ligand of BCA is dissolved in the solution, the rate of protein efflux is accelerated significantly. This paper develops a reaction-diffusion (RD) model to explain both this observation, and the physical-organic chemistry that underlies it. Using this model, we calculate the dissociation constants of several unlabeled ligands from BCA, using plots of levitation height versus time. Notably, although this method requires no electricity, and only a single piece of inexpensive equipment, it can measure accurately the binding of unlabeled proteins to small molecules over a wide range of dissociation constants (Kd’s within the range of ~ 10 nM to 100 µM are measured easily). Assays performed using this method generally can be completed within a relatively short time period (20 minutes – 2 hours). A deficiency of this system is that it is not, in its present form, applicable to proteins with molecular weight

  11. Development of a Thermofluor assay for stability determination of membrane proteins using the Na(+)/H(+) antiporter NhaA and cytochrome c oxidase.

    PubMed

    Kohlstaedt, Martin; von der Hocht, Iris; Hilbers, Florian; Thielmann, Yvonne; Michel, Hartmut

    2015-05-01

    Crystallization of membrane proteins is very laborious and time-consuming, yielding well diffracting crystals in only a minority of projects. Therefore, a rapid and easy method is required to optimize the conditions for initial crystallization trials. The Thermofluor assay has been developed as such a tool. However, its applicability to membrane proteins is still limited because either large hydrophilic extramembranous regions or cysteine residues are required for the available dyes to bind and therefore act as reporters in this assay. No probe has been characterized to discriminate between the hydrophobic surfaces of detergent micelles, folded and detergent-covered membrane proteins and denatured membrane proteins. Of the four dyes tested, the two dyes 1-anilinonaphthalene-8-sulfonic acid (ANS) and SYPRO Orange were systematically screened for compatibility with five detergents commonly used in the crystallization of membrane proteins. ANS showed the weakest interactions with all of the detergents screened. It was possible to determine the melting temperature of the sodium ion/proton antiporter NhaA, a small membrane protein without large hydrophilic domains, over a broad pH range using ANS. Furthermore, cytochrome c oxidase (CcO) was used to apply the method to a four-subunit membrane protein complex. It was possible to obtain preliminary information on the temperature-dependent denaturation of this complex using the dye ANS. Application of the dye 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) to CcO in the Thermofluor assay enabled the determination of the melting temperatures of distinct subunits of the complex. PMID:25945577

  12. Should South Africa Be Performing Nucleic Acid Testing on HIV Enzyme-Linked Immunosorbent Assay-Negative Samples?▿

    PubMed Central

    Gous, Natasha; Scott, Lesley; Perovic, Olga; Venter, Francios; Stevens, Wendy

    2010-01-01

    The frequency of acute HIV infection (AHI) among HIV-1 enzyme-linked immunosorbent assay (ELISA)-negative samples received from general hospital patient admissions was assessed. Of 3,005 samples pooled for nucleic acid testing, a prevalence of 0.13% was found. Pooled nucleic acid testing may be feasible for low-cost identification of AHI in high-prevalence settings. PMID:20610671

  13. [Amino acid composition of rice grain proteins].

    PubMed

    Peruanskiĭ, Iu V; Savich, I M

    1976-01-01

    The composition of the major reserve proteins of rice grain--globulins, prolamines and glutelins--was examined in four rice varieties (Dubovsky 129, Kuban 3, Alakul, Ushtobinsky). Globulins proved to be most heterogeneous whereas glutelins appeared to be least heterogeneous. In regards to the ratio of components globulins showed high variability and glutelins displayed high stability. PMID:1005365

  14. Development of a fusion protein SNVP as substrate for assaying multi-serotype botulinum neurotoxins.

    PubMed

    Luo, Sen; Li, Tao; Wang, Qin; Tian, Renmao; Liu, Hao; Fang, Huali; Chen, Fanghong; Wang, Hui

    2014-10-15

    The SNARE super family has three core members, namely SNAP-25, VAMP-2, and syntaxin. SNAP-25 is cleaved by botulinum toxins (BoNTs)/A, /C, and /E, whereas VAMP-2 is the substrate for proteolytic BoNTs/B, /D, /F, and /G. In this study, we constructed a hybrid gene encoding the fusion protein SNVP that encompasses SNAP-25 residues Met1 to Gly206 and VAMP-2 residues Met1 to Lys94. The hybrid gene was cloned in a prokaryotic vector carrying an N-terminal pelB signal sequence and overexpressed in Escherichia coli BL21(DE3) Rosetta. To easily purify the protein, 6× His double-affinity tags were designed as the linker and C terminus of the fusion protein. SNVP was purified to homogeneity by affinity chromatography on a HisTrap FF column and determined to be more than 97% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal sequencing of the purified protein showed that signal peptide was successfully removed. The fusion protein SNVP contained the protease cleavage sites of all seven serotypes of BoNTs. SNVP was also proved to be recognized and cleaved by the endopeptidase of BoNTs (BoNT/A-LC, BoNT/B-LC, BoNT/E-LC, and BoNT/G-LC). The novel fusion substrate SNVP exhibited high biological activity under the optimal conditions, suggesting its potential use as a reagent for BoNT assay. PMID:23851341

  15. Nucleic acid compositions and the encoding proteins

    DOEpatents

    Preston, III, James F.; Chow, Virginia; Nong, Guang; Rice, John D.; St. John, Franz J.

    2014-09-02

    The subject invention provides at least one nucleic acid sequence encoding an aldouronate-utilization regulon isolated from Paenibacillus sp. strain JDR-2, a bacterium which efficiently utilizes xylan and metabolizes aldouronates (methylglucuronoxylosaccharides). The subject invention also provides a means for providing a coordinately regulated process in which xylan depolymerization and product assimilation are coupled in Paenibacillus sp. strain JDR-2 to provide a favorable system for the conversion of lignocellulosic biomass to biobased products. Additionally, the nucleic acid sequences encoding the aldouronate-utilization regulon can be used to transform other bacteria to form organisms capable of producing a desired product (e.g., ethanol, 1-butanol, acetoin, 2,3-butanediol, 1,3-propanediol, succinate, lactate, acetate, malate or alanine) from lignocellulosic biomass.

  16. Guanine nanowire based amplification strategy: Enzyme-free biosensing of nucleic acids and proteins.

    PubMed

    Gao, Zhong Feng; Huang, Yan Li; Ren, Wang; Luo, Hong Qun; Li, Nian Bing

    2016-04-15

    Sensitive and specific detection of nucleic acids and proteins plays a vital role in food, forensic screening, clinical and environmental monitoring. There remains a great challenge in the development of signal amplification method for biomolecules detection. Herein, we describe a novel signal amplification strategy based on the formation of guanine nanowire for quantitative detection of nucleic acids and proteins (thrombin) at room temperature. In the presence of analytes and magnesium ions, the guanine nanowire could be formed within 10 min. Compared to the widely used single G-quadruplex biocatalytic label unit, the detection limits are improved by two orders of magnitude in our assay. The proposed enzyme-free method avoids fussy chemical label-ling process, complex programming task, and sophisticated equipment, which might provide an ideal candidate for the fabrication of selective and sensitive biosensing platform. PMID:26649493

  17. Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production.

    PubMed

    Michelucci, Alessandro; Cordes, Thekla; Ghelfi, Jenny; Pailot, Arnaud; Reiling, Norbert; Goldmann, Oliver; Binz, Tina; Wegner, André; Tallam, Aravind; Rausell, Antonio; Buttini, Manuel; Linster, Carole L; Medina, Eva; Balling, Rudi; Hiller, Karsten

    2013-05-01

    Immunoresponsive gene 1 (Irg1) is highly expressed in mammalian macrophages during inflammation, but its biological function has not yet been elucidated. Here, we identify Irg1 as the gene coding for an enzyme producing itaconic acid (also known as methylenesuccinic acid) through the decarboxylation of cis-aconitate, a tricarboxylic acid cycle intermediate. Using a gain-and-loss-of-function approach in both mouse and human immune cells, we found Irg1 expression levels correlating with the amounts of itaconic acid, a metabolite previously proposed to have an antimicrobial effect. We purified IRG1 protein and identified its cis-aconitate decarboxylating activity in an enzymatic assay. Itaconic acid is an organic compound that inhibits isocitrate lyase, the key enzyme of the glyoxylate shunt, a pathway essential for bacterial growth under specific conditions. Here we show that itaconic acid inhibits the growth of bacteria expressing isocitrate lyase, such as Salmonella enterica and Mycobacterium tuberculosis. Furthermore, Irg1 gene silencing in macrophages resulted in significantly decreased intracellular itaconic acid levels as well as significantly reduced antimicrobial activity during bacterial infections. Taken together, our results demonstrate that IRG1 links cellular metabolism with immune defense by catalyzing itaconic acid production. PMID:23610393

  18. Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production

    PubMed Central

    Michelucci, Alessandro; Cordes, Thekla; Ghelfi, Jenny; Pailot, Arnaud; Reiling, Norbert; Goldmann, Oliver; Binz, Tina; Wegner, André; Tallam, Aravind; Rausell, Antonio; Buttini, Manuel; Linster, Carole L.; Medina, Eva; Balling, Rudi; Hiller, Karsten

    2013-01-01

    Immunoresponsive gene 1 (Irg1) is highly expressed in mammalian macrophages during inflammation, but its biological function has not yet been elucidated. Here, we identify Irg1 as the gene coding for an enzyme producing itaconic acid (also known as methylenesuccinic acid) through the decarboxylation of cis-aconitate, a tricarboxylic acid cycle intermediate. Using a gain-and-loss-of-function approach in both mouse and human immune cells, we found Irg1 expression levels correlating with the amounts of itaconic acid, a metabolite previously proposed to have an antimicrobial effect. We purified IRG1 protein and identified its cis-aconitate decarboxylating activity in an enzymatic assay. Itaconic acid is an organic compound that inhibits isocitrate lyase, the key enzyme of the glyoxylate shunt, a pathway essential for bacterial growth under specific conditions. Here we show that itaconic acid inhibits the growth of bacteria expressing isocitrate lyase, such as Salmonella enterica and Mycobacterium tuberculosis. Furthermore, Irg1 gene silencing in macrophages resulted in significantly decreased intracellular itaconic acid levels as well as significantly reduced antimicrobial activity during bacterial infections. Taken together, our results demonstrate that IRG1 links cellular metabolism with immune defense by catalyzing itaconic acid production. PMID:23610393

  19. Efficacy of reducing sugar and phenol-sulfuric acid assays for analysis of soluble carbohydrates in feedstuffs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Reducing sugar (RSA) and phenol–sulfuric acid (PSA) assays are commonly used to analyze water-soluble carbohydrates. However, questions have arisen as to their accuracy for measurement of feedstuffs with diverse carbohydrate profiles. This study evaluated the efficacy of RSA and PSA as they would co...

  20. SENSITIVE ASSAY, BASED ON HYDROXY FATTY ACIDS FROM LIPOPOLYSACCHARIDE LIPID A, FOR GRAM-NEGATIVE BACTERIA IN SEDIMENTS

    EPA Science Inventory

    Biochemical measures have provided insight into the biomass and community structure of sedimentary microbiota without the requirement of selection by growth or quantitative removal from the sediment grains. This study used the assay of the hydroxy fatty acids released from the li...

  1. Molecular Assay for Detection of Ciprofloxacin Resistance in Neisseria gonorrhoeae Isolates from Cultures and Clinical Nucleic Acid Amplification Test Specimens

    PubMed Central

    Peterson, S. W.; Martin, I.; Demczuk, W.; Bharat, A.; Hoang, L.; Wylie, J.; Allen, V.; Lefebvre, B.; Tyrrell, G.; Horsman, G.; Haldane, D.; Garceau, R.; Wong, T.

    2015-01-01

    We developed a real-time PCR assay to detect single nucleotide polymorphisms associated with ciprofloxacin resistance in specimens submitted for nucleic acid amplification testing (NAAT). All three single nucleotide polymorphism (SNP) targets produced high sensitivity and specificity values. The presence of ≥2 SNPs was sufficient to predict ciprofloxacin resistance in an organism. PMID:26292300

  2. Development of a precision-fed ileal amino acid digestibility assay using 3-week-old broiler chicks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of these studies was to develop a precision-fed ileal digestibility assay, primarily for amino acids (AA), using 3-wk-old broiler chicks. For all experiments, day-old Ross × Ross 708 broiler chicks were fed a standard corn-soybean meal starter diet until 21 d of age. In experiment 1, f...

  3. Whole-cell microtiter plate screening assay for terminal hydroxylation of fatty acids by P450s.

    PubMed

    Weissenborn, Martin J; Notonier, Sandra; Lang, Sarah-Luise; Otte, Konrad B; Herter, Susanne; Turner, Nicholas J; Flitsch, Sabine L; Hauer, Bernhard

    2016-05-01

    A readily available galactose oxidase (GOase) variant was used to develop a whole cell screening assay. This endpoint detection system was applied in a proof-of-concept approach by screening a focussed mutant library. This led to the discovery of the thus far most active P450 Marinobacter aquaeolei mutant catalysing the terminal hydroxylation of fatty acids. PMID:27074906

  4. A protocol for the systematic and quantitative measurement of protein-lipid interactions using the liposome-microarray-based assay.

    PubMed

    Saliba, Antoine-Emmanuel; Vonkova, Ivana; Deghou, Samy; Ceschia, Stefano; Tischer, Christian; Kugler, Karl G; Bork, Peer; Ellenberg, Jan; Gavin, Anne-Claude

    2016-06-01

    Lipids organize the activity of the cell's proteome through a complex network of interactions. The assembly of comprehensive atlases embracing all protein-lipid interactions is an important challenge that requires innovative methods. We recently developed a liposome-microarray-based assay (LiMA) that integrates liposomes, microfluidics and fluorescence microscopy and which is capable of measuring protein recruitment to membranes in a quantitative and high-throughput manner. Compared with previous assays that are labor-intensive and difficult to scale up, LiMA improves the protein-lipid interaction assay throughput by at least three orders of magnitude. Here we provide a step-by-step LiMA protocol that includes the following: (i) the serial and generic production of the liposome microarray; (ii) its integration into a microfluidic format; (iii) the measurement of fluorescently labeled protein (either purified proteins or from cell lysate) recruitment to liposomal membranes using high-throughput microscopy; (iv) automated image analysis pipelines to quantify protein-lipid interactions; and (v) data quality analysis. In addition, we discuss the experimental design, including the relevant quality controls. Overall, the protocol-including device preparation, assay and data analysis-takes 6-8 d. This protocol paves the way for protein-lipid interaction screens to be performed on the proteome and lipidome scales. PMID:27149326

  5. FLU, an amino acid substitution model for influenza proteins

    PubMed Central

    2010-01-01

    Background The amino acid substitution model is the core component of many protein analysis systems such as sequence similarity search, sequence alignment, and phylogenetic inference. Although several general amino acid substitution models have been estimated from large and diverse protein databases, they remain inappropriate for analyzing specific species, e.g., viruses. Emerging epidemics of influenza viruses raise the need for comprehensive studies of these dangerous viruses. We propose an influenza-specific amino acid substitution model to enhance the understanding of the evolution of influenza viruses. Results A maximum likelihood approach was applied to estimate an amino acid substitution model (FLU) from ~113, 000 influenza protein sequences, consisting of ~20 million residues. FLU outperforms 14 widely used models in constructing maximum likelihood phylogenetic trees for the majority of influenza protein alignments. On average, FLU gains ~42 log likelihood points with an alignment of 300 sites. Moreover, topologies of trees constructed using FLU and other models are frequently different. FLU does indeed have an impact on likelihood improvement as well as tree topologies. It was implemented in PhyML and can be downloaded from ftp://ftp.sanger.ac.uk/pub/1000genomes/lsq/FLU or included in PhyML 3.0 server at http://www.atgc-montpellier.fr/phyml/. Conclusions FLU should be useful for any influenza protein analysis system which requires an accurate description of amino acid substitutions. PMID:20384985

  6. Acid Cleavable Surface enhanced Raman Tagging for Protein Detection

    PubMed Central

    Zhang, Dongmao; Vangala, Karthikeshwar; Li, Shaoyong; Yanney, Michael; Xia, Hao; Zou, Sige; Sygula, Andrzej

    2010-01-01

    Dye conjugation is a common strategy improving the surface enhanced Raman detection sensitivity of biomolecules. Reported is a proof-of-concept study of a novel surface enhanced Raman spectroscopic tagging strategy termed as acid-cleavable SERS tag (ACST) method. Using Rhodamine B as the starting material, we prepared the first ACST prototype that consisted of, from the distal end, a SERS tag moiety (STM), an acid-cleavable linker, and a protein reactive moiety. Complete acid cleavage of the ACST tags was achieved at a very mild condition that is 1.5% trifluoroacetic acid (TFA) aqueous solution at room temperature. SERS detection of this ACST tagged protein was demonstrated using bovine serum albumin (BSA) as the model protein. While the SERS spectrum of intact ACST-BSA was entirely dominated by the fluorescent signal of STM, quality SERS spectra can be readily obtained with the acid cleaved ACST-BSA conjugates. Separation of the acid cleaved STM from protein further enhances the SERS sensitivity. Current SERS detection sensitivity, achieved with the acid cleaved ACST-BSA conjugate is ~5 nM in terms of the BSA concentration and ~1.5 nM in ACST content. The linear dynamic range of the cleaved ACST-BSA conjugate spans four orders of magnitudes from ~10 nM to ~100 μM in protein concentrations. Further improvement in the SERS sensitivity can be achieved with resonance Raman acquisition. This cleavable tagging strategy may also be used for elimination of protein interference in fluorescence based biomolecule detection. PMID:21109888

  7. A fluorescence-based high throughput assay for the determination of small molecule–human serum albumin protein binding

    PubMed Central

    McCallum, Megan M.; Pawlak, Alan J.; Shadrick, William R.; Simeonov, Anton; Jadhav, Ajit; Yasgar, Adam; Maloney, David J.; Arnold, Leggy A.

    2014-01-01

    Herein, we describe the development of a fluorescence-based high throughput assay to determine the small molecule binding towards human serum albumin (HSA). This innovative competition assay is based on the use of a novel fluorescent small molecule Red Mega 500 with unique spectroscopic and binding properties. The commercially available probe displays a large fluorescence intensity difference between the protein-bound and protein-unbound state. The competition of small molecules for HSA binding in the presence of probe resulted in low fluorescence intensities. The assay was evaluated with the LOPAC small molecule library of 1280 compounds identifying known high protein binders. The small molecule competition of HSA–Red Mega 500 binding was saturable at higher compound concentrations and exhibited IC50 values between 3–24 μM. The compound affinity towards HSA was confirmed by isothermal titration calorimetry indicating that the new protein binding assay is a valid high throughput assay to determine plasma protein binding. PMID:24390461

  8. A Sensitive Peptide Nucleic Acid Probe Assay for Detection of BRAF V600 Mutations in Melanoma.

    PubMed

    Chen, Tai-Long; Chang, John Wen-Cheng; Hsieh, Jia-Juan; Cheng, Hsin-Yi; Chiou, Chiuan-Chian

    Mutated v-Raf murine sarcoma viral oncogene homolog B (BRAF) is an important biomarker for the prediction of therapeutic efficacy of several anticancer drugs. The detection of BRAF mutation faces two challenges: Firstly, there are multiple types of mutations, and secondly, tumor samples usually contain various amounts of wild-type, normal tissues. Here, we describe a newly established method for sensitive detection of multiple types of BRAF V600 mutations in excess wild-type background. The method introduced a fluorophore-tagged peptide nucleic acid (PNA) to serve as both polymerase chain reaction (PCR) clamp and sensor probe, which inhibited the amplification of wild-type templates during PCR and revealed multiple types of mutant signals during melting analysis. We demonstrated the design and optimization process of the method, and applied it in the detection of BRAF mutations in 49 melanoma samples. This PNA probe assay method detected three types of mutations in 17 samples, and was much more sensitive than conventional PCR plus Sanger sequencing. PMID:27566656

  9. Mechanism of Nucleic Acid Chaperone Function of Retroviral Nuceleocapsid (NC) Proteins

    NASA Astrophysics Data System (ADS)

    Rouzina, Ioulia; Vo, My-Nuong; Stewart, Kristen; Musier-Forsyth, Karin; Cruceanu, Margareta; Williams, Mark

    2006-03-01

    Recent studies have highlighted two main activities of HIV-1 NC protein contributing to its function as a universal nucleic acid chaperone. Firstly, it is the ability of NC to weakly destabilize all nucleic acid,(NA), secondary structures, thus resolving the kinetic traps for NA refolding, while leaving the annealed state stable. Secondly, it is the ability of NC to aggregate NA, facilitating the nucleation step of bi-molecular annealing by increasing the local NA concentration. In this work we use single molecule DNA stretching and gel-based annealing assays to characterize these two chaperone activities of NC by using various HIV-1 NC mutants and several other retroviral NC proteins. Our results suggest that two NC functions are associated with its zinc fingers and cationic residues, respectively. NC proteins from other retroviruses have similar activities, although expressed to a different degree. Thus, NA aggregating ability improves, and NA duplex destabilizing activity decreases in the sequence: MLV NC, HIV NC, RSV NC. In contrast, HTLV NC protein works very differently from other NC proteins, and similarly to typical single stranded NA binding proteins. These features of retroviral NCs co-evolved with the structure of their genomes.

  10. Identification of novel PTEN-binding partners: PTEN interaction with fatty acid binding protein FABP4.

    PubMed

    Gorbenko, O; Panayotou, G; Zhyvoloup, A; Volkova, D; Gout, I; Filonenko, V

    2010-04-01

    PTEN is a tumor suppressor with dual protein and lipid-phosphatase activity, which is frequently deleted or mutated in many human advanced cancers. Recent studies have also demonstrated that PTEN is a promising target in type II diabetes and obesity treatment. Using C-terminal PTEN sequence in pEG202-NLS as bait, yeast two-hybrid screening on Mouse Embryo, Colon Cancer, and HeLa cDNA libraries was carried out. Isolated positive clones were validated by mating assay and identified through automated DNA sequencing and BLAST database searches. Sequence analysis revealed a number of PTEN-binding proteins linking this phosphatase to a number of different signaling cascades, suggesting that PTEN may perform other functions besides tumor-suppressing activity in different cell types. In particular, the interplay between PTEN function and adipocyte-specific fatty-acid-binding protein FABP4 is of notable interest. The demonstrable tautology of PTEN to FABP4 suggested a role for this phosphatase in the regulation of lipid metabolism and adipocyte differentiation. This interaction was further studied using coimmunoprecipitation and gel-filtration assays. Finally, based on Biacore assay, we have calculated the K(D) of PTEN-FABP4 complex, which is around 2.8 microM. PMID:19911253

  11. Proteins, Peptides and Amino Acids: Role in Infant Nutrition.

    PubMed

    Nutten, Sophie

    2016-01-01

    Proteins are polymers composed of 30 or more amino acids; some of them are essential dietary components, since they are not synthetized by human metabolic processes. They are crucial for healthy growth and development and influence major functions of the body. The infant's first year is a critical time of rapid growth and development, which must be supported by a high rate of protein synthesis. Breast milk, as a single specific food source in the first months of life, is providing the total protein and essential amino acids required. Infant formulas have been designed for infants who cannot be breastfed. They should be similar to breast milk in their composition and their functional outcomes, insuring appropriate growth, optimal development, maturation of the immune system, easy digestion and healthy metabolic programming. By modifying their protein components, specific infant formulas have also been developed for specific needs. For example, partially hydrolyzed (prevention of atopic dermatitis) and extensively hydrolyzed or amino-acid-based infant formulas (reduction in allergy symptoms) have been designed for the management of cow's milk protein allergy. In conclusion, proteins provided via breast milk or infant formula are essential components of the infant's diet; therefore, the specific quality, quantity and conformation of proteins are of utmost importance for healthy growth and development. PMID:27336588

  12. Fatty acid induced remodeling within the human liver fatty acid-binding protein.

    PubMed

    Sharma, Ashwani; Sharma, Amit

    2011-09-01

    We crystallized human liver fatty acid-binding protein (LFABP) in apo, holo, and intermediate states of palmitic acid engagement. Structural snapshots of fatty acid recognition, entry, and docking within LFABP support a heads-in mechanism for ligand entry. Apo-LFABP undergoes structural remodeling, where the first palmitate ingress creates the atomic environment for placement of the second palmitate. These new mechanistic insights will facilitate development of pharmacological agents against LFABP. PMID:21757748

  13. A novel protease activity assay method based on an engineered autoinhibited protein using an enzyme-linked immunoassay.

    PubMed

    Yoon, Hyun Kyung; Yoo, Tae Hyeon

    2013-12-01

    Proteases are involved in various biological phenomena, and their aberrant activity can be an important indicator of disease. Thus, various methods have been developed to analyze the activities of proteases, but their wide application has been hampered because each method has drawbacks. In this report, we propose a new protease assay method based on an engineered autoinhibited protein and enzyme-linked immunoassay (ELISA) in which a protease of interest activates the autoinhibited protein and the signal is amplified via ELISA. Using this concept a sensitive assay method for MMP2 and caspase-3 was developed. The limit of detection for the two proteases was as low as 7 pM for MMP2 and 0.1 pM for caspase-3. The autoinhibited protein is designed modularly, and the new platform is general enough for the development of assay methods for other proteases with minimal modification. PMID:24106734

  14. Systematic Evaluation of Different Nucleic Acid Amplification Assays for Cytomegalovirus Detection: Feasibility of Blood Donor Screening.

    PubMed

    Vollmer, T; Knabbe, C; Dreier, J

    2015-10-01

    Acute primary cytomegalovirus (CMV) infections, which commonly occur asymptomatically among blood donors, represent a significant risk for serious morbidity in immunocompromised patients (a major group of transfusion recipients). We implemented a routine CMV pool screening procedure for plasma for the identification of CMV DNA-positive donors, and we evaluated the sensitivities and performance of different CMV DNA amplification systems. Minipools (MPs) of samples from 18,405 individual donors (54,451 donations) were screened for CMV DNA using the RealStar CMV PCR assay (Altona Diagnostic Technologies), with a minimum detection limit of 11.14 IU/ml. DNA was extracted with a high-volume protocol (4.8 ml, Chemagic Viral 5K kit; PerkinElmer) for blood donor pool screening (MP-nucleic acid testing [NAT]) and with the Nuclisens easyMAG system (0.5 ml; bioMérieux) for individual donation (ID)-NAT. In total, six CMV DNA-positive donors (0.03%) were identified by routine CMV screening, with DNA concentrations ranging from 4.35 × 10(2) to 4.30 × 10(3) IU/ml. Five donors already showed seroconversion and detectable IgA, IgM, and/or IgG antibody titers (IgA(+)/IgM(+)/IgG(-) or IgA(+)/IgM(+)/IgG(+)), and one donor showed no CMV-specific antibodies. Comparison of three commercial assays, i.e., the RealStar CMV PCR kit, the Sentosa SA CMV quantitative PCR kit (Vela Diagnostics), and the CMV R-gene PCR kit (bioMérieux), for MP-NAT and ID-NAT showed comparably good analytical sensitivities, ranging from 10.23 to 11.14 IU/ml (MP-NAT) or from 37.66 to 57.94 IU/ml (ID-NAT). The clinical relevance of transfusion-associated CMV infections requires further investigation, and the evaluated methods present powerful basic tools providing sensitive possibilities for viral testing. The application of CMV MP-NAT facilitated the identification of one donor with a window-phase donation during acute primary CMV infection. PMID:26202109

  15. Gambogic acid is an antagonist of anti-apoptotic Bcl-2-family proteins

    PubMed Central

    Zhai, Dayong; Jin, Chaofang; Shiau, Chung-wai; Kitada, Shinichi; Satterthwait, Arnold C; Reed, John C.

    2008-01-01

    The natural product Gambogic acid (GA) has been reported to have cytotoxic activity against tumor cells in culture, and was identified as an active compound in a cell-based high-throughput screening (HTS) assay for activators of caspases, proteases involved in apoptosis. Using the anti-apoptotic Bcl-2-family protein, Bfl-1, as a target for screening of a library of natural products, we identified GA as a competitive inhibitor that displaced BH3 peptides from Bfl-1 in a fluorescent polarization assay (FPA). Analysis of competition for BH3 peptide binding revealed that GA inhibits all 6 human Bcl-2-family proteins to various extents, with Mcl-1 and Bcl-B the most potently inhibited (concentrations required for 50% inhibition [IC50] <1 μM). Competition for BH3 peptide binding was also confirmed using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. GA functionally inhibited the anti-apoptotic Bcl-2-family proteins, as demonstrated by experiments using isolated mitochondria in which recombinant purified Bcl-2-family proteins suppress SMAC release in vitro, showing that GA neutralizes their suppressive effects on mitochondria in a concentration-dependent manner. GA killed tumor cell lines via an apoptotic mechanism, whereas analogs of GA with greatly reduced potency at BH3 peptide displacement showed little or no cytotoxic activity. However, GA retained cytotoxic activity against bax−/− bak−/− cells in which anti-apoptotic Bcl-2-family proteins lack a cytoprotective phenotype, implying that GA also has additional targets that contribute to its cytotoxic mechanism. Altogether, the findings suggest that suppression of anti-apoptotic Bcl-2-family proteins may be among the cytotoxic mechanisms by which GA kills tumor cells. PMID:18566235

  16. Comparison of the Simplexa™ Flu A/B & RSV kit (nucleic acid extraction-dependent assay) and the Prodessa ProFlu+™ assay for detecting influenza and respiratory syncytial viruses.

    PubMed

    Selvaraju, Suresh B; Bambach, Adrienne V; Leber, Amy L; Patru, Maria-Magdalena; Patel, Anami; Menegus, Marilyn A

    2014-09-01

    The relative performance of 2 widely used reverse transcription polymerase chain reaction (RT-PCR) assays, the Focus diagnostics Simplexa™ Flu A/B & RSV kit (nucleic acid extraction-dependent assay) and the Prodessa Proflu+™ assay, was evaluated using 735 prospectively and retrospectively collected nasopharyngeal swab specimens. Overall, the assays showed positive and negative agreements of 100% and 99.7% for influenza A, 98.1% and 99.9% for influenza B, and 99.3% and 99.5% for respiratory syncytial virus. The relative analytical sensitivity of the 2 assays was also similar. PMID:25209363

  17. Functional gold nanoparticles coupled with microporous membranes: a flow controlled assay for colorimetric visualization of proteins.

    PubMed

    Chen, Yu-Yuan; Unnikrishnan, Binesh; Li, Yu-Jia; Huang, Chih-Ching

    2014-11-21

    We report a rapid and simple assay for colorimetric visualization of thrombin at nanomolar levels using functional gold nanoparticles (FAuNPs) coupled with microporous membranes. We used a 29-mer thiolated-thrombin-binding-aptamer (TBA29) to prepare TBA29 functionalized AuNPs (TBA29-AuNPs) for the selective detection of human thrombin. The sensing mechanism is based on the principle of TBA29-AuNPs flowing down through the nitrocellulose membrane (NCM) pores at different flow rates after binding to thrombin. Compared with free TBA29-AuNPs, when thrombin-TBA29-AuNPs were dropped on the NCM, the particles flowed down more easily through the NCM pores along with the buffer solution due to the increase in the gravity of particles. Therefore, color intensities of TBA29-AuNPs on the NCM depended on the concentration of thrombin; the color intensity was lighter when the concentration of thrombin was higher. Thrombin can be detected at the nanomolar level with the naked eye using this colorimetric probe. A protein G modified AuNP based probe (PG-AuNPs/NCM) was employed to detect human immunoglobulin G (hIgG) in plasma samples to demonstrate the practicality of our sensing system. Also, fibrinogen modified Au NPs were analyzed to demonstrate that this concept of detection could be extended to other proteins or systems, by functionalizing with suitable molecules. PMID:25267979

  18. New high-performance liquid chromatography assay for glycosyltransferases based on derivatization with anthranilic acid and fluorescence detection.

    PubMed

    Anumula, Kalyan Rao

    2012-07-01

    Assays were developed using the unique labeling chemistry of 2-aminobenzoic acid (2AA; anthranilic acid, AA) for measuring activities of both β1-4 galactosyltransferase (GalT-1) and α2-6 sialyltransferase (ST-6) by high-performance liquid chromatography (HPLC) with fluorescence detection (Anumula KR. 2006. Advances in fluorescence derivatization methods for high-performance liquid chromatographic analysis of glycoprotein carbohydrates. Anal Biochem. 350:1-23). N-Acetylglucosamine (GlcNAc) and N-acetyllactosamine were used as acceptors and uridine diphosphate (UDP)-galactose and cytidine monophosphate (CMP)-N-acetylneuraminic acid (NANA) as donors for GalT-1 and ST-6, respectively. Enzymatic products were labeled in situ with AA and were separated from the substrates on TSKgel Amide 80 column using normal-phase conditions. Enzyme units were determined from the peak areas by comparison with the concomitantly derivatized standards Gal-β1-4GlcNAc and NANA-α2-6 Gal-β1-4GlcNAc. Linearity (time and enzyme concentration), precision (intra- and interassay) and reproducibility for the assays were established. The assays were found to be useful in monitoring the enzyme activities during isolation and purification. The assays were highly sensitive and performed equal to or better than the traditional radioactive sugar-based measurements. The assay format can also be used for measuring the activity of other transferases, provided that the carbohydrate acceptors contain a reducing end for labeling. An assay for glycoprotein acceptors was developed using IgG. A short HPLC profiling method was developed for the separation of IgG glycans (biantennary G0, G1, G2, mono- and disialylated), which facilitated the determination of GalT-1 and ST-6 activities in a rapid manner. Furthermore, this profiling method should prove useful for monitoring the changes in IgG glycans in clinical settings. PMID:22459802

  19. Relationship between binding affinities to cellular retinoic acid-binding protein and biological potency of a new series of retinoids.

    PubMed

    Sani, B P; Dawson, M I; Hobbs, P D; Chan, R L; Schiff, L J

    1984-01-01

    Binding affinities of a new and unusual series of retinoic acid analogues to cellular retinoic acid-binding protein, a possible mediator of their biological function in the control of differentiation and tumorigenesis, and to serum albumin, their plasma transport protein, were determined. Also, biological activity of these retinoids in the reversal of keratinization in hamster tracheal organ cultures was assessed and compared with their binding affinities. Analogues that possessed high biological activity showed high binding efficiency to cellular retinoic acid-binding protein. Those that were biologically less active were poor binders to the binding protein. Three retinoids, 4657-57, 3920-59, and 4445-75, which showed 90 to 100% binding efficiency of that of retinoic acid for cellular retinoic acid-binding protein expressed high biological activity detectable in the range of 10(-10) M as against 10(-11) M for retinoic acid. The correlation noticed in these two activities not only enhances the confidence in the two assay procedures but also paves the way for design and development of potential chemopreventive agents. No apparent differences were observed in the binding affinities of the retinoids to binding proteins of a normal tissue or a tumor tissue. No correlation existed between the binding affinities of these retinoids to serum albumin and their biological activity. Structure-activity relationships of the retinoids in relation to their binding affinities and biological activities have been discussed. PMID:6317169

  20. Characterisation of a fatty acid and retinol binding protein orthologue from the hookworm Ancylostoma ceylanicum.

    PubMed

    Fairfax, Keke C; Vermeire, Jon J; Harrison, Lisa M; Bungiro, Richard D; Grant, Wayne; Husain, Sohail Z; Cappello, Michael

    2009-12-01

    Hookworms, bloodfeeding intestinal nematodes, infect nearly one billion people in resource limited countries and are a leading cause of anaemia and malnutrition. Like other nematodes, hookworms lack the capacity to synthesise essential fatty acids de novo and therefore must acquire those from exogenous sources. The cDNA corresponding to a putative Ancylostoma ceylanicum fatty acid and retinol binding protein-1 (AceFAR-1) was amplified from adult hookworm mRNA. Studies using quantitative reverse transcriptase real-time PCR demonstrate that AceFAR-1 transcripts are most abundant in the earliest developmental stages of the parasite, and greater in females than males. Using in vitro assays, the recombinant AceFAR-1 (rAceFAR-1) was shown to bind individual fatty acids with equilibrium dissociation constants in the low micromolar range. The pattern of fatty acid uptake by live adult worms cultured ex vivo was similar to the in vitro binding profile of rAceFAR-1, raising the possibility that the native protein may be involved in acquisition of fatty acids by A. ceylanicum. Animals vaccinated orally with rAceFAR-1 and the mucosal adjuvant cholera toxin exhibited a statistically significant (40-47%) reduction in intestinal worm burden compared with controls immunized with antigen or adjuvant alone. Together, these data suggest a potential role for AceFAR-1 in hookworm biology, making it a potentially valuable target for drug and vaccine development. PMID:19591834

  1. A macromolecular delivery vehicle for protein-based vaccines: Acid-degradable protein-loaded microgels

    PubMed Central

    Murthy, Niren; Xu, Mingcheng; Schuck, Stephany; Kunisawa, Jun; Shastri, Nilabh; Fréchet, Jean M. J.

    2003-01-01

    The development of protein-based vaccines remains a major challenge in the fields of immunology and drug delivery. Although numerous protein antigens have been identified that can generate immunity to infectious pathogens, the development of vaccines based on protein antigens has had limited success because of delivery issues. In this article, an acid-sensitive microgel material is synthesized for the development of protein-based vaccines. The chemical design of these microgels is such that they degrade under the mildly acidic conditions found in the phagosomes of antigen-presenting cells (APCs). The rapid cleavage of the microgels leads to phagosomal disruption through a colloid osmotic mechanism, releasing protein antigens into the APC cytoplasm for class I antigen presentation. Ovalbumin was encapsulated in microgel particles, 200–500 nm in diameter, prepared by inverse emulsion polymerization with a synthesized acid-degradable crosslinker. Ovalbumin is released from the acid-degradable microgels in a pH-dependent manner; for example, microgels containing ovalbumin release 80% of their encapsulated proteins after 5 h at pH 5.0, but release only 10% at pH 7.4. APCs that phagocytosed the acid-degradable microgels containing ovalbumin were capable of activating ovalbumin-specific cytoxic T lymphocytes. The acid-degradable microgels developed in this article should therefore find applications as delivery vehicles for vaccines targeted against viruses and tumors, where the activation of cytoxic T lymphocytes is required for the development of immunity. PMID:12704236

  2. Amino acids interference on the quantification of reducing sugars by the 3,5-dinitrosalicylic acid assay mislead carbohydrase activity measurements.

    PubMed

    Teixeira, Ricardo Sposina Sobral; da Silva, Ayla Sant'Ana; Ferreira-Leitão, Viridiana Santana; da Silva Bon, Elba Pinto

    2012-12-01

    This study evaluated the interference of the amino acids tryptophan, cysteine, histidine, tyrosine, hydroxyproline, leucine, proline, serine, glycine, valine, glutamic acid, phenylalanine, and methionine on the measurement of reducing sugars using a phenol-free 3,5-dinitrosalicylic acid (DNS) reagent. It was found that in reaction mixtures containing 20mM of either tryptophan, cysteine, histidine, tyrosine, or hydroxyproline the measurement of 3.7 mM glucose was overestimated by 76%, 50%, 35%, 18%, and 10%, respectively. The amino acids valine, glutamic acid, and phenylalanine did not affect the DNS reaction, while methionine decreased the color development by 5%. The measurement of glucose, xylose, arabinose, and cellobiose at the 3.7-12.4 mM range in the presence of 20 mM cysteine resulted in an overestimated concentration of 34.8-50%. Enzymatic assays for measuring xylanolytic and filter paper activity (FPAse) were conducted in the presence of 20-60 mM cysteine, and compared to cysteine-free assays. In the presence of cysteine, the measured xylanase activity increased threefold and the FPAse activity increased twofold due to the overestimation of the reducing sugar concentrations in the assays. The interference from cysteine was reduced to a maximum of 8.6% when a DNS reagent containing phenol was used. PMID:23103512

  3. Nucleic Acid Programmable Protein Array: A Just-In-Time Multiplexed Protein Expression and Purification Platform

    PubMed Central

    Qiu, Ji; LaBaer, Joshua

    2012-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. PMID:21943897

  4. Amino acid repeats and the structure and evolution of proteins.

    PubMed

    Albà, M M; Tompa, P; Veitia, R A

    2007-01-01

    Many proteins have repeats or runs of single amino acids. The pathogenicity of some repeat expansions has fueled proteomic, genomic and structural explorations of homopolymeric runs not only in human but in a wide variety of other organisms. Other types of amino acid repetitive structures exhibit more complex patterns than homopeptides. Irrespective of their precise organization, repetitive sequences are defined as low complexity or simple sequences, as one or a few residues are particularly abundant. Prokaryotes show a relatively low frequency of simple sequences compared to eukaryotes. In the latter the percentage of proteins containing homopolymeric runs varies greatly from one group to another. For instance, within vertebrates, amino acid repeat frequency is much higher in mammals than in amphibians, birds or fishes. For some repeats, this is correlated with the GC-richness of the regions containing the corresponding genes. Homopeptides tend to occur in disordered regions of transcription factors or developmental proteins. They can trigger the formation of protein aggregates, particularly in 'disease' proteins. Simple sequences seem to evolve more rapidly than the rest of the protein/gene and may have a functional impact. Therefore, they are good candidates to promote rapid evolutionary changes. All these diverse facets of homopolymeric runs are explored in this review. PMID:18753788

  5. Purification and assay of secretory lithostathine in human pancreatic juice by fast protein liquid chromatography.

    PubMed Central

    Mariani, A; Mezzi, G; Malesci, A

    1995-01-01

    Impaired secretion of lithostathine, a pancreatic glycoprotein capable of inhibiting the growth of CaCO3 crystals, has been reported in chronic calcifying pancreatitis. Controversial results were obtained, however, using immunoassays with different antibodies. The aim of this study was to purify and to measure juice lithostathine by a non-immunological method. Fast protein liquid chromatography (FPLC) on a cation exchange column eluted by a sodium chloride gradient, was used. The conditions appropriate to separate secretory (S) from hydrolysed (H) isoforms of immunopurified lithostathine were also used for juice analysis. Pancreatic juice was collected by endoscopic cannulation of the major pancreatic duct, after secretin stimulation, from eight patients with chronic pancreatitis (CP) and from eight controls. In all samples, S-isoforms of lithostathine (ranging from 16 to 19 Mr at SDS-PAGE) were the only constituent of two of the 15 peaks in which FPLC resolved the pancreatic proteins. The nature of these two peaks was confirmed by their coelution with immunopurified S-lithostathine and by immunoblot analysis with polyclonal anti-lithostathine antibodies. The ratio between the area of S-lithostathine peaks and the total area of proteic eluates, was always lower in CP patients (5.3 micrograms/mg of protein, median value; 0.2-15.4, range) than in controls (35.2 micrograms/mg; 16.6-55.9). It is concluded that lithostathine can be purified and measured in pancreatic juice by FPLC. Our results with a nonimmunological assay confirm a reduced secretion of lithostathine in patients with CP. Images Figure 2 Figure 4 Figure 5 PMID:7737574

  6. (-)-Hydroxycitric Acid Nourishes Protein Synthesis via Altering Metabolic Directions of Amino Acids in Male Rats.

    PubMed

    Han, Ningning; Li, Longlong; Peng, Mengling; Ma, Haitian

    2016-08-01

    (-)-Hydroxycitric acid (HCA), a major active ingredient of Garcinia Cambogia extracts, had shown to suppress body weight gain and fat accumulation in animals and humans. While, the underlying mechanism of (-)-HCA has not fully understood. Thus, this study was aimed to investigate the effects of long-term supplement with (-)-HCA on body weight gain and variances of amino acid content in rats. Results showed that (-)-HCA treatment reduced body weight gain and increased feed conversion ratio in rats. The content of hepatic glycogen, muscle glycogen, and serum T4 , T3 , insulin, and Leptin were increased in (-)-HCA treatment groups. Protein content in liver and muscle were significantly increased in (-)-HCA treatment groups. Amino acid profile analysis indicated that most of amino acid contents in serum and liver, especially aromatic amino acid and branched amino acid, were higher in (-)-HCA treatment groups. However, most of the amino acid contents in muscle, especially aromatic amino acid and branched amino acid, were reduced in (-)-HCA treatment groups. These results indicated that (-)-HCA treatment could reduce body weight gain through promoting energy expenditure via regulation of thyroid hormone levels. In addition, (-)-HCA treatment could promote protein synthesis by altering the metabolic directions of amino acids. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27145492

  7. Suppression of muscle protein turnover and amino acid degradation by dietary protein deficiency

    NASA Technical Reports Server (NTRS)

    Tawa, N. E. Jr; Goldberg, A. L.

    1992-01-01

    To define the adaptations that conserve amino acids and muscle protein when dietary protein intake is inadequate, rats (60-70 g final wt) were fed a normal or protein-deficient (PD) diet (18 or 1% lactalbumin), and their muscles were studied in vitro. After 7 days on the PD diet, both protein degradation and synthesis fell 30-40% in skeletal muscles and atria. This fall in proteolysis did not result from reduced amino acid supply to the muscle and preceded any clear decrease in plasma amino acids. Oxidation of branched-chain amino acids, glutamine and alanine synthesis, and uptake of alpha-aminoisobutyrate also fell by 30-50% in muscles and adipose tissue of PD rats. After 1 day on the PD diet, muscle protein synthesis and amino acid uptake decreased by 25-40%, and after 3 days proteolysis and leucine oxidation fell 30-45%. Upon refeeding with the normal diet, protein synthesis also rose more rapidly (+30% by 1 day) than proteolysis, which increased significantly after 3 days (+60%). These different time courses suggest distinct endocrine signals for these responses. The high rate of protein synthesis and low rate of proteolysis during the first 3 days of refeeding a normal diet to PD rats contributes to the rapid weight gain ("catch-up growth") of such animals.

  8. Structural Assessment of the Effects of Amino Acid Substitutions on Protein Stability and Protein-Protein Interaction

    PubMed Central

    Teng, Shaolei; Wang, Liangjiang; Srivastava, Anand K.; Schwartz, Charles E.; Alexov, Emil

    2012-01-01

    A structure-based approach is described for predicting the effects of amino acid substitutions on protein function. Structures were predicted using a homology modelling method. Folding and binding energy differences between wild-type and mutant structures were computed to quantitatively assess the effects of amino acid substitutions on protein stability and protein–protein interaction, respectively. We demonstrated that pathogenic mutations at the interaction interface could affect binding energy and destabilise protein complex, whereas mutations at the non-interface might reduce folding energy and destabilise monomer structure. The results suggest that the structure-based analysis can provide useful information for understanding the molecular mechanisms of diseases. PMID:21297231

  9. Quantitative evaluation of proteins with bicinchoninic acid (BCA): resonance Raman and surface-enhanced resonance Raman scattering-based methods.

    PubMed

    Chen, Lei; Yu, Zhi; Lee, Youngju; Wang, Xu; Zhao, Bing; Jung, Young Mee

    2012-12-21

    A rapid and highly sensitive bicinchoninic acid (BCA) reagent-based protein quantitation tool was developed using competitive resonance Raman (RR) and surface-enhanced resonance Raman scattering (SERRS) methods. A chelation reaction between BCA and Cu(+), which is reduced by protein in an alkaline environment, is exploited to create a BCA-Cu(+) complex that has strong RR and SERRS activities. Using these methods, protein concentrations in solutions can be quantitatively measured at concentrations as low as 50 μg mL(-1) and 10 pg mL(-1). There are many advantages of using RR and SERRS-based assays. These assays exhibit a much wider linear concentration range and provide an additional one (RR method) to four (SERRS method) orders of magnitude increase in detection limits relative to UV-based methods. Protein-to-protein variation is determined using a reference to a standard curve at concentrations of BSA that exhibits excellent recoveries. These novel methods are extremely accurate in detecting total protein concentrations in solution. This improvement in protein detection sensitivity could yield advances in the biological sciences and medical diagnostic field and extend the applications of reagent-based protein assay techniques. PMID:23099478

  10. Characterization and amino acid sequence of a fatty acid-binding protein from human heart.

    PubMed

    Offner, G D; Brecher, P; Sawlivich, W B; Costello, C E; Troxler, R F

    1988-05-15

    The complete amino acid sequence of a fatty acid-binding protein from human heart was determined by automated Edman degradation of CNBr, BNPS-skatole [3'-bromo-3-methyl-2-(2-nitrobenzenesulphenyl)indolenine], hydroxylamine, Staphylococcus aureus V8 proteinase, tryptic and chymotryptic peptides, and by digestion of the protein with carboxypeptidase A. The sequence of the blocked N-terminal tryptic peptide from citraconylated protein was determined by collisionally induced decomposition mass spectrometry. The protein contains 132 amino acid residues, is enriched with respect to threonine and lysine, lacks cysteine, has an acetylated valine residue at the N-terminus, and has an Mr of 14768 and an isoelectric point of 5.25. This protein contains two short internal repeated sequences from residues 48-54 and from residues 114-119 located within regions of predicted beta-structure and decreasing hydrophobicity. These short repeats are contained within two longer repeated regions from residues 48-60 and residues 114-125, which display 62% sequence similarity. These regions could accommodate the charged and uncharged moieties of long-chain fatty acids and may represent fatty acid-binding domains consistent with the finding that human heart fatty acid-binding protein binds 2 mol of oleate or palmitate/mol of protein. Detailed evidence for the amino acid sequences of the peptides has been deposited as Supplementary Publication SUP 50143 (23 pages) at the British Library Lending Division, Boston Spa, Yorkshire LS23 7BQ, U.K., from whom copies may be obtained as indicated in Biochem. J. (1988) 249, 5. PMID:3421901

  11. The Citrus transcription factor, CitERF13, regulates citric acid accumulation via a protein-protein interaction with the vacuolar proton pump, CitVHA-c4.

    PubMed

    Li, Shao-jia; Yin, Xue-ren; Xie, Xiu-lan; Allan, Andrew C; Ge, Hang; Shen, Shu-ling; Chen, Kun-song

    2016-01-01

    Organic acids are essential to fruit flavor. The vacuolar H(+) transporting adenosine triphosphatase (V-ATPase) plays an important role in organic acid transport and accumulation. However, less is known of V-ATPase interacting proteins and their relationship with organic acid accumulation. The relationship between V-ATPase and citric acid was investigated, using the citrus tangerine varieties 'Ordinary Ponkan (OPK)' and an early maturing mutant 'Zaoshu Ponkan (ZPK)'. Five V-ATPase genes (CitVHA) were predicted as important to citric acid accumulation. Among the genes, CitVHA-c4 was observed, using a yeast two-hybrid screen, to interact at the protein level with an ethylene response factor, CitERF13. This was verified using bimolecular fluorescence complementation assays. A similar interaction was also observed between Arabidopsis AtERF017 (a CitERF13 homolog) and AtVHA-c4 (a CitVHA-c4 homolog). A synergistic effect on citric acid levels was observed between V-ATPase proteins and interacting ERFs when analyzed using transient over-expression in tobacco and Arabidopsis mutants. Furthermore, the transcript abundance of CitERF13 was concomitant with CitVHA-c4. CitERF13 or AtERF017 over-expression leads to significant citric acid accumulation. This accumulation was abolished in an AtVHA-c4 mutant background. ERF-VHA interactions appear to be involved in citric acid accumulation, which was observed in both citrus and Arabidopsis. PMID:26837571

  12. Molecular cloning of an immunogenic protein of Baylisascaris procyonis and expression in Escherichia coli for use in developing improved serodiagnostic assays.

    PubMed

    Dangoudoubiyam, Sriveny; Vemulapalli, Ramesh; Hancock, Kathy; Kazacos, Kevin R

    2010-12-01

    Larva migrans caused by Baylisascaris procyonis is an important zoonotic disease. Current serological diagnostic assays for this disease depend on the use of the parasite's larval excretory-secretory (ES) antigens. In order to identify genes encoding ES antigens and to generate recombinant antigens for use in diagnostic assays, construction and immunoscreening of a B. procyonis third-stage larva cDNA expression library was performed and resulted in identification of a partial-length cDNA clone encoding an ES antigen, designated repeat antigen 1 (RAG1). The full-length rag1 cDNA contained a 753-bp open reading frame that encoded a protein of 250 amino acids with 12 tandem repeats of a 12-amino-acid long sequence. The rag1 genomic DNA revealed a single intron of 837 bp that separated the 753-bp coding sequence into two exons delimited by canonical splice sites. No nucleotide or amino acid sequences present in the GenBank databases had significant similarity with those of RAG1. We have cloned, expressed, and purified the recombinant RAG1 (rRAG1) and analyzed its diagnostic potential by enzyme-linked immunosorbent assay. Anti-Baylisascaris species-specific rabbit serum showed strong reactivity to rRAG1, while only minimal to no reactivity was observed with sera against the related ascarids Toxocara canis and Ascaris suum, strongly suggesting the specificity of rRAG1. On the basis of these results, the identified RAG1 appears to be a promising diagnostic antigen for the development of serological assays for specific detection of B. procyonis larva migrans. PMID:20926699

  13. Optimization of time-resolved fluorescence assay for detection of europium-tetraazacyclododecyltetraacetic acid-labeled ligand-receptor interactions.

    PubMed

    De Silva, Channa R; Vagner, Josef; Lynch, Ronald; Gillies, Robert J; Hruby, Victor J

    2010-03-01

    Lanthanide-based luminescent ligand binding assays are superior to traditional radiolabel assays due to improving sensitivity and affordability in high-throughput screening while eliminating the use of radioactivity. Despite significant progress using lanthanide(III)-coordinated chelators such as diethylenetriaminepentaacetic acid (DTPA) derivatives, dissociation-enhanced lanthanide fluoroimmunoassays (DELFIAs) have not yet been successfully used with more stable chelators (e.g., tetraazacyclododecyltetraacetic acid [DOTA] derivatives) due to the incomplete release of lanthanide(III) ions from the complex. Here a modified and optimized DELFIA procedure incorporating an acid treatment protocol is introduced for use with Eu(III)-DOTA-labeled peptides. Complete release of Eu(III) ions from DOTA-labeled ligands was observed using hydrochloric acid (2.0M) prior to the luminescent enhancement step. [Nle(4),d-Phe(7)]-alpha-melanocyte-stimulating hormone (NDP-alpha-MSH) labeled with Eu(III)-DOTA was synthesized, and the binding affinity to cells overexpressing the human melanocortin-4 (hMC4) receptor was evaluated using the modified protocol. Binding data indicate that the Eu(III)-DOTA-linked peptide bound to these cells with an affinity similar to its DTPA analogue. The modified DELFIA procedure was further used to monitor the binding of an Eu(III)-DOTA-labeled heterobivalent peptide to the cells expressing both hMC4 and cholecystokinin-2 (CCK-2) receptors. The modified assay provides superior results and is appropriate for high-throughput screening of ligand libraries. PMID:19852924

  14. Automated assay of methylmalonic acid in serum and urine by derivatization with 1-pyrenyldiazomethane, liquid chromatography, and fluorescence detection.

    PubMed

    Schneede, J; Ueland, P M

    1993-03-01

    Determination of methylmalonic acid (MMA) in serum has been established as an accurate test for the diagnosis of cobalamin deficiency. We describe here the development and performance of a liquid-chromatographic assay of MMA in blood and urine. The assay is based on our recent finding that one of the carboxylic acid moieties of some short-chain dicarboxylic acids reacts with the fluorogenic reagent 1-pyrenyldiazomethane in an aqueous medium, whereas the other remains underivatized (Anal Chem 1992; 63:315-9). The pH-dependent ionization of the free carboxylic acid group of 1-pyrenylmethyl monoesters permits retention on anion-exchange columns, which are used for solid-phase extraction. The analysis is done with a cyanopropyl column coupled in series with an octadecyldimethylsilyl column. Solid-phase extraction and sample injection are carried out automatically by a Gilson ASPEC sample processor. The assay response varies linearly with MMA concentration in the range 0.1-1000 mumol/L in serum. The within-day and between-day CVs are 2.8-10.9%, and the detection limit of 5 fmol injected (approximately 20 nmol/L in serum) is sufficiently low to determine MMA in serum (mean 0.187 mumol/L, SD 0.084, range 0.044-0.431, n = 44) and urine from healthy subjects. PMID:8448848

  15. INCAP studies of energy, amino acids, and protein.

    PubMed

    Viteri, Fernando E

    2010-03-01

    This Special Issue summarizes the results of several studies aimed at providing information on a series of questions related to the adequate protein and energy intakes that allow adequate growth and function in children and work performance and productivity in adults. The effect of different sources of protein on nitrogen balance and the requirements of essential amino acids in young children were also explored in fully recovered, previously malnourished children housed in the Metabolic Ward of the Biomedical Division of INCAP. The following are the main results of these investigations: Animal experiments and studies in children recovering from protein-energy malnutrition (PEM) strongly suggest that even when requirements of all nutrients are satisfied, inactivity reduces the rate of linear growth and physical activity improves it as well as lean body mass repletion. The effects of different energy intakes on nitrogen balance demonstrated how energy intake modifies the need to ingest different amounts of protein to satisfy protein requirements. Insensible nitrogen losses in preschool children and their relation to protein intake was demonstrated. The quality of even "good protein sources" modifies the amount needed to satisfy nitrogen requirements, and corn and bean-based diets can satisfy protein needs for health and even growth of young children. Essential amino acid requirements of 2-year-old children was assessed by diverse measurements of nitrogen metabolism and amino acid levels in blood, and were found lower than those recommended by FAO-WHO. In rural adult populations the relationship between energy and protein intake, productivity and body composition, and the impact of environmental hygiene on nitrogen balance was demonstrated and measured. PMID:20461903

  16. An electrophoretic mobility shift assay identifies a mechanistically unique inhibitor of protein sumoylation.

    PubMed

    Kim, Yeong Sang; Nagy, Katelyn; Keyser, Samantha; Schneekloth, John S

    2013-04-18

    The dynamic, posttranslational modification of proteins with a small ubiquitin-like modifier (SUMO) tag has been recognized as an important cellular regulatory mechanism relevant to a number of cancers as well as normal embryonic development. As part of a program aimed toward the identification of inhibitors of SUMO-conjugating enzymes, we developed a microfluidic electrophoretic mobility shift assay to monitor sumoylation events in real time. We disclose herein the use of this assay to identify a cell-permeable compound capable of blocking the transfer of SUMO-1 from the E2 enzyme Ubc9 to the substrate. We screened a small collection of compounds and identified an oxygenated flavonoid derivative that inhibits sumoylation in vitro. Next, we carried out an in-depth mechanistic analysis that ruled out many common false-positive mechanisms such as aggregation or alkylation. Furthermore, we report that this flavonoid inhibits a single step in the sumoylation cascade: the transfer of SUMO from the E2 enzyme (Ubc9) thioester conjugate to the substrate. In addition to having a unique mechanism of action, this inhibitor has a discrete structure-activity relationship uncharacteristic of a promiscuous inhibitor. Cell-based studies showed that the flavonoid inhibits the sumoylation of topoisomerase-I in response to camptothecin treatment in two different breast cancer cell lines, while isomeric analogs are inactive. Importantly, this compound blocks sumoylation while not affecting ubiquitylation in cells. This work identifies a point of entry for pharmacologic inhibition of the sumoylation cascade and may serve as the basis for continued study of additional pharmacophores that modulate SUMO-conjugating enzymes such as Ubc9. PMID:23601649

  17. Application of microchip assay system for the measurement of C-reactive protein in human saliva.

    PubMed

    Christodoulides, Nicolaos; Mohanty, Sanghamitra; Miller, Craig S; Langub, M Chris; Floriano, Pierre N; Dharshan, Priya; Ali, Mehnaaz F; Bernard, Bruce; Romanovicz, Dwight; Anslyn, Eric; Fox, Philip C; McDevitt, John T

    2005-03-01

    In the last decade, saliva has been advocated as a non-invasive alternative to blood as a diagnostic fluid. However, use of saliva has been hindered by the inadequate sensitivity of current methods to detect the lower salivary concentrations of many constituents compared to serum. Furthermore, developments in the areas related to lab-on-a-chip systems for saliva-based point of care diagnostics are complicated by the high viscosity and heterogeneous properties associated with this diagnostic fluid. The biomarker C-reactive protein (CRP) is an acute phase reactant and a well-accepted indicator of inflammation. Numerous clinical studies have established elevated serum CRP as a strong, independent risk factor for the development of cardiovascular disease (CVD). CVD has also been associated with oral infections (i.e. periodontal diseases) and there is evidence that systemic CRP may be a link between the two. Clinical measurements of CRP in serum are currently performed with "high sensitivity" CRP (hsCRP) enzyme-linked immunosorbent assay (ELISA) tests that lack the sensitivity for the detection of this important biomarker in saliva. Because measurement of salivary CRP may represent a novel approach for diagnosing and monitoring chronic inflammatory disease, including CVD and periodontal diseases, the objective of this study was to apply an ultra-sensitive microchip assay system for the measurement of CRP in human saliva. Here, we describe this novel lab-on-a-chip system in its first application for the measurement of CRP in saliva and demonstrate its advantages over the traditional ELISA method. The increased sensitivity of the microchip system (10 pg ml(-1) of CRP with 1000-fold dilution of saliva sample) is attributed to its inherent increased signal to noise ratio, resulting from the higher bead surface area available for antigen/antibody interactions and the high stringency washes associated with this approach. Finally, the microchip assay system was utilized in

  18. Studies on fatty acid-binding proteins. The diurnal variation shown by rat liver fatty acid-binding protein.

    PubMed Central

    Wilkinson, T C; Wilton, D C

    1987-01-01

    The concentration of fatty acid-binding protein in rat liver was examined by SDS/polyacrylamide-gel electrophoresis, by Western blotting and by quantifying the fluorescence enhancement achieved on the binding of the fluorescent probe 11-(dansylamino)undecanoic acid. A 2-3-fold increase in the concentration of this protein produced by treatment of rats with the peroxisome proliferator tiadenol was readily detected; however, only a small variation in the concentration of the protein due to a diurnal rhythm was observed. This result contradicts the 7-10-fold variation previously reported for this protein [Hargis, Olson, Clarke & Dempsey (1986) J. Biol. Chem. 261, 1988-1991]. Images Fig. 1. Fig. 3. PMID:3593284

  19. A J-Like Protein Influences Fatty Acid Composition of Chloroplast Lipids in Arabidopsis

    PubMed Central

    Ajjawi, Imad; Coku, Ardian; Froehlich, John E.; Yang, Yue; Osteryoung, Katherine W.; Benning, Christoph; Last, Robert L.

    2011-01-01

    A comprehensive understanding of the lipid and fatty acid metabolic machinery is needed for optimizing production of oils and fatty acids for fuel, industrial feedstocks and nutritional improvement in plants. T-DNA mutants in the poorly annotated Arabidopsis thaliana gene At1g08640 were identified as containing moderately high levels (50–100%) of 16∶1Δ7 and 18∶1Δ9 leaf fatty acids and subtle decreases (5–30%) of 16∶3 and 18∶3 (http://www.plastid.msu.edu/). TLC separation of fatty acids in the leaf polar lipids revealed that the chloroplastic galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were the main lipid types affected by this mutation. Analysis of the inferred amino acid sequence of At1g08640 predicted the presence of a transit peptide, three transmembrane domains and an N-terminal J-like domain, and the gene was named CJD1 for Chloroplast J-like Domain 1. GFP reporter experiments and in vitro chloroplast import assays demonstrated CJD1 is a chloroplast membrane protein. Screening of an Arabidopsis cDNA library by yeast-2-hybrid (Y2H) using the J-like domain of CJD1 as bait identified a plastidial inner envelope protein (Accumulation and Replication of Chloroplasts 6, ARC6) as the primary interacting partner in the Y2H assay. ARC6 plays a central role in chloroplast division and binds CJD1 via its own J-like domain along with an adjacent conserved region whose function is not fully known. These results provide a starting point for future investigations of how mutations in CJD1 affect lipid composition. PMID:22028775

  20. Protein and amino acid metabolism in the human newborn

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Birth and adaptation to extrauterine life involve major shifts in the protein and energy metabolism of the human newborn. These include a shift from a state of continuous supply of nutrients including amino acids from the mother to cyclic periodic oral intake, a change in the redox state of organs, ...

  1. Identification of multiple salicylic acid-binding proteins using two high throughput screens

    PubMed Central

    Manohar, Murli; Tian, Miaoying; Moreau, Magali; Park, Sang-Wook; Choi, Hyong Woo; Fei, Zhangjun; Friso, Giulia; Asif, Muhammed; Manosalva, Patricia; von Dahl, Caroline C.; Shi, Kai; Ma, Shisong; Dinesh-Kumar, Savithramma P.; O'Doherty, Inish; Schroeder, Frank C.; van Wijk, Klass J.; Klessig, Daniel F.

    2014-01-01

    Salicylic acid (SA) is an important hormone involved in many diverse plant processes, including floral induction, stomatal closure, seed germination, adventitious root initiation, and thermogenesis. It also plays critical functions during responses to abiotic and biotic stresses. The role(s) of SA in signaling disease resistance is by far the best studied process, although it is still only partially understood. To obtain insights into how SA carries out its varied functions, particularly in activating disease resistance, two new high throughput screens were developed to identify novel SA-binding proteins (SABPs). The first utilized crosslinking of the photo-reactive SA analog 4-AzidoSA (4AzSA) to proteins in an Arabidopsis leaf extract, followed by immuno-selection with anti-SA antibodies and then mass spectroscopy-based identification. The second utilized photo-affinity crosslinking of 4AzSA to proteins on a protein microarray (PMA) followed by detection with anti-SA antibodies. To determine whether the candidate SABPs (cSABPs) obtained from these screens were true SABPs, recombinantly-produced proteins were generated and tested for SA-inhibitable crosslinking to 4AzSA, which was monitored by immuno-blot analysis, SA-inhibitable binding of the SA derivative 3-aminoethylSA (3AESA), which was detected by a surface plasmon resonance (SPR) assay, or SA-inhibitable binding of [3H]SA, which was detected by size exclusion chromatography. Based on our criteria that true SABPs must exhibit SA-binding activity in at least two of these assays, nine new SABPs are identified here; nine others were previously reported. Approximately 80 cSABPs await further assessment. In addition, the conflicting reports on whether NPR1 is an SABP were addressed by showing that it bound SA in all three of the above assays. PMID:25628632

  2. Real-time assays for monitoring the influence of sulfide and sulfane sulfur species on protein thiol redox states.

    PubMed

    Greiner, Romy; Dick, Tobias P

    2015-01-01

    Hydrogen sulfide (H2S) is known to induce persulfidation of protein thiols. However, the process of H2S-induced persulfidation is not fully understood as it requires an additional oxidant. There are several mechanistic possibilities and it is of interest to determine which pathway is kinetically most relevant. Here, we detail in vitro assays for the real-time monitoring of thiol redox states in two model proteins with oxidizable cysteines, PTEN, and roGFP2. These allow kinetic measurements of the response of defined protein thiols (or disulfides) to sulfide and sulfane sulfur species. The combination of these assays with cold cyanolysis reveals the role of intermediary sulfane sulfur species in H2S-induced protein thiol oxidation. PMID:25747475

  3. In vitro protein degradation of 38 sainfoin accessions and its relationship to tannin content by different assays.

    PubMed

    Lorenz, Martin M; Hayot Carbonero, Christine; Smith, Lydia; Udén, Peter

    2012-05-23

    This study compared 38 sainfoin and 2 Lotus accessions to their respective tannin contents, N buffer solubility, and in vitro protein degradation. Tannin contents were measured by a protein precipitation method using either bovine serum albumin or Rubisco and by the colorimetric HCl/butanol method. Precipitation of bovine serum albumin and Rubisco was highly correlated (R(2) = 0.939). Correlations between the protein precipitation variants and the HCl/butanol method were relatively low (R(2) < 0.6). Protein degradation was measured at 4 h of incubation in an inhibited in vitro system and could not be explained by any of the tannin assays (R(2) < 0.03) and only partially by N buffer solubility (R(2) ≤ 0.433). Decisive factors other than the quantity of tannins or their ability to precipitate proteins must be considered. Resistance of soluble protein toward degradation can possibly be caused by tannin protein binding. PMID:22494200

  4. Should digestion assays be used to estimate persistence of potential allergens in tests for safety of novel food proteins?

    PubMed Central

    Schnell, Santiago; Herman, Rod A

    2009-01-01

    Food allergies affect an estimated 3 to 4% of adults and up to 8% of children in developed western countries. Results from in vitro simulated gastric digestion studies with purified proteins are routinely used to assess the allergenic potential of novel food proteins. The digestion of purified proteins in simulated gastric fluid typically progresses in an exponential fashion allowing persistence to be quantified using pseudo-first-order rate constants or half lives. However, the persistence of purified proteins in simulated gastric fluid is a poor predictor of the allergenic status of food proteins, potentially due to food matrix effects that can be significant in vivo. The evaluation of the persistence of novel proteins in whole, prepared food exposed to simulated gastric fluid may provide a more correlative result, but such assays should be thoroughly validated to demonstrate a predictive capacity before they are accepted to predict the allergenic potential of novel food proteins. PMID:19146693

  5. Fabrication of Uniform DNA-Conjugated Hydrogel Microparticles via Replica Molding for Facile Nucleic Acid Hybridization Assays

    PubMed Central

    Lewis, Christina L.; Choi, Chang-Hyung; Lin, Yan; Lee, Chang-Soo; Yi, Hyunmin

    2010-01-01

    We identify and investigate several critical parameters in the fabrication of single-stranded DNA conjugated poly(ethylene glycol) (PEG) microparticles based on replica molding (RM) for highly uniform and robust nucleic acid hybridization assays. The effects of PEG-diacrylate, probe DNA, and photoinitiator concentrations on the overall fluorescence and target DNA penetration depth upon hybridization are examined. Fluorescence and confocal microscopy results illustrate high conjugation capacity of probe and target DNA, femtomole sensitivity, and sequence specificity. Combined these findings demonstrate a significant step toward simple, robust, and scalable procedures to manufacture highly uniform and high capacity hybridization assay particles in a well-controlled manner by exploiting many advantages that the batch processing-based RM technique offers. We envision that the results presented here may be readily applied to rapid and high throughput hybridization assays for a wide variety of applications in bioprocess monitoring, food safety, and biological threat detection. PMID:20527819

  6. Fabrication of uniform DNA-conjugated hydrogel microparticles via replica molding for facile nucleic acid hybridization assays.

    PubMed

    Lewis, Christina L; Choi, Chang-Hyung; Lin, Yan; Lee, Chang-Soo; Yi, Hyunmin

    2010-07-01

    We identify and investigate several critical parameters in the fabrication of single-stranded DNA conjugated poly(ethylene glycol) (PEG) microparticles based on replica molding (RM) for highly uniform and robust nucleic acid hybridization assays. The effects of PEG-diacrylate, probe DNA, and photoinitiator concentrations on the overall fluorescence and target DNA penetration depth upon hybridization are examined. Fluorescence and confocal microscopy results illustrate high conjugation capacity of the probe and target DNA, femtomole sensitivity, and sequence specificity. Combined, these findings demonstrate a significant step toward simple, robust, and scalable procedures to manufacture highly uniform and high-capacity hybridization assay particles in a well-controlled manner by exploiting many advantages that the batch processing-based RM technique offers. We envision that the results presented here may be readily applied to rapid and high-throughput hybridization assays for a wide variety of applications in bioprocess monitoring, food safety, and biological threat detection. PMID:20527819

  7. Nucleic acid-protein interactions: Wedding for love or circumstances?

    PubMed

    Lavelle, Christophe; Buckle, Malcolm

    2009-08-01

    The sixth Figeac meeting on nucleic acid-protein interactions was held in Figeac, France, from September 26th to October 1st, 2008. It was organized by the working group "nucleic acid-protein interactions and gene expression" from the French Society for Biochemistry and Molecular Biology. This report briefly summarizes the presentations by 40 speakers during the four plenary sessions, which were organised as follows: (1) nucleic acids: targets and tools, (2) RNA superstar, (3) nuclear structure and dynamics, and (4) new concepts - new approaches. A total of 22 plenary lectures, 18 oral communications and 40 posters were presented over the 5 days, providing a highly stimulating environment for scientific exchange between the approximately 80 participants (biochemists, physicists, bio-informaticians and molecular and cellular biologists). PMID:19422875

  8. Highly Effective Protein Converting Strategy for Ultrasensitive Electrochemical Assay of Cystatin C.

    PubMed

    Yang, Zhe-Han; Zhuo, Ying; Yuan, Ruo; Chai, Ya-Qin

    2016-05-17

    In this work, a highly effective protein converting strategy based on immunoreaction-induced DNA strand displacement and T7 Exonuclease (T7 Exo)-assisted protein cyclic enzymatic amplification for ultrasensitive detection of cystatin C was described. Herein, Au@Fe3O4 as magnetic separator was labeled by antibody 1-conjungated DNA (DNA1) and the DNA substrate of T7 Exo (DNA3) which initially hybridized with output DNA (S1) to form a stable S1/DNA1 duplex (S1/DNA3). Antibody 2 was labeled by competing DNA (DNA 2). In the presence of cystatin C, sandwich immunoreaction would induce proximity hybridization between DNA2 and DNA3 and thus displace S1 from the S1/DNA3 duplex with formation of a stable DNA2/DNA3 duplex, realizing the conversion of input target cystatin C into output S1. To enhance the conversion ratio, the DNA2/DNA3 duplex was then digested by T7 Exo with release of DNA2 which could act as competing DNA again to displace S1 from the S1/DNA3 duplex in adjacent locations and initiate another cleavage reaction. Through such a cyclic process, each input cystatin C could induce more than one output S1, enhancing detection sensitivity. A hairpin DNA modified electrode was used to capture the output S1, and then, a hybridization chain reaction is triggered on the biosensor surface. Then, thionine as electron mediator was embedded into the dsDNA polymers to produce a detection signal. The electrochemical biosensor exhibited a much wider linear range of 0.01 pg mL(-1) to 30 ng mL(-1) with low detection limit of 3 fg mL(-1). Moreover, this method introduced protein unrelated to nucleic acids into the realm of potential inputs for translation, which might create a new immunoassay method for sensitive detection of protein. PMID:27104623

  9. Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity.

    PubMed

    Okada, Motohiro; Sangadala, Sreedhara; Liu, Yunshan; Yoshida, Munehito; Reddy, Boojala Vijay B; Titus, Louisa; Boden, Scott D

    2009-12-01

    The requirement of large amounts of the recombinant human bone morphogenetic protein-2 (BMP-2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP-2. In this study, we describe the development and optimization of a cell-based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP-responsive Smad1-driven luciferase reporter gene. LIM mineralization protein-1 (LMP-1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP-2. Our previous report elucidated that the binding of LMP-1 with the WW2 domain in Smad ubiquitin regulatory factor-1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell-based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP-1, and its mutant (LMP-1DeltaSmurf1) that lacks the Smurf1-WW2 domain-binding motif. Both the wild type and the mutant proteins were engineered to contain an 11-amino acid HIV-TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell-based reporter assay confirmed that LMP-1 potentiates the BMP-induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP-1 was significantly reduced when a specific-motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and

  10. Homogeneous time-resolved G protein-coupled receptor-ligand binding assay based on fluorescence cross-correlation spectroscopy.

    PubMed

    Antoine, Thomas; Ott, David; Ebell, Katharina; Hansen, Kerrin; Henry, Luc; Becker, Frank; Hannus, Stefan

    2016-06-01

    G protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic target classes for a wide spectrum of diseases. Drug discovery projects generally benefit from a broad range of experimental approaches for screening compound libraries and for the characterization of binding modes of drug candidates. Owing to the difficulties in solubilizing and purifying GPCRs, assay formats have been so far mainly limited to cell-based functional assays and radioligand binding assays. In this study, we used fluorescence cross-correlation spectroscopy (FCCS) to analyze the interaction of detergent-solubilized receptors to various types of GPCR ligands: endogenous peptides, small molecules, and a large surrogate antagonist represented by a blocking monoclonal antibody. Our work demonstrates the suitability of the homogeneous and time-resolved FCCS assay format for a robust, high-throughput determination of receptor-ligand binding affinities and kinetic rate constants for various therapeutically relevant GPCRs. PMID:26954998

  11. Point-of-care multiplexed assays of nucleic acids using microcapillary-based loop-mediated isothermal amplification.

    PubMed

    Zhang, Yi; Zhang, Lu; Sun, Jiashu; Liu, Yulei; Ma, Xingjie; Cui, Shangjin; Ma, Liying; Xi, Jianzhong Jeff; Jiang, Xingyu

    2014-07-15

    This report demonstrates a straightforward, robust, multiplexed and point-of-care microcapillary-based loop-mediated isothermal amplification (cLAMP) for assaying nucleic acids. This assay integrates capillaries (glass or plastic) to introduce and house sample/reagents, segments of water droplets to prevent contamination, pocket warmers to provide heat, and a hand-held flashlight for a visual readout of the fluorescent signal. The cLAMP system allows the simultaneous detection of two RNA targets of human immunodeficiency virus (HIV) from multiple plasma samples, and achieves a high sensitivity of two copies of standard plasmid. As few nucleic acid detection methods can be wholly independent of external power supply and equipment, our cLAMP holds great promise for point-of-care applications in resource-poor settings. PMID:24937125

  12. Effects of hemolysis, lipemia, hyperbilirrubinemia, and anticoagulants in canine C-reactive protein, serum amyloid A, and ceruloplasmin assays

    PubMed Central

    2005-01-01

    Abstract The objective of the present study was to determine the effects that hemolysis, lipemia, bilirubinemia, and anticoagulants might have on the most commonly used assays for C-reactive protein and serum amyloid A, and determination of ceruloplasmin values in dogs. Solutions of hemoglobin, lipid, and bilirubin were added to serum aliquots. Additionally, serum and plasma samples with different anticoagulants (heparin, EDTA, and citrate) were obtained from healthy dogs. Hemolysis, lipemia, and hyperbilirubinemia interfered significantly with the C-reactive protein and ceruloplasmin results, but not with those for the serum amyloid A assay. The use of anticoagulants produced significant changes in the results for the assays tested. However, the magnitude of the differences caused by the interfering substances does not appear to have an important impact on the clinical interpretation of the tests. PMID:16152718

  13. Correlation between centromere protein-F autoantibodies and cancer analyzed by enzyme-linked immunosorbent assay

    PubMed Central

    2013-01-01

    Background Centromere protein-F (CENP-F) is a large nuclear protein of 367 kDa, which is involved in multiple mitosis-related events such as proper assembly of the kinetochores, stabilization of heterochromatin, chromosome alignment and mitotic checkpoint signaling. Several studies have shown a correlation between CENP-F and cancer, e.g. the expression of CENP-F has been described to be upregulated in cancer cells. Furthermore, several studies have described a significant correlation between the expression of autoantibodies to CENP-F and cancer. Methods Autoantibodies to CENP-F were detected in a small number of samples during routine indirect immunofluorescence (IIF) analysis for anti-nuclear antibodies (ANA) using HEp-2 cells as substrate. Using overlapping synthetic peptides covering a predicted structural maintenance of chromosomes (SMC) domain, we developed an enzyme-linked immunosorbent assay (ELISA) for detection of CENP-F antibodies. Results Analyzing the reactivity of the sera positive in IIF for CENP-F antibodies to overlapping CENP-F peptides, we showed that autoantibodies to several peptides correlate with the presence of antibodies to CENP-F and a diagnosis of cancer, as increased CENP-F antibody expression specific for malignant cancer patients to five peptides was found (A9, A12, A14, A16, A27). These antibodies to CENP-F in clinical samples submitted for ANA analysis were found to have a positive predictive value for cancer of 50%. Furthermore, the expression of cancer-correlated CENP-F antibodies seemed to increase as a function of time from diagnosis. Conclusion These results conform to previous findings that approximately 50% of those patients clinically tested for ANA analyses who express CENP-F antibodies are diagnosed with cancer, confirming that these antibodies may function as circulating tumor markers. Thus, a peptide-based CENP-F ELISA focused on the SMC domain may aid in identifying individuals with a potential cancer. PMID:23978088

  14. Detection and characterisation of Complement protein activity in bovine milk by bactericidal sequestration assay.

    PubMed

    Maye, Susan; Stanton, Catherine; Fitzgerald, Gerald F; Kelly, Philip M

    2015-08-01

    While the Complement protein system in human milk is well characterised, there is little information on its presence and activity in bovine milk. Complement forms part of the innate immune system, hence the importance of its contribution during milk ingestion to the overall defences of the neonate. A bactericidal sequestration assay, featuring a Complement sensitive strain, Escherichia coli 0111, originally used to characterise Complement activity in human milk was successfully applied to freshly drawn bovine milk samples, thus, providing an opportunity to compare Complement activities in both human and bovine milks. Although not identical in response, the levels of Complement activity in bovine milk were found to be closely comparable with that of human milk. Differential counts of Esch. coli 0111 after 2 h incubation were 6.20 and 6.06 log CFU/ml, for raw bovine and human milks, respectively - the lower value representing a stronger Complement response. Exposing bovine milk to a range of thermal treatments e.g. 42, 45, 65, 72, 85 or 95 °C for 10 min, progressively inhibited Complement activity by increasing temperature, thus confirming the heat labile nature of this immune protein system. Low level Complement activity was found, however, in 65 and 72 °C heat treated samples and in retailed pasteurised milk which highlights the outer limit to which high temperature, short time (HTST) industrial thermal processes should be applied if retention of activity is a priority. Concentration of Complement in the fat phase was evident following cream separation, and this was also reflected in the further loss of activity recorded in low fat variants of retailed pasteurised milk. Laboratory-based churning of the cream during simulated buttermaking generated an aqueous (buttermilk) phase with higher levels of Complement activity than the fat phase, thus pointing to a likely association with the milk fat globule membrane (MFGM) layer. PMID:26119290

  15. Screening Carbohydrate Libraries for Protein Interactions Using the Direct ESI-MS Assay. Applications to Libraries of Unknown Concentration

    NASA Astrophysics Data System (ADS)

    Kitova, Elena N.; El-Hawiet, Amr; Klassen, John S.

    2014-08-01

    A semiquantitative electrospray ionization mass spectrometry (ESI-MS) binding assay suitable for analyzing mixtures of oligosaccharides, at unknown concentrations, for interactions with target proteins is described. The assay relies on the differences in the ratio of the relative abundances of the ligand-bound and free protein ions measured by ESI-MS at two or more initial protein concentrations to distinguish low affinity (≤103 M-1) ligands from moderate and high affinity (>105 M-1) ligands present in the library and to rank their affinities. Control experiments were performed on solutions of a single chain antibody and a mixture of synthetic oligosaccharides, with known affinities, in the absence and presence of a 40-component carbohydrate library to demonstrate the implementation and reliability of the assay. The application of the assay for screening natural libraries of carbohydrates against proteins is also demonstrated using mixtures of human milk oligosaccharides, isolated from breast milk, and fragments of a bacterial toxin and human galectin 3.

  16. Multiparameter Analysis-Based Electrochemiluminescent Assay for Simultaneous Detection of Multiple Biomarker Proteins on a Single Interface.

    PubMed

    Liang, Wenbin; Fan, Chenchen; Zhuo, Ying; Zheng, Yingning; Xiong, Chengyi; Chai, Yaqin; Yuan, Ruo

    2016-05-01

    Electrochemiluminescent (ECL) assay with high sensitivity has been considered as one of the potential strategies to simultaneously detect multiple biomarker proteins. However, it was essential, but full of challenges, to overcome the limitation caused by cross reactions among different ECL indicators. Herein, the multiparameter analysis of ECL-potential signals demonstrated by multivariate linear algebraic equations was first employed in the simultaneous ECL assay to realize multiple detection of biomarker proteins on a single interface. Additionally, owing to the exponential amplification of self-synthesized nucleotide dendrimer by hybridization chain reaction (HCR) and rolling circle amplification (RCA), the developed simultaneous ECL assay showed improved sensitivity and satisfactory accuracy for the detection of N-terminal of the prohormone brain natriuretic peptide (BNPT) and cardiac troponin I (cTnI). Furthermore, a self-designed magnetic beads-based flow system was also employed to improve the feasibility and analysis speed of the simultaneous ECL assay. Importantly, the proposed strategy enabled simultaneous detection of multiple biomarker proteins simply, which could be readily expanded for the multiplexed estimation of various kinds of proteins and nucleotide sequence also, revealing a new avenue for early disease diagnosis with higher efficiency. PMID:27064937

  17. An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates.

    PubMed

    Bhattacharyya, Sucharita; Renn, Jonathan P; Yu, Houqing; Marko, John F; Matouschek, Andreas

    2016-09-15

    The 26S proteasome is the molecular machine at the center of the ubiquitin proteasome system and is responsible for adjusting the concentrations of many cellular proteins. It is a drug target in several human diseases, and assays for the characterization of modulators of its activity are valuable. The 26S proteasome consists of two components: a core particle, which contains the proteolytic sites, and regulatory caps, which contain substrate receptors and substrate processing enzymes, including six ATPases. Current high-throughput assays of proteasome activity use synthetic fluorogenic peptide substrates that report directly on the proteolytic activity of the proteasome, but not on the activities of the proteasome caps that are responsible for protein recognition and unfolding. Here, we describe a simple and robust assay for the activity of the entire 26S proteasome using fluorescence anisotropy to follow the degradation of fluorescently labeled protein substrates. We describe two implementations of the assay in a high-throughput format and show that it meets the expected requirement of ATP hydrolysis and the presence of a canonical degradation signal or degron in the target protein. PMID:27296635

  18. CMP-N-acetylneuraminic acid synthetase interacts with fragile X related protein 1

    PubMed Central

    Ma, Yun; Tian, Shuai; Wang, Zongbao; Wang, Changbo; Chen, Xiaowei; Li, Wei; Yang, Yang; He, Shuya

    2016-01-01

    Fragile X mental retardation protein (FMRP), fragile X related 1 protein (FXR1P) and FXR2P are the members of the FMR protein family. These proteins contain two KH domains and a RGG box, which are characteristic of RNA binding proteins. The absence of FMRP, causes fragile X syndrome (FXS), the leading cause of hereditary mental retardation. FXR1P is expressed throughout the body and important for normal muscle development, and its absence causes cardiac abnormality. To investigate the functions of FXR1P, a screen was performed to identify FXR1P-interacting proteins and determine the biological effect of the interaction. The current study identified CMP-N-acetylneuraminic acid synthetase (CMAS) as an interacting protein using the yeast two-hybrid system, and the interaction between FXR1P and CMAS was validated in yeast using a β-galactosidase assay and growth studies with selective media. Furthermore, co-immunoprecipitation was used to analyze the FXR1P/CMAS association and immunofluorescence microscopy was performed to detect expression and intracellular localization of the proteins. The results of the current study indicated that FXR1P and CMAS interact, and colocalize in the cytoplasm and the nucleus of HEK293T and HeLa cells. Accordingly, a fragile X related 1 (FXR1) gene overexpression vector was constructed to investigate the effect of FXR1 overexpression on the level of monosialotetrahexosylganglioside 1 (GM1). The results of the current study suggested that FXR1P is a tissue-specific regulator of GM1 levels in SH-SY5Y cells, but not in HEK293T cells. Taken together, the results initially indicate that FXR1P interacts with CMAS, and that FXR1P may enhance the activation of sialic acid via interaction with CMAS, and increase GM1 levels to affect the development of the nervous system, thus providing evidence for further research into the pathogenesis of FXS. PMID:27357083

  19. Microfluidic molecular assay platform for the detection of miRNAs, mRNAs, proteins, and post-translational modifications at single-cell resolution

    SciTech Connect

    Wu, Meiye; Singh, Anup K.

    2014-07-15

    In this study, cell signaling is a dynamic and complex process. A typical signaling pathway may begin with activation of cell surface receptors, leading to activation kinase cascade that culminates in induction of mRNA and non-coding miRNA production in the nucleus, followed by modulation of mRNA expression by miRNAs in the cytosol, and end with production of proteins in response to the signaling pathway. Signaling pathways involve proteins, miRNA, and mRNAs, along with various forms of transient post-translational modifications, and detecting each type of signaling molecule requires categorically different sample preparation methods such as Western blotting for proteins, PCR for nucleic acids, and flow cytometry for post-translational modifications. Since we know that cells in populations behave heterogeneously1, especially in the cases of stem cells, cancer, and hematopoiesis, there is need for a new technology that provides capability to detect and quantify multiple categories of signaling molecules in intact single cells to provide a comprehensive view of the cell’s physiological state. In this technical brief, we describe our microfluidic platform with a portfolio of customized molecular assays that can detect nucleic acids, proteins, and post-translational modifications in single intact cells with >95% reduction in reagent requirement in under 8 hours.

  20. Microfluidic molecular assay platform for the detection of miRNAs, mRNAs, proteins, and post-translational modifications at single-cell resolution

    DOE PAGESBeta

    Wu, Meiye; Singh, Anup K.

    2014-07-15

    In this study, cell signaling is a dynamic and complex process. A typical signaling pathway may begin with activation of cell surface receptors, leading to activation kinase cascade that culminates in induction of mRNA and non-coding miRNA production in the nucleus, followed by modulation of mRNA expression by miRNAs in the cytosol, and end with production of proteins in response to the signaling pathway. Signaling pathways involve proteins, miRNA, and mRNAs, along with various forms of transient post-translational modifications, and detecting each type of signaling molecule requires categorically different sample preparation methods such as Western blotting for proteins, PCR formore » nucleic acids, and flow cytometry for post-translational modifications. Since we know that cells in populations behave heterogeneously1, especially in the cases of stem cells, cancer, and hematopoiesis, there is need for a new technology that provides capability to detect and quantify multiple categories of signaling molecules in intact single cells to provide a comprehensive view of the cell’s physiological state. In this technical brief, we describe our microfluidic platform with a portfolio of customized molecular assays that can detect nucleic acids, proteins, and post-translational modifications in single intact cells with >95% reduction in reagent requirement in under 8 hours.« less

  1. Structure and Function of the E. coli Protein YmgB: a Protein Critical for Biofilm Formation and Acid-resistance

    PubMed Central

    Lee, Jintae; Page, Rebecca; García-Contreras, Rodolfo; Palermino, Jeanne-Marie; Zhang, Xue-Song; Doshi, Ojus; Wood, Thomas K.; Peti, Wolfgang

    2007-01-01

    The Escherichia coli gene cluster ymgABC was identified in transcriptome studies to play a role in biofilm development and stability. In this study we show that YmgB represses biofilm formation in rich medium containing glucose, decreases cellular motility, and protects the cell from acid indicating that YmgB plays a major role in acid-resistance in E. coli. Our data also shows that these phenotypes are potentially mediated through interactions with the important cell signal indole. In addition, gel shift assays suggest that YmgB may be a non-specific DNA-binding protein. Using nickel-enrichment DNA microarrays, we show that YmgB binds, either directly or indirectly via a second protein, genes important for biofilm formation. To advance our understanding of the function of YmgB, we used X-ray crystallography to solve the structure of the protein to 1.8 Å resolution. YmgB is a biological dimer that is structurally homologous to the E. coli gene regulatory protein Hha, despite its low sequence identity of only 5%. This supports our DNA microarray data that YmgB is a gene regulatory protein. Therefore, this protein, which clearly has a critical role in acid-resistance in E. coli, has been renamed as AriR for regulator of acid-resistance influenced by indole. PMID:17765265

  2. Acid extraction and purification of recombinant spider silk proteins.

    PubMed

    Mello, Charlene M; Soares, Jason W; Arcidiacono, Steven; Butler, Michelle M

    2004-01-01

    A procedure has been developed for the isolation of recombinant spider silk proteins based upon their unique stability and solubilization characteristics. Three recombinant silk proteins, (SpI)7, NcDS, and [(SpI)4/(SpII)1]4, were purified by extraction with organic acids followed by affinity or ion exchange chromatography resulting in 90-95% pure silk solutions. The protein yield of NcDS (15 mg/L culture) and (SpI)7 (35 mg/L) increased 4- and 5-fold, respectively, from previously reported values presumably due to a more complete solubilization of the expressed recombinant protein. [(SpI)4/(SpII)1]4, a hybrid protein based on the repeat sequences of spidroin I and spidroin II, had a yield of 12.4 mg/L. This method is an effective, reproducible technique that has broad applicability for a variety of silk proteins as well as other acid stable biopolymers. PMID:15360297

  3. Nucleic acid-binding properties of the RRM-containing protein RDM1

    SciTech Connect

    Hamimes, Samia; Bourgeon, Dominique; Stasiak, Alicja Z.; Stasiak, Andrzej; Van Dyck, Eric . E-mail: Vandyck@iarc.fr

    2006-05-26

    RDM1 (RAD52 Motif 1) is a vertebrate protein involved in the cellular response to the anti-cancer drug cisplatin. In addition to an RNA recognition motif, RDM1 contains a small amino acid motif, named RD motif, which it shares with the recombination and repair protein, RAD52. RDM1 binds to single- and double-stranded DNA, and recognizes DNA distortions induced by cisplatin adducts in vitro. Here, we have performed an in-depth analysis of the nucleic acid-binding properties of RDM1 using gel-shift assays and electron microscopy. We show that RDM1 possesses acidic pH-dependent DNA-binding activity and that it binds RNA as well as DNA, and we present evidence from competition gel-shift experiments that RDM1 may be capable of discrimination between the two nucleic acids. Based on reported studies of RAD52, we have generated an RDM1 variant mutated in its RD motif. We find that the L{sub 119}GF {sup {yields}} AAA mutation affects the mode of RDM1 binding to single-stranded DNA.

  4. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    PubMed

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins. PMID:27080133

  5. Buffer interference with protein dynamics: a case study on human liver fatty acid binding protein.

    PubMed

    Long, Dong; Yang, Daiwen

    2009-02-18

    Selection of suitable buffer types is often a crucial step for generating appropriate protein samples for NMR and x-ray crystallographic studies. Although the possible interaction between MES buffer (2-(N-morpholino)ethanesulfonic acid) and proteins has been discussed previously, the interaction is usually thought to have no significant effects on the structures of proteins. In this study, we demonstrate the direct, albeit weak, interaction between MES and human liver fatty acid binding protein (hLFABP). Rather than affecting the structure of hLFABP, we found that the dynamics of hLFABP, which were previously proposed to be relevant to its functions, were significantly affected by the binding of hLFABP with MES. Buffer interference with protein dynamics was also demonstrated with Bis-Tris buffer, which is quite different from MES and fatty acids in terms of their molecular structures and properties. This result, to our knowledge, is the first published report on buffer interference with protein dynamics on a microsecond to millisecond timescale and could represent a generic problem in the studies of functionally relevant protein dynamics. Although being a fortuity, our finding of buffer-induced changes in protein dynamics offers a clue to how hLFABP accommodates its ligands. PMID:19217864

  6. Properties of whey protein-based films containing organic acids and nisin to control Listeria monocytogenes.

    PubMed

    Pintado, Cristina M B S; Ferreira, Maria A S S; Sousa, Isabel

    2009-09-01

    Whey protein isolate and glycerol were mixed to form a matrix to incorporate antimicrobial agents and produce edible films with antimicrobial activity against Listeria monocytogenes strains isolated from cheeses. Various organic acids were used to decrease pH down to approximately 3. In a preliminary assay without nisin, the effect of each organic acid was evaluated with respect to the rheological properties of the film solutions and the inhibitory and mechanical properties of the films. Lactic, malic, and citric acids (3%, wt/vol), which were used in a subsequent study of their combined inhibitory effect with nisin (50 IU/ml), had significantly higher antilisterial activity (P < 0.05) compared with the control (2 N HCl, 3% [wt/vol], with nisin). The largest mean zone of inhibition was 4.00 +/- 0.92 mm for malic acid with nisin. Under small-amplitude oscillatory stress, the protein-glycerol-acid film solutions exhibited a predominantly viscous behavior or a weak gel behavior, with the storage modulus (G') slightly higher than the loss modulus (G"). The malic acid-based solution was the only one whose viscosity was not influenced by the addition of nisin. The addition of nisin resulted in a nonsignificant (P > 0.05) increase in the percentage of elongation at break. Results from tensile and puncture stress were variable, but in general no significant differences were found after the incorporation of nisin. The overall results support the use of malic acid with nisin to produce effective antimicrobial films to control L. monocytogenes growth. PMID:19777891

  7. Bile acids influence the growth, oestrogen receptor and oestrogen-regulated proteins of MCF-7 human breast cancer cells.

    PubMed Central

    Baker, P. R.; Wilton, J. C.; Jones, C. E.; Stenzel, D. J.; Watson, N.; Smith, G. J.

    1992-01-01

    The effects of the major human serum bile acid, glycochenodeoxycholic acid (GCDC), as well as unconjugated chenodeoxycholic acid (CDC), on the MCF-7 human breast cancer cell line have been studied in vitro under oestrogen and bile acid deprived culture conditions. GCDC increased the growth of the breast cancer cells over the range 10-300 microM. At concentrations in excess of the bile acid binding capacity of the medium cell growth was prevented. In contrast 10 microM CDC tended to reduce cell growth. Oestrogen (ER) and progesterone (PgR) receptors, pS2 and total cathepsin D were quantified by monoclonal antibody based immunoassays. Ten to 100 microM GCDC and 10 microM CDC down-regulated ER protein and this was accompanied by induction of the oestrogen-regulated proteins PgR, pS2 and possibly cathepsin D, including increased secretion of the latter two proteins into the culture medium. All these changes were quantitatively similar to those observed with 10 nM oestradiol. The bile acid effects on ER and PgR were not due to interference with the assay procedures. Cells incubated with 50 microM GCDC or 10 microM CDC had higher pmolar concentrations of the bile acids than controls. This study suggests that naturally occurring bile acids influence the growth and steroid receptor function of human breast cancer cells. PMID:1562465

  8. Use of Modern Chemical Protein Synthesis and Advanced Fluorescent Assay Techniques to Experimentally Validate the Functional Annotation of Microbial Genomes

    SciTech Connect

    Kent, Stephen

    2012-07-20

    The objective of this research program was to prototype methods for the chemical synthesis of predicted protein molecules in annotated microbial genomes. High throughput chemical methods were to be used to make large numbers of predicted proteins and protein domains, based on microbial genome sequences. Microscale chemical synthesis methods for the parallel preparation of peptide-thioester building blocks were developed; these peptide segments are used for the parallel chemical synthesis of proteins and protein domains. Ultimately, it is envisaged that these synthetic molecules would be ‘printed’ in spatially addressable arrays. The unique ability of total synthesis to precision label protein molecules with dyes and with chemical or biochemical ‘tags’ can be used to facilitate novel assay technologies adapted from state-of-the art single molecule fluorescence detection techniques. In the future, in conjunction with modern laboratory automation this integrated set of techniques will enable high throughput experimental validation of the functional annotation of microbial genomes.

  9. Advances in protein-amino acid nutrition of poultry.

    PubMed

    Baker, David H

    2009-05-01

    The ideal protein concept has allowed progress in defining requirements as well as the limiting order of amino acids in corn, soybean meal, and a corn-soybean meal mixture for growth of young chicks. Recent evidence suggests that glycine (or serine) is a key limiting amino acid in reduced protein [23% crude protein (CP) reduced to 16% CP] corn-soybean meal diets for broiler chicks. Research with sulfur amino acids has revealed that small excesses of cysteine are growth depressing in chicks fed methionine-deficient diets. Moreover, high ratios of cysteine:methionine impair utilization of the hydroxy analog of methionine, but not of methionine itself. A high level of dietary L: -cysteine (2.5% or higher) is lethal for young chicks, but a similar level of DL: -methionine, L: -cystine or N-acetyl-L: -cysteine causes no mortality. A supplemental dietary level of 3.0% L: -cysteine (7x requirement) causes acute metabolic acidosis that is characterized by a striking increase in plasma sulfate and decrease in plasma bicarbonate. S-Methylmethionine, an analog of S-adenosylmethionine, has been shown to have choline-sparing activity, but it only spares methionine when diets are deficient in choline and(or) betaine. Creatine, or its precursor guanidinoacetic acid, can spare dietary arginine in chicks. PMID:19009229

  10. Interaction of Ku protein and DNA-dependent protein kinase catalytic subunit with nucleic acids.

    PubMed Central

    Dynan, W S; Yoo, S

    1998-01-01

    The Ku protein-DNA-dependent protein kinase system is one of the major pathways by which cells of higher eukaryotes respond to double-strand DNA breaks. The components of the system are evolutionarily conserved and homologs are known from a number of organisms. The Ku protein component binds directly to DNA ends and may help align them for ligation. Binding of Ku protein to DNA also nucleates formation of an active enzyme complex containing the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The interaction between Ku protein, DNA-PKcs and nucleic acids has been extensively investigated. This review summarizes the results of these biochemical investigations and relates them to recent molecular genetic studies that reveal highly characteristic repair and recombination defects in mutant cells lacking Ku protein or DNA-PKcs. PMID:9512523

  11. Comparative Analysis of Human, Mouse, and Pig Glial Fibrillary Acidic Protein Gene Structures.

    PubMed

    Eun, Kiyoung; Hwang, Seon-Ung; Jeon, Hye-Min; Hyun, Sang-Hwan; Kim, Hyunggee

    2016-01-01

    Comparing the coding and regulatory sequences of genes in different species provides information on whether proteins translated from genes have conserved functions or gene expressions are regulated by analogical mechanisms. Herein, we compared the coding and regulatory sequences of glial fibrillary acidic protein (GFAP) from humans, mice, and pigs. The GFAP gene encodes a class III intermediate filament protein expressed specifically in astrocytes of the central nervous system. On comparing the mRNA, regulatory region (promoter), and protein sequences of GFAP gene in silico, we found that GFAP mRNA 3'-untranslated region (3'-UTR), promoter, and amino acid sequences showed higher similarities between humans and pigs than between humans and mice. In addition, the promoter-luciferase reporter gene assay revealed that the pig GFAP promoter functioned in human astrocytes. Notably, the 1.8-kb promoter fragment upstream from transcription initiation site showed strongest transcriptional activity compared to 5.2-kb DNA fragment or other regions of GFAP promoter. We also found that pig GFAP mRNA and promoter activity increased in pig fibroblasts by human IL-1β treatment. Taken together, these results suggest that the regulatory mechanisms and functions of pig genes might be more similar to those of humans than mice, indicating that pigs, particularly miniature pigs, are a useful model for studying human biological and pathological events. PMID:26913554

  12. Disrupting Protein Expression with Peptide Nucleic Acids Reduces Infection by Obligate Intracellular Rickettsia

    PubMed Central

    Pelc, Rebecca S.; McClure, Jennifer C.; Kaur, Simran J.; Sears, Khandra T.; Rahman, M. Sayeedur; Ceraul, Shane M.

    2015-01-01

    Peptide Nucleic Acids (PNAs) are single-stranded synthetic nucleic acids with a pseudopeptide backbone in lieu of the phosphodiester linked sugar and phosphate found in traditional oligos. PNA designed complementary to the bacterial Shine-Dalgarno or start codon regions of mRNA disrupts translation resulting in the transient reduction in protein expression. This study examines the use of PNA technology to interrupt protein expression in obligate intracellular Rickettsia sp. Their historically intractable genetic system limits characterization of protein function. We designed PNA targeting mRNA for rOmpB from Rickettsia typhi and rickA from Rickettsia montanensis, ubiquitous factors important for infection. Using an in vitro translation system and competitive binding assays, we determined that our PNAs bind target regions. Electroporation of R. typhi and R. montanensis with PNA specific to rOmpB and rickA, respectively, reduced the bacteria’s ability to infect host cells. These studies open the possibility of using PNA to suppress protein synthesis in obligate intracellular bacteria. PMID:25781160

  13. Incorporation of Unnatural Amino Acids into Proteins Expressed in Mammalian Cells.

    PubMed

    Serfling, R; Coin, I

    2016-01-01

    The site-specific incorporation of unnatural amino acids (Uaas) via genetic code expansion provides a powerful method to introduce synthetic moieties into specific positions of a protein directly in the live cell. The technique, first developed in bacteria, is nowadays widely applicable in mammalian cells. In general, different Uaas are incorporated with different efficiency. By comparing the incorporation efficiency of several Uaas recently designed for bioorthogonal chemistry, we present here a facile dual-fluorescence assay to evaluate relative yields of Uaa incorporation. Several biological questions can be addressed using Uaas tools. In recent years, photo-cross-linking Uaas have been extensively applied to map ligand-binding sites on G protein-coupled receptors (GPCRs). We describe a simple and efficient two-plasmid system to incorporate a photoactivatable Uaa into a class B GPCR, and demonstrate cross-linking to its nonmodified natural ligand. PMID:27586329

  14. Identification of Biofilm Matrix-Associated Proteins from an Acid Mine Drainage Microbial Community ▿ †

    PubMed Central

    Jiao, Yongqin; D'haeseleer, Patrik; Dill, Brian D.; Shah, Manesh; VerBerkmoes, Nathan C.; Hettich, Robert L.; Banfield, Jillian F.; Thelen, Michael P.

    2011-01-01

    In microbial communities, extracellular polymeric substances (EPS), also called the extracellular matrix, provide the spatial organization and structural stability during biofilm development. One of the major components of EPS is protein, but it is not clear what specific functions these proteins contribute to the extracellular matrix or to microbial physiology. To investigate this in biofilms from an extremely acidic environment, we used shotgun proteomics analyses to identify proteins associated with EPS in biofilms at two developmental stages, designated DS1 and DS2. The proteome composition of the EPS was significantly different from that of the cell fraction, with more than 80% of the cellular proteins underrepresented or undetectable in EPS. In contrast, predicted periplasmic, outer membrane, and extracellular proteins were overrepresented by 3- to 7-fold in EPS. Also, EPS proteins were more basic by ∼2 pH units on average and about half the length. When categorized by predicted function, proteins involved in motility, defense, cell envelope, and unknown functions were enriched in EPS. Chaperones, such as histone-like DNA binding protein and cold shock protein, were overrepresented in EPS. Enzymes, such as protein peptidases, disulfide-isomerases, and those associated with cell wall and polysaccharide metabolism, were also detected. Two of these enzymes, identified as β-N-acetylhexosaminidase and cellulase, were confirmed in the EPS fraction by enzymatic activity assays. Compared to the differences between EPS and cellular fractions, the relative differences in the EPS proteomes between DS1 and DS2 were smaller and consistent with expected physiological changes during biofilm development. PMID:21685158

  15. Identification of Biofilm Matrix-Associated Proteins from an Acid Mine Drainage Microbial Community

    SciTech Connect

    Jiao, Yongqin; D'Haeseleer, Patrik M; Dill, Brian; Shah, Manesh B; Verberkmoes, Nathan C; Hettich, Robert {Bob} L; Banfield, Jillian F.; Thelen, Michael P.

    2011-01-01

    In microbial communities, extracellular polymeric substances (EPS), also called the extracellular matrix, provide the spatial organization and structural stability during biofilm development. One of the major components of EPS is protein, but it is not clear what specific functions these proteins contribute to the extracellular matrix or to microbial physiology. To investigate this in biofilms from an extremely acidic environment, we used shotgun proteomics analyses to identify proteins associated with EPS in biofilms at two developmental stages, designated DS1 and DS2. The proteome composition of the EPS was significantly different from that of the cell fraction, with more than 80% of the cellular proteins underrepresented or undetectable in EPS. In contrast, predicted periplasmic, outer membrane, and extracellular proteins were overrepresented by 3- to 7-fold in EPS. Also, EPS proteins were more basic by 2 pH units on average and about half the length. When categorized by predicted function, proteins involved in motility, defense, cell envelope, and unknown functions were enriched in EPS. Chaperones, such as histone-like DNA binding protein and cold shock protein, were overrepresented in EPS. Enzymes, such as protein peptidases, disulfide-isomerases, and those associated with cell wall and polysaccharide metabolism, were also detected. Two of these enzymes, identified as -N-acetylhexosaminidase and cellulase, were confirmed in the EPS fraction by enzymatic activity assays. Compared to the differences between EPS and cellular fractions, the relative differences in the EPS proteomes between DS1 and DS2 were smaller and consistent with expected physiological changes during biofilm development.

  16. Leukocyte Protease Binding to Nucleic Acids Promotes Nuclear Localization and Cleavage of Nucleic Acid Binding Proteins

    PubMed Central

    Thomas, Marshall P.; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

    2014-01-01

    Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. Here we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein (RBP) targets, while adding RNA to recombinant RBP substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Pre-incubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G (CATG). During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps (NETs), which bind NE and CATG. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and NETs in a DNA-dependent manner. Thus, high affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation. PMID:24771851

  17. Nanoparticle-Based Histidine-Rich Protein-2 Assay for the Detection of the Malaria Parasite Plasmodium falciparum.

    PubMed

    Castro-Sesquen, Yagahira E; Kim, Chloe; Gilman, Robert H; Sullivan, David J; Searson, Peter C

    2016-08-01

    A nanoparticle-based assay for detection and quantification of Plasmodium falciparum histidine-rich protein 2 (HRP2) in urine and serum is reported. The assay uses magnetic beads conjugated with anti-HRP2 antibody for protein capture and concentration, and antibody-conjugated quantum dots for optical detection. Western blot analysis demonstrated that magnetic beads allow the concentration of HRP2 protein in urine by 20-fold. The concentration effect was achieved because large volume of urine can be incubated with beads, and magnetic separation can be easily performed in minutes to isolate beads containing HRP2 protein. Magnetic beads and quantum dots conjugated to anti-HRP2 antibodies allows the detection of low concentrations of HRP2 protein (0.5 ng/mL), and quantification in the range of 33-2,000 ng/mL corresponding to the range associated with non-severe to severe malaria. This assay can be easily adapted to a noninvasive point-of-care test for classification of severe malaria. PMID:27185769

  18. Technical note: A portable on-chip assay system for absorbance and plasmonic detection of protein hormone in milk.

    PubMed

    Ozhikandathil, Jayan; Badilescu, Simona; Packirisamy, Muthukumaran

    2015-07-01

    This paper reports a portable device and method to extract and detect protein hormone in milk samples. Recombinant protein hormone spiked into milk samples was extracted by solid-phase extraction, and detection was carried out using the plasmonic property of gold nanoislands deposited on a glass substrate. Trace levels of hormone spiked in milk were analyzed by their optical absorbance property using a microfluidic chip. We built a portable assay system using disposable lab-on-chip devices. The proposed method is able to detect spiked recombinant protein hormone in milk at concentrations as low as 5ng/mL. PMID:25497819

  19. Modeling nucleic acid structure in the presence of single-stranded binding proteins

    NASA Astrophysics Data System (ADS)

    Forties, Robert; Bundschuh, Ralf

    2009-03-01

    There are many important proteins which bind single-stranded nucleic acids, such as the nucleocapsid protein in HIV, the RecA DNA repair protein in bacteria, and all proteins involved in mRNA splicing and translation. We extend the Vienna Package for quantitatively modeling the secondary structure of nucleic acids to include proteins which bind to unpaired portions of the nucleic acid. All parameters needed to model nucleic acid secondary structures in the absence of proteins have been previously measured. This leaves the footprint and sequence dependent binding affinity of the protein as adjustable parameters of our model. Using this model we are able to predict the probability of the protein binding at any position in the nucleic acid sequence, the impact of the protein on nucleic acid base pairing, the end-to-end distance distribution for the nucleic acid, and FRET distributions for fluorophores attached to the nucleic acid.

  20. Multicenter evaluation of the Quidel Lyra Direct C. difficile nucleic acid amplification assay.

    PubMed

    Beck, Eric T; Buchan, Blake W; Riebe, Katherine M; Alkins, Brenda R; Pancholi, Preeti; Granato, Paul A; Ledeboer, Nathan A

    2014-06-01

    Clostridium difficile is a Gram-positive bacterium commonly found in health care and long-term-care facilities and is the most common cause of antibiotic-associated diarrhea. Rapid detection of this bacterium can assist physicians in implementing contact precautions and appropriate antibiotic therapy in a timely manner. The purpose of this study was to compare the clinical performance of the Quidel Lyra Direct C. difficile assay (Lyra assay) (Quidel, San Diego, CA) to that of a direct cell culture cytotoxicity neutralization assay (CCNA) and enhanced toxigenic culture. This study was performed at three geographically diverse laboratories within the United States using residual stool specimens submitted for routine C. difficile testing. Residual samples were tested using the Lyra assay on three real-time PCR platforms, and results were compared to those for direct CCNA and enhanced toxigenic culture. The test results for all platforms were consistent across all three test sites. The sensitivity and specificity of the Lyra assay on the SmartCycler II, ABI 7500 Fast DX, and ABI QuantStudio DX instruments compared to CCNA were 90.0% and 93.3%, 95.0% and 94.2%, and 93.8% and 95.0%, respectively. Compared to enhanced toxigenic culture, the sensitivity and specificity of the Lyra assay on the SmartCycler II, ABI 7500, and QuantStudio instruments were 82.1% and 96.9%, 89.3% and 98.8%, and 85.7% and 99.0%, respectively. Overall, the Lyra assay is easy to use and versatile and compares well to C. difficile culture methods. PMID:24671790

  1. Multicenter Evaluation of the Quidel Lyra Direct C. difficile Nucleic Acid Amplification Assay

    PubMed Central

    Beck, Eric T.; Buchan, Blake W.; Riebe, Katherine M.; Alkins, Brenda R.; Pancholi, Preeti; Granato, Paul A.

    2014-01-01

    Clostridium difficile is a Gram-positive bacterium commonly found in health care and long-term-care facilities and is the most common cause of antibiotic-associated diarrhea. Rapid detection of this bacterium can assist physicians in implementing contact precautions and appropriate antibiotic therapy in a timely manner. The purpose of this study was to compare the clinical performance of the Quidel Lyra Direct C. difficile assay (Lyra assay) (Quidel, San Diego, CA) to that of a direct cell culture cytotoxicity neutralization assay (CCNA) and enhanced toxigenic culture. This study was performed at three geographically diverse laboratories within the United States using residual stool specimens submitted for routine C. difficile testing. Residual samples were tested using the Lyra assay on three real-time PCR platforms, and results were compared to those for direct CCNA and enhanced toxigenic culture. The test results for all platforms were consistent across all three test sites. The sensitivity and specificity of the Lyra assay on the SmartCycler II, ABI 7500 Fast DX, and ABI QuantStudio DX instruments compared to CCNA were 90.0% and 93.3%, 95.0% and 94.2%, and 93.8% and 95.0%, respectively. Compared to enhanced toxigenic culture, the sensitivity and specificity of the Lyra assay on the SmartCycler II, ABI 7500, and QuantStudio instruments were 82.1% and 96.9%, 89.3% and 98.8%, and 85.7% and 99.0%, respectively. Overall, the Lyra assay is easy to use and versatile and compares well to C. difficile culture methods. PMID:24671790

  2. Nucleomorphin. A novel, acidic, nuclear calmodulin-binding protein from dictyostelium that regulates nuclear number.

    PubMed

    Myre, Michael A; O'Day, Danton H

    2002-05-31

    Probing of Dictyostelium discoideum cell extracts after SDS-PAGE using (35)S-recombinant calmodulin (CaM) as a probe has revealed approximately three-dozen Ca(2+)-dependent calmodulin binding proteins. Here, we report the molecular cloning, expression, and subcellular localization of a gene encoding a novel calmodulin-binding protein (CaMBP); we have called nucleomorphin, from D. discoideum. A lambdaZAP cDNA expression library of cells from multicellular development was screened using a recombinant calmodulin probe ((35)S-VU1-CaM). The open reading frame of 1119 nucleotides encodes a polypeptide of 340 amino acids with a calculated molecular mass of 38.7 kDa and is constitutively expressed throughout the Dictyostelium life cycle. Nucleomorphin contains a highly acidic glutamic/aspartic acid inverted repeat (DEED) with significant similarity to the conserved nucleoplasmin domain and a putative transmembrane domain in the carboxyl-terminal region. Southern blotting reveals that nucleomorphin exists as a single copy gene. Using gel overlay assays and CaM-agarose we show that bacterially expressed nucleomorphin binds to bovine CaM in a Ca(2+)-dependent manner. Amino-terminal fusion to the green fluorescence protein (GFP) showed that GFP-NumA localized to the nucleus as distinct arc-like patterns similar to heterochromatin regions. GFP-NumA lacking the acidic DEED repeat still showed arc-like accumulations at the nuclear periphery, but the number of nuclei in these cells was increased markedly compared with control cells. Cells expressing GFP-NumA lacking the transmembrane domain localized to the nuclear periphery but did not affect nuclear number or gross morphology. Nucleomorphin is the first nuclear CaMBP to be identified in Dictyostelium. Furthermore, these data present the first identification of a member of the nucleoplasmin family as a calmodulin-binding protein and suggest nucleomorphin has a role in nuclear structure in Dictyostelium. PMID:11919178

  3. Interaction of aurintricarboxylic acid (ATA) with four nucleic acid binding proteins DNase I, RNase A, reverse transcriptase and Taq polymerase

    NASA Astrophysics Data System (ADS)

    Ghosh, Utpal; Giri, Kalyan; Bhattacharyya, Nitai P.

    2009-12-01

    In the investigation of interaction of aurintricarboxylic acid (ATA) with four biologically important proteins we observed inhibition of enzymatic activity of DNase I, RNase A, M-MLV reverse transcriptase and Taq polymerase by ATA in vitro assay. As the telomerase reverse transcriptase (TERT) is the main catalytic subunit of telomerase holoenzyme, we also monitored effect of ATA on telomerase activity in vivo and observed dose-dependent inhibition of telomerase activity in Chinese hamster V79 cells treated with ATA. Direct association of ATA with DNase I ( Kd = 9.019 μM)), RNase A ( Kd = 2.33 μM) reverse transcriptase ( Kd = 0.255 μM) and Taq polymerase ( Kd = 81.97 μM) was further shown by tryptophan fluorescence quenching studies. Such association altered the three-dimensional conformation of DNase I, RNase A and Taq polymerase as detected by circular dichroism. We propose ATA inhibits enzymatic activity of the four proteins through interfering with DNA or RNA binding to the respective proteins either competitively or allosterically, i.e. by perturbing three-dimensional structure of enzymes.

  4. Quantitative analysis of G-protein-coupled receptor internalization using DnaE intein-based assay.

    PubMed

    Lu, Bin; Chen, Linjie; Zhang, Yaping; Shi, Ying; Zhou, Naiming

    2016-01-01

    G-protein-coupled receptors (GPCRs), the largest family of cell surface receptors, are involved in many physiological processes. They represent highly important therapeutic targets for drug discovery. Currently, there are numerous cell-based assays developed for the pharmacological profiling of GPCRs and the identification of novel agonists and antagonists. However, the development of new, faster, easier, and more cost-effective approaches to detect GPCR activity remains highly desirable. β-arrestin-dependent internalization has been demonstrated to be a common mechanism for most GPCRs. Here we describe a novel assay for quantitative analysis of GPCR internalization based on DnaE intein-mediated reconstitution of fragmented Renilla luciferase or Firefly luciferase when activated GPCRs interact with β-arrestin2 or Rab5. Further validation, using functionally divergent GPCRs, showed that EC50 values obtained for the known agonists and antagonists were in close agreement with the results of previous reports. This suggests that this assay is sensitive enough to permit quantification of GPCR internalization. Compared with conventional assays, this novel assay system is cost-effective, rapid, and easy to manipulate. These advantages may allow this assay to be used universally as a functional cell-based system for GPCR characterization and in the screening process of drug discovery. PMID:26928549

  5. Polyunsaturated Branched-Chain Fatty Acid Geranylgeranoic Acid Induces Unfolded Protein Response in Human Hepatoma Cells

    PubMed Central

    Iwao, Chieko; Shidoji, Yoshihiro

    2015-01-01

    The acyclic diterpenoid acid geranylgeranoic acid (GGA) has been reported to induce autophagic cell death in several human hepatoma-derived cell lines; however, the molecular mechanism for this remains unknown. In the present study, several diterpenoids were examined for ability to induce XBP1 splicing and/or lipotoxicity for human hepatoma cell lines. Here we show that three groups of diterpenoids emerged: 1) GGA, 2,3-dihydro GGA and 9-cis retinoic acid induce cell death and XBP1 splicing; 2) all-trans retinoic acid induces XBP1 splicing but little cell death; and 3) phytanic acid, phytenic acid and geranylgeraniol induce neither cell death nor XBP1 splicing. GGA-induced ER stress/ unfolded protein response (UPR) and its lipotoxicity were both blocked by co-treatment with oleic acid. The blocking activity of oleic acid for GGA-induced XBP1 splicing was not attenuated by methylation of oleic acid. These findings strongly suggest that GGA at micromolar concentrations induces the so-called lipid-induced ER stress response/UPR, which is oleate-suppressive, and shows its lipotoxicity in human hepatoma cells. PMID:26186544

  6. The alpha-helical domain of liver fatty acid binding protein is responsible for the diffusion-mediated transfer of fatty acids to phospholipid membranes.

    PubMed

    Córsico, Betina; Liou, Heng Ling; Storch, Judith

    2004-03-30

    Intestinal fatty acid binding protein (IFABP) and liver FABP (LFABP), homologous proteins expressed at high levels in intestinal absorptive cells, employ markedly different mechanisms for the transfer of fatty acids (FAs) to acceptor membranes. Transfer from IFABP occurs during protein-membrane collisional interactions, while for LFABP, transfer occurs by diffusion through the aqueous phase. Earlier, we had shown that the helical domain of IFABP is critical in determining its collisional FA transfer mechanism. In the study presented here, we have engineered a pair of chimeric proteins, one with the "body" (ligand binding domain) of IFABP and the alpha-helical region of LFABP (alphaLbetaIFABP) and the other with the ligand binding pocket of LFABP and the helical domain of IFABP (alphaIbetaLFABP). The objective of this work was to determine whether the change in the alpha-helical domain of each FABP would alter the rate and mechanism of transfer of FA from the chimeric proteins in comparison with those of the wild-type proteins. The fatty acid transfer properties of the FABP chimeras were examined using a fluorescence resonance transfer assay. The results showed a significant modification of the absolute rate of FA transfer from the chimeric proteins compared to that of the wild type, indicating that the slower rate of FA transfer observed for wild-type LFABP relative to that of wild-type IFABP is, in part, determined by the helical domain of the proteins. In addition to these quantitative changes, it was of great interest to observe that the apparent mechanism of FA transfer also changed when the alpha-helical domain was exchanged, with transfer from alphaLbetaIFABP occurring by aqueous diffusion and transfer from alphaIbetaLFABP occurring via protein-membrane collisional interactions. These results demonstrate that the alpha-helical region of LFABP is responsible for its diffusional mechanism of fatty acid transfer to membranes. PMID:15035630

  7. Structure and Function of the Escherichia coli Protein YmgB: A Protein Critical for Biofilm Formation and Acid-resistance

    SciTech Connect

    Lee,J.; Page, R.; Garcia-Contreras, R.; Palermino, J.; Zhang, X.; Doshi, O.; Wood, T.; Peti, W.

    2007-01-01

    The Escherichia coli gene cluster ymgABC was identified in transcriptome studies to have a role in biofilm development and stability. In this study, we showed that YmgB represses biofilm formation in rich medium containing glucose, decreases cellular motility, and protects the cell from acid indicating that YmgB has a major role in acid-resistance in E. coli. Our data show that these phenotypes are potentially mediated through interactions with the important cell signal indole. In addition, gel mobility-shift assays suggest that YmgB may be a non-specific DNA-binding protein. Using nickel-enrichment DNA microarrays, we showed that YmgB binds, either directly or indirectly, via a probable ligand, genes important for biofilm formation. To advance our understanding of the function of YmgB, we used X-ray crystallography to solve the structure of the protein to 1.8 A resolution. YmgB is a biological dimer that is structurally homologous to the E. coli gene regulatory protein Hha, despite having only 5% sequence identity. This supports our DNA microarray data showing that YmgB is a gene regulatory protein. Therefore, this protein, which clearly has a critical role in acid-resistance in E. coli, has been renamed as AriR for regulator of acid resistance influenced by indole.

  8. Use of agar diffusion assay to measure bactericidal activity of alkaline salts of fatty acids against bacteria associated with poultry processing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The agar diffusion assay was used to examine antibacterial activity of alkaline salts of caproic, caprylic, capric, lauric, and myristic acids. A 0.5M concentration of each fatty acid was dissolved in 1.0 M potassium hydroxide (KOH), and pH of the mixtures was adjusted to 10.5 with citric acid. Solu...

  9. Quantifying Protein-Fatty Acid Interactions Using Electrospray Ionization Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Liu, Lan; Kitova, Elena N.; Klassen, John S.

    2011-02-01

    The application of the direct electrospray ionization mass spectrometry (ESI-MS) assay to quantify interactions between bovine β-lactoglobulin (Lg) and a series of fatty acids (FA), CH3(CH2)xCOOH, where x = 6 (caprylic acid, CpA), 8 (capric acid, CA), 10 (lauric acid, LA), 12 (myristic acid, MA), 14 (palmitic acid, PA) and 16 (stearic acid, SA), is described. Control ESI-MS binding measurements performed on the Lg-PA interaction revealed that both the protonated and deprotonated gas phase ions of the (Lg + PA) complex are prone to dissociate in the ion source, which leads to artificially small association constants ( K a ). The addition of imidazole, a stabilizing solution additive, at high concentration (10 mM) increased the relative abundance of (Lg + PA) complex measured by ESI-MS in both positive and negative ion modes. The K a value measured in negative ion mode and using sampling conditions that minimize in-source dissociation is in good agreement with a value determined using a competitive fluorescence assay. The K a values measured by ESI-MS for the Lg interactions with MA and SA are also consistent with values expected based on the fluorescence measurements. However, the K a values measured using optimal sampling conditions in positive ion mode are significantly lower than those measured in negative ion mode for all of the FAs investigated. It is concluded that the protonated gaseous ions of the (Lg + FA) complexes are kinetically less stable than the deprotonated ions. In-source dissociation was significant for the complexes of Lg with the shorter FAs (CpA, CA, and LA) in both modes and, in the case of CpA, no binding could be detected by ESI-MS. The affinities of Lg for CpA, CA, and LA determined using the reference ligand ESI-MS assay, a method for quantifying labile protein-ligand complexes that are prone to in-source dissociation, were found to be in good agreement with reported values.

  10. A novel human T-leukemia virus type 1 cell-to-cell transmission assay permits definition of SU glycoprotein amino acids important for infectivity.

    PubMed Central

    Delamarre, L; Rosenberg, A R; Pique, C; Pham, D; Dokhélar, M C

    1997-01-01

    Human T-leukemia virus type 1 (HTLV-1) envelope glycoproteins play a major role in viral transmission, which in the case of this virus occurs almost exclusively via cell-to-cell contact. Until very recently, the lack of an HTLV-1 infectivity assay precluded the determination of the HTLV-1 protein domains required for infectivity. Here, we describe an assay which allows the quantitative evaluation of HTLV-1 cell-to-cell transmission in a single round of infection. Using this assay, we demonstrate that in this system, cell-to-cell transmission is at least 100 times more efficient than transmission with free viral particles. We have examined 46 surface (SU) glycoprotein mutants in order to define the amino acids of the HTLV-1 SU glycoprotein required for full infectivity. We demonstrate that these amino acids are distributed along the entire length of the SU glycoprotein, including the N-terminus and C-terminus regions, which have not been previously defined as being important for HTLV-1 glycoprotein function. For most of the mutated glycoproteins, the capacity to mediate cell-to-cell transmission is correlated with the ability to induce formation of syncytia. This result indicates that the fusion capacity is the main factor responsible for infectivity mediated by the HTLV-1 SU envelope glycoprotein, as is the case for other retroviral glycoproteins. However, other factors must also intervene, since two of the mutated glycoproteins were correctly fusogenic but could not mediate cell-to-cell transmission. Existence of this phenotype shows that capacity for fusion is not sufficient to confer infectivity, even in cell-to-cell transmission, and could suggest that postfusion events involve the SU. PMID:8985345

  11. Comparison of peptide cocktails and purified protein derivatives for use in the Bovigam assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Currently, the Bovigam™ assay is used as an official complementary test within the bovine tuberculosis eradication program. This assay measures Interferon-gamma (IFN-') produced by lymphocytes in response to specific antigens. The objectives of the present study were to compare in vitro antigen prep...

  12. Development and validation of a robust and sensitive assay for the discovery of selective inhibitors for serine/threonine protein phosphatases PP1α (PPP1C) and PP5 (PPP5C).

    PubMed

    Swingle, Mark R; Honkanen, Richard E

    2014-10-01

    Protein phosphatase types 1 α (PP1α/PPP1C) and 5 (PP5/PPP5C) are members of the PPP family of serine/threonine protein phosphatases. PP1 and PP5 share a common catalytic mechanism, and several natural compounds, including okadaic acid, microcystin, and cantharidin, act as strong inhibitors of both enzymes. However, to date there have been no reports of compounds that can selectively inhibit PP1 or PP5, and specific or highly selective inhibitors for either PP1 or PP5 are greatly desired by both the research and pharmaceutical communities. Here we describe the development and optimization of a sensitive and robust (representative PP5C assay data: Z'=0.93; representative PP1Cα assay data: Z'=0.90) fluorescent phosphatase assay that can be used to simultaneously screen chemical libraries and natural product extracts for the presence of catalytic inhibitors of PP1 and PP5. PMID:25383722

  13. Development and Validation of a Robust and Sensitive Assay for the Discovery of Selective Inhibitors for Serine/Threonine Protein Phosphatases PP1α (PPP1C) and PP5 (PPP5C)

    PubMed Central

    Swingle, Mark R.

    2014-01-01

    Abstract Protein phosphatase types 1 α (PP1α/PPP1C) and 5 (PP5/PPP5C) are members of the PPP family of serine/threonine protein phosphatases. PP1 and PP5 share a common catalytic mechanism, and several natural compounds, including okadaic acid, microcystin, and cantharidin, act as strong inhibitors of both enzymes. However, to date there have been no reports of compounds that can selectively inhibit PP1 or PP5, and specific or highly selective inhibitors for either PP1 or PP5 are greatly desired by both the research and pharmaceutical communities. Here we describe the development and optimization of a sensitive and robust (representative PP5C assay data: Z′=0.93; representative PP1Cα assay data: Z′=0.90) fluorescent phosphatase assay that can be used to simultaneously screen chemical libraries and natural product extracts for the presence of catalytic inhibitors of PP1 and PP5. PMID:25383722

  14. Development of a Highly Automated and Multiplexed Targeted Proteome Pipeline and Assay for 112 Rat Brain Synaptic Proteins

    PubMed Central

    Colangelo, Christopher M.; Ivosev, Gordana; Chung, Lisa; Abbott, Thomas; Shifman, Mark; Sakaue, Fumika; Cox, David; Kitchen, Rob R.; Burton, Lyle; Tate, Stephen A; Gulcicek, Erol; Bonner, Ron; Rinehart, Jesse; Nairn, Angus C.; Williams, Kenneth R.

    2015-01-01

    We present a comprehensive workflow for large scale (>1000 transitions/run) label-free LC-MRM proteome assays. Innovations include automated MRM transition selection, intelligent retention time scheduling (xMRM) that improves Signal/Noise by >2-fold, and automatic peak modeling. Improvements to data analysis include a novel Q/C metric, Normalized Group Area Ratio (NGAR), MLR normalization, weighted regression analysis, and data dissemination through the Yale Protein Expression Database. As a proof of principle we developed a robust 90 minute LC-MRM assay for Mouse/Rat Post-Synaptic Density (PSD) fractions which resulted in the routine quantification of 337 peptides from 112 proteins based on 15 observations per protein. Parallel analyses with stable isotope dilution peptide standards (SIS), demonstrate very high correlation in retention time (1.0) and protein fold change (0.94) between the label-free and SIS analyses. Overall, our first method achieved a technical CV of 11.4% with >97.5% of the 1697 transitions being quantified without user intervention, resulting in a highly efficient, robust, and single injection LC-MRM assay. PMID:25476245

  15. Re-exploration of the Codon Context Effect on Amber Codon-Guided Incorporation of Noncanonical Amino Acids in Escherichia coli by the Blue-White Screening Assay.

    PubMed

    Xu, Huan; Wang, Yan; Lu, Jiaqi; Zhang, Bo; Zhang, Ziwei; Si, Longlong; Wu, Ling; Yao, Tianzhuo; Zhang, Chuanling; Xiao, Sulong; Zhang, Lihe; Xia, Qing; Zhou, Demin

    2016-07-01

    The effect of codon context on amber codon-guided incorporation of noncanonical amino acids (NAAs) has been previously examined by antibiotic selection. Here, we re-explored this effect by screening a library in which three nucleotides upstream and downstream of the amber codon were randomised, and inserted within the lacZ-α gene. Thousands of clones were obtained and distinguished by the depth of blue colour upon exposure to X-gal. Large-scale sequencing revealed remarkable preferences in nucleotides downstream of the amber codon, and moderate preferences for upstream nucleotides. Nucleotide preference was quantified by a dual-luciferase assay, which verified that the optimum context for NAA incorporation, AATTAGACT, was applicable to different proteins. Our work provides a general guide for engineering amber codons into genes of interest in bacteria. PMID:27028123

  16. Hyperdimensional analysis of amino acid pair distributions in proteins.

    PubMed

    Henriksen, Svend B; Mortensen, Rasmus J; Geertz-Hansen, Henrik M; Neves-Petersen, Maria Teresa; Arnason, Omar; Söring, Jón; Petersen, Steffen B

    2011-01-01

    Our manuscript presents a novel approach to protein structure analyses. We have organized an 8-dimensional data cube with protein 3D-structural information from 8706 high-resolution non-redundant protein-chains with the aim of identifying packing rules at the amino acid pair level. The cube contains information about amino acid type, solvent accessibility, spatial and sequence distance, secondary structure and sequence length. We are able to pose structural queries to the data cube using program ProPack. The response is a 1, 2 or 3D graph. Whereas the response is of a statistical nature, the user can obtain an instant list of all PDB-structures where such pair is found. The user may select a particular structure, which is displayed highlighting the pair in question. The user may pose millions of different queries and for each one he will receive the answer in a few seconds. In order to demonstrate the capabilities of the data cube as well as the programs, we have selected well known structural features, disulphide bridges and salt bridges, where we illustrate how the queries are posed, and how answers are given. Motifs involving cysteines such as disulphide bridges, zinc-fingers and iron-sulfur clusters are clearly identified and differentiated. ProPack also reveals that whereas pairs of Lys residues virtually never appear in close spatial proximity, pairs of Arg are abundant and appear at close spatial distance, contrasting the belief that electrostatic repulsion would prevent this juxtaposition and that Arg-Lys is perceived as a conservative mutation. The presented programs can find and visualize novel packing preferences in proteins structures allowing the user to unravel correlations between pairs of amino acids. The new tools allow the user to view statistical information and visualize instantly the structures that underpin the statistical information, which is far from trivial with most other SW tools for protein structure analysis. PMID:22174733

  17. Development of a suspension array assay in multiplex for simultaneous measurement of serum levels of four eosinophil granule proteins

    PubMed Central

    Makiya, Michelle A.; Herrick, Jesica A.; Khoury, Paneez; Prussin, Calman P.; Nutman, Thomas B.; Klion, Amy D.

    2014-01-01

    The concentrations of major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO) have been associated with eosinophilic disease severity. Whereas a variety of techniques have been used to measure individual eosinophil granule protein concentration, none of these methods efficiently measures MBP, ECP, EDN and EPO simultaneously. A multiplex suspension array system was developed to simultaneously measure the concentration of MBP, ECP, EDN and EPO in serum. The assay showed excellent inter- and intra-assay reliability, and serum levels of MBP, ECP and EDN from eosinophilic subjects analyzed by ELISA and multiplex were highly correlated (r = 0.8579; P < 0.0001, r = 0.6356; P = 0.0006 and r = 0.8600; P < 0.0001, respectively, Spearman rank correlation). Moreover, the multiplex assay required 500-fold less serum than a single ELISA to achieve comparable sensitivity. Absolute eosinophil count and eosinophil surface expression of the activation marker, CD69, were significantly correlated with concentrations of MBP, EDN and EPO, but not ECP, in serum from eosinophilic subjects. Furthermore, subjects with eosinophilic gastrointestinal disorder and normal peripheral absolute eosinophil counts (< 0.5 × 109/L) had significantly increased concentrations of MBP (P < 0.0001), ECP (P < 0.0001), EDN (P = 0.0001) and EPO (P < 0.0001) compared to normal donors. In summary, the eosinophil granule protein multiplex assay provides a rapid and reliable way to measure eosinophil granule protein levels and should prove useful in assessing patterns of degranulation in patients with eosinophilic disorders. PMID:24914990

  18. Quantitation of 5-Methyltetrahydrofolic Acid in Dried Blood Spots and Dried Plasma Spots by Stable Isotope Dilution Assays

    PubMed Central

    Kopp, Markus; Rychlik, Michael

    2015-01-01

    Because of minimal data available on folate analysis in dried matrix spots (DMSs), we combined the advantages of stable isotope dilution assays followed by LC-MS/MS analysis with DMS sampling to develop a reliable method for the quantitation of plasma 5-methyltetrahydrofolic acid in dried blood spots (DBSs) and dried plasma spots (DPSs) as well as for the quantitation of whole blood 5-methyltetrahydrofolic acid in DBSs. We focused on two diagnostically conclusive parameters exhibited by the plasma and whole blood 5-methyltetrahydrofolic acid levels that reflect both temporary and long-term folate status. The method is performed using the [2H4]-labeled isotopologue of the vitamin as the internal standard, and three steps are required for the extraction procedure. Elution of the punched out matrix spots was performed using stabilization buffer including Triton X-100 in a standardized ultrasonication treatment followed by enzymatic digestion (whole blood only) and solid-phase extraction with SAX cartridges. This method is sensitive enough to quantify 27 nmol/L whole blood 5-methyltetrahydrofolic acid in DBSs and 6.3 and 4.4 nmol/L plasma 5-methyltetrahydrofolic acid in DBSs and DPSs, respectively. The unprecedented accurate quantification of plasma 5-methyltetrahydrofolic acid in DBSs was achieved by thermal treatment prior to ultrasonication, inhibiting plasma conjugase activity. Mass screenings are more feasible and easier to facilitate for this method in terms of sample collection and storage compared with conventional clinical sampling for the assessment of folate status. PMID:26605791

  19. Sensitive assay, based on hydroxy fatty acids from lipopolysaccharide lipid A, for Gram-negative bacteria in sediments.

    PubMed Central

    Parker, J H; Smith, G A; Fredrickson, H L; Vestal, J R; White, D C

    1982-01-01

    Biochemical measures have provided insight into the biomass and community structure of sedimentary microbiota without the requirement of selection by growth or quantitative removal from the sediment grains. This study used the assay of the hydroxy fatty acids released from the lipid A of the lipopolysaccharide in sediments to provide an estimate of the gram-negative bacteria. The method was sensitive to picomolar amounts of hydroxy fatty acids. The recovery of lipopolysaccharide hydroxy fatty acids from organisms added to sediments was quantitative. The lipids were extracted from the sediments with single-phase chloroform-methanol extraction. The lipid-extraction residue was hydrolyzed in 1 N HCl, and the hydroxy fatty acids of the lipopolysaccharide were recovered in chloroform for analysis by gas-liquid chromatography. This method proved to be about fivefold more sensitive than the classical phenol-water or trichloroacetic acid methods when applied to marine sediments. By examination of the patterns of hydroxy fatty acids, it was also possible to help define the community structure of the sedimentary gram-negative bacteria. PMID:6817712

  20. Validation and application of a liquid-chromatographic/enzymatic assay for individual bile acids in the serum of rats.

    PubMed

    Thompson, M B; Blair, P C; Morris, R W; Neptun, D A; Deyo, D F; Popp, J A

    1987-10-01

    A liquid-chromatographic technique with a post-column enzymatic reaction and fluorescence detection was validated for analysis of individual bile acids in the serum of rats. Extraction recoveries averaged 91.1% (SD 6.9%) for all bile acids. The assay was sensitive (minimum detection of 16.8 pmol per 100-microL injection), linear (r greater than 0.999 for concentrations ranging between 45 and 112,500 pmol per 100-microL injection), and reproducible (mean CVs for three different concentrations of standards and a serum pool ranged from 4.4% to 12.2%). In rats treated for three days with either neomycin, carbon tetrachloride, alpha-naphthylisothiocyanate, or total bile-duct ligation (five animals per group), total concentrations of bile acids were significantly increased (P less than 0.004). Concentrations of 16 of 17 individual bile acids differed significantly between groups (P less than 0.04). Examination of the relative concentrations (percent of total) of individual bile acids by canonical discriminant analysis placed each animal into the appropriate treatment or control group. Use of this technique in toxicological studies can help detect and identify specific types of disruptions in the enterohepatic circulation of bile acids. PMID:3665040

  1. Fatty acids exacerbate tubulointerstitial injury in protein-overload proteinuria.

    PubMed

    Thomas, Mark E; Harris, Kevin P G; Walls, John; Furness, Peter N; Brunskill, Nigel J

    2002-10-01

    The role of the albumin-carried fatty acids in the induction of tubulointerstitial injury was studied in protein-overload proteinuria. Rats were injected with fatty acid-carrying BSA [FA(+)BSA], fatty acid-depleted BSA [FA(-)BSA], or saline. Macrophage infiltration was measured by immunohistochemical staining, apoptotic cells were detected by in situ end labeling, and proliferating cells were identified by in situ hybridization for histone mRNA. Macrophage infiltration was significantly greater in the FA(+)BSA group than in the FA(-)BSA and saline groups. The infiltrate was largely restricted to the outer cortex. Apoptosis was greater in the FA(+)BSA group than in the FA(-)BSA and saline groups. Compared with the saline group, apoptosis was significantly increased in the FA(+)BSA group but not in the FA(-)BSA group. Cortical cells proliferated significantly more in the FA(+)BSA and FA(-)BSA groups than in the saline group. FA(+)BSA is therefore a more potent inducer of macrophage infiltration and cell death than FA(-)BSA. The fatty acids carried on albumin may be the chief instigators of tubulointerstitial injury in protein-overload proteinuria. PMID:12217854

  2. The biological activities of protein/oleic acid complexes reside in the fatty acid.

    PubMed

    Fontana, Angelo; Spolaore, Barbara; Polverino de Laureto, Patrizia

    2013-06-01

    A complex formed by human α-lactalbumin (α-LA) and oleic acid (OA), named HAMLET, has been shown to have an apoptotic activity leading to the selective death of tumor cells. In numerous publications it has been reported that in the complex α-LA is monomeric and adopts a partly folded or "molten globule" state, leading to the idea that partly folded proteins can have "beneficial effects". The protein/OA molar ratio initially has been reported to be 1:1, while recent data have indicated that the OA-complex is given by an oligomeric protein capable of binding numerous OA molecules per protein monomer. Proteolytic fragments of α-LA, as well as other proteins unrelated to α-LA, can form OA-complexes with biological activities similar to those of HAMLET, thus indicating that a generic protein can form a cytotoxic complex under suitable experimental conditions. Moreover, even the selective tumoricidal activity of HAMLET-like complexes has been questioned. There is recent evidence that the biological activity of long chain unsaturated fatty acids, including OA, can be ascribed to their effect of perturbing the structure of biological membranes and consequently the function of membrane-bound proteins. In general, it has been observed that the cytotoxic effects exerted by HAMLET-like complexes are similar to those reported for OA alone. Overall, these findings can be interpreted by considering that the protein moiety does not have a toxic effect on its own, but merely acts as a solubilising agent for the inherently toxic fatty acid. PMID:23499846

  3. Bacterial interactomes: Interacting protein partners share similar function and are validated in independent assays more frequently than previously reported.

    DOE PAGESBeta

    Shatsky, Maxim; Allen, Simon; Gold, Barbara; Liu, Nancy L.; Juba, Thomas R.; Elias, Dwayne A; Reveco, Sonia A.; Prathapam, Ramadevi; He, Jennifer; Yang, Wenhong; et al

    2016-05-01

    Numerous affinity purification – mass-spectrometry (AP-MS) and yeast two hybrid (Y2H) screens have each defined thousands of pairwise protein-protein interactions (PPIs), most between functionally unrelated proteins. The accuracy of these networks, however, is under debate. Here we present an AP-MS survey of the bacterium Desulfovibrio vulgaris together with a critical reanalysis of nine published bacterial Y2H and AP-MS screens. We have identified 459 high confidence PPIs from D. vulgaris and 391 from Escherichia coli. Compared to the nine published interactomes, our two networks are smaller; are much less highly connected; have significantly lower false discovery rates; and are much moremore » enriched in protein pairs that are encoded in the same operon, have similar functions, and are reproducibly detected in other physical interaction assays. Lastly, our work establishes more stringent benchmarks for the properties of protein interactomes and suggests that bona fide PPIs much more frequently involve protein partners that are annotated with similar functions or that can be validated in independent assays than earlier studies suggested.« less

  4. Bacterial Interactomes: Interacting Protein Partners Share Similar Function and Are Validated in Independent Assays More Frequently Than Previously Reported.

    PubMed

    Shatsky, Maxim; Allen, Simon; Gold, Barbara L; Liu, Nancy L; Juba, Thomas R; Reveco, Sonia A; Elias, Dwayne A; Prathapam, Ramadevi; He, Jennifer; Yang, Wenhong; Szakal, Evelin D; Liu, Haichuan; Singer, Mary E; Geller, Jil T; Lam, Bonita R; Saini, Avneesh; Trotter, Valentine V; Hall, Steven C; Fisher, Susan J; Brenner, Steven E; Chhabra, Swapnil R; Hazen, Terry C; Wall, Judy D; Witkowska, H Ewa; Biggin, Mark D; Chandonia, John-Marc; Butland, Gareth

    2016-05-01

    Numerous affinity purification-mass spectrometry (AP-MS) and yeast two-hybrid screens have each defined thousands of pairwise protein-protein interactions (PPIs), most of which are between functionally unrelated proteins. The accuracy of these networks, however, is under debate. Here, we present an AP-MS survey of the bacterium Desulfovibrio vulgaris together with a critical reanalysis of nine published bacterial yeast two-hybrid and AP-MS screens. We have identified 459 high confidence PPIs from D. vulgaris and 391 from Escherichia coli Compared with the nine published interactomes, our two networks are smaller, are much less highly connected, and have significantly lower false discovery rates. In addition, our interactomes are much more enriched in protein pairs that are encoded in the same operon, have similar functions, and are reproducibly detected in other physical interaction assays than the pairs reported in prior studies. Our work establishes more stringent benchmarks for the properties of protein interactomes and suggests that bona fide PPIs much more frequently involve protein partners that are annotated with similar functions or that can be validated in independent assays than earlier studies suggested. PMID:26873250

  5. Ultrasensitive biotin assay of a noncompetitive format in a homogeneous solution based on resonance energy transfer induced by a protein-protein interaction.

    PubMed

    Ikeda, Tomohiro; Miyao, Hiroki; Sueda, Shinji

    2014-06-17

    Biotin is a water-soluble vitamin serving as a cofactor for several metabolic enzymes and plays crucial roles in every living cell. In the present study, we describe a noncompetitive assay for determination of biotin in a homogeneous solution. Our assay is based on a biotinylation reaction from archaeon Sulfolobus tokodaii. S. tokodaii biotinylation has a unique property that biotin protein ligase (BPL) forms a stable complex with its biotinylated substrate protein (BCCP). Determination of biotin was performed by monitoring the complexation reaction between BPL and BCCP through biotinylation, based on luminescence resonance energy transfer (LRET) from a Tb(3+) complex to fluorescein, where BPL and BCCP were labeled with a Tb(3+) complex and fluorescein, respectively. Our assay allows for ultrasensitive detection of biotin with a detection limit of approximately 1 pM (or 0.2 fmol in a 0.2 mL sample volume) by a simple procedure without use of radioactive materials or enzymatic signal amplification. In addition, owing to its noncompetitive format, our assay has a very wide measurement range of at least 3 orders of magnitude. Our assay is also beneficial as a model system for interaction analysis based on LRET. PMID:24824306

  6. Decarboxylation of malonyl-(acyl carrier protein) by 3-oxoacyl-(acyl carrier protein) synthases in plant fatty acid biosynthesis.

    PubMed Central

    Winter, E; Brummel, M; Schuch, R; Spener, F

    1997-01-01

    In order to identify regulatory steps in fatty acid biosynthesis, the influence of intermediate 3-oxoacyl-(acyl carrier proteins) (3-oxoacyl-ACPs) and end-product acyl-ACPs of the fatty acid synthase reaction on the condensation reaction was investigated in vitro, using total fatty acid synthase preparations and purified 3-oxoacyl-ACP synthases (KASs; EC 2.3.1.41) from Cuphea lanceolata seeds. KAS I and II in the fatty acid synthase preparations were assayed for the elongation of octanoyl- and hexadecanoyl-ACP respectively, and the accumulation of the corresponding condensation product 3-oxoacyl-ACP was studied by modulating the content of the reducing equivalentS NADH and NADPH. Complete omission of reducing equivalents resulted with either KAS in the abnormal synthesis of acetyl-ACP from malonyl-ACP by a decarboxylation reaction. Supplementation with NADPH or NADH, separately or in combination with recombinant 3-oxoacyl-ACP reductase (EC 1.1.1.100), led to a decrease in the amount of acetyl-ACP and a simultaneous increase in elongation products. This demonstrates that the accumulation of 3-oxoacyl-ACP inhibits the condensation reaction on the one hand, and induces the decarboxylation of malonyl-ACP on the other. By carrying out similar experiments with purified enzymes, this decarboxylation was attributed to the action of KAS. Our data point to a regulatory mechanism for the degradation of malonyl-ACP in plants which is activated by the accumulation of the fatty acid synthase intermediate 3-oxoacyl-ACP. PMID:9020860

  7. Screening metagenomic data for viruses using the E-Probe Diagnostic Nucleic Acid Assay (EDNA)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are many plant pathogen-specific diagnostic assays, based on PCR and immune-detection. However, the ability to test for large numbers of pathogens simultaneously is lacking. Next generation sequencing (NGS) allows one to detect all organisms within a given sample, but has computational limitat...

  8. A functional assay to measure postsynaptic gamma-aminobutyric acidB responses in cultured spinal cord neurons: Heterologous regulation of the same K+ channel

    SciTech Connect

    Kamatchi, G.L.; Ticku, M.K. )

    1991-02-01

    The stimulation of postsynaptic gamma-aminobutyric acid (GABA)B receptors leads to slow inhibitory postsynaptic potentials due to the influx of K(+)-ions. This was studied biochemically, in vitro in mammalian cultured spinal cord neurons by using 86Rb as a substitute for K+. (-)-Baclofen, a GABAB receptor agonist, produced a concentration-dependent increase in the 86Rb-influx. This effect was stereospecific and blocked by GABAB receptor antagonists like CGP 35 348 (3-aminopropyl-diethoxymethyl-phosphonic acid) and phaclofen. Apart from the GABAB receptors, both adenosine via adenosine1 receptors and 5-hydroxytryptamine (5-HT) via 5-HT1 alpha agonists also increased the 86Rb-influx. These agonists failed to show any additivity between them when they were combined in their maximal concentration. In addition, their effect was antagonized specifically by their respective antagonists without influencing the others. These findings suggest the presence of GABAB, adenosine1 and 5-HT1 alpha receptors in the cultured spinal cord neurons, which exhibit a heterologous regulation of the same K(+)-channel. The effect of these agonists were antagonized by phorbol 12,13-didecanoate, an activator of protein kinase C, and pretreatment with pertussis toxin. This suggests that these agonists by acting on their own receptors converge on the same K(+)-channel through the Gi/Go proteins. In summary, we have developed a biochemical functional assay for studying and characterizing GABAB synaptic pharmacology in vitro, using spinal cord neurons.

  9. The acid tolerance response of Salmonella typhimurium involves transient synthesis of key acid shock proteins.

    PubMed Central

    Foster, J W

    1993-01-01

    Although Salmonella typhimurium prefers neutral-pH environments, it can adapt to survive conditions of severe low-pH stress (pH 3.3). The process, termed the acid tolerance response (ATR), includes two distinct stages. The first stage, called pre-acid shock, is induced at pH 5.8 and involves the production of an inducible pH homeostasis system functional at external pH values below 4.0. The second stage occurs following an acid shock shift to pH 4.5 or below and is called the post-acid shock stage. During this stage of the ATR, 43 acid shock proteins (ASPs) are synthesized. The present data reveal that several ASPs important for pH 3.3 acid tolerance are only transiently produced. Their disappearance after 30 to 40 min of pH 4.4 acid shock coincides with an inability to survive subsequent pH 3.3 acid challenge. Clearly, an essential feature of inducible acid tolerance is an ability to synthesize these key ASPs. The pre-acid shock stage, with its inducible pH homeostasis system, offers the cell an enhanced ability to synthesize ASPs following rapid shifts to conditions below pH 4.0, an external pH that normally prevents ASP synthesis. The data also address possible signals for ASP synthesis. The inducing signal for 22 ASPs appears to be internal acidification, while external pH serves to induce 13 others. Of the 14 transient ASPs, 10 are induced in response to changes in internal pH. Mutations in the fur (ferric uptake regulator) locus that produce an Atr- acid-sensitive phenotype also eliminate induction of six transiently induced ASPs. Images PMID:8458840

  10. High-throughput and functional SNP detection assays for oleic and linolenic acids in soybean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean is a primary source of vegetable oil, accounting for 53% of the total vegetable oil consumption in the USA in 2013. Soybean oil with high oleic acid and low linolenic acid content is desired, because it not only improves the oxidative stability of the oil, but also reduces the amount of unde...

  11. [Identification of the protein partners of the human nucleolar protein SURF6 in HeLa cells by GST pull-down assay].

    PubMed

    Kordiukova, M Iu; Polzikov, M A; Shishova, K V; Zatsepina, O V

    2014-01-01

    The eukaryotic proteins comprising the SURF6 protein family are evolutionary conservative and housekeeping proteins however, functional roles of human SURF6 have not been studied so far. To shed light to this question in the present work we applied GST pull-down assay and used two proteins fused with GST, namely human GST-SURF6 and the conservative C-terminal domain of mouse Surf6 that has 85% homology with the C-terminus of the human SURF6 conservative domain (GST-Surf6-dom), to identify SURF6-interacting proteins in human HeLa cells. The results obtained showed that GST-SURF6 interacts with several key nucleolar RNA processing factors (B23/nucleophosmin, nucleolin, EBP2), and also with the specific cofactor of RNA polymerase I, protein UBE These results are the first experimental evidences in favor of participation of the human SURF6 protein in ribosome biogenesis, including transcription of rDNA and processing of rRNAs. The same results were obtained, when GST-Surf6-dom was used to pull-down proteins in HeLa cells. In addition, the panel of the GST-Surf6-dom protein partners, which were identified by mass-spectrometry, points to putative interactions of human SURF6 with a number of nuclear and nucleolar, proteins of other functional groups, i.e. to the protein plurifunctionality. PMID:25898752

  12. The Extended ToxTracker Assay Discriminates Between Induction of DNA Damage, Oxidative Stress, and Protein Misfolding.

    PubMed

    Hendriks, Giel; Derr, Remco S; Misovic, Branislav; Morolli, Bruno; Calléja, Fabienne M G R; Vrieling, Harry

    2016-03-01

    Chemical exposure of cells may damage biomolecules, cellular structures, and organelles thereby jeopardizing cellular homeostasis. A multitude of defense mechanisms have evolved that can recognize specific types of damaged molecules and will initiate distinct cellular programs aiming to remove the damage inflicted and prevent cellular havoc. As a consequence, quantitative assessment of the activity of the cellular stress responses may serve as a sensitive reporter for the induction of specific types of damage. We have previously developed the ToxTracker assay, a mammalian stem cell-based genotoxicity assay employing two green fluorescent protein reporters specific for DNA damage and oxidative stress. We have now expanded the ToxTracker assay with an additional four reporter cell lines to include monitoring of additional stress signaling pathways. This panel of six green fluorescent protein reporters is able to discriminate between different primary reactivity of chemicals being their ability to react with DNA and block DNA replication, induce oxidative stress, activate the unfolded protein response, or cause a general P53-dependent cellular stress response. Extensive validation using the compound library suggested by the European Centre for the Validation of Alternative Methods (ECVAM) and a large panel of reference chemicals shows that the ToxTracker assay has an outstanding sensitivity and specificity. In addition, we developed Toxplot, a dedicated software tool for automated data analysis and graphical representation of the test results. Rapid and reliable identification by the ToxTracker assay of specific biological reactivity can significantly improve in vitro human hazard assessment of chemicals. PMID:26719371

  13. A 32,000-molecular weight protein from bovine placenta with placental lactogen-like activity in radioreceptor assays

    SciTech Connect

    Eakle, K.A.; Arima, Y.; Swanson, P.; Grimek, H.; Bremel, R.D.

    1982-05-01

    Considerable discrepancies exist in the literature concerning the size and activity of bovine placental lactogen. Our bovine placental lactogen purification preparations were 20% as active as bovine PRL (bPRL) on a per weight basis when compared to bPRL in a lactogenic radioreceptor assay. To identify the active component in these preparations, the proteins were radioiodinated and bound to membrane receptors in the presence and absence of competing hormones, bPRL, and bovine GH (bGH). After centrifugation, membrane pellets were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gels were autoradiographed. Only one radioiodinated protein band was present. This protein was displaced in the presence of competing hormone and comigrated with a major component of the purification preparations. In radioreceptor assays the active component was as active as bPRL and bGH. From the migration of protein standards included with the radioiodinated purification preparations in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we estimate that the protein is 32,000 mol wt--about 10,000 mol wt larger than placental lactogens isolated in other species. The possibility that the active molecule was a precursor protein was investigated by examining proteins secreted by bovine placenta tissue cultures. The binding activity in these secretions, as well as in the purification preparations, eluted between ovalbumin (43,000 mol wt) and bPRL (22,000 mol wt) under nondenaturing conditions using high performance gel filtration chromatography. Analysis of this secreted protein, also by binding to membrane receptors, showed that the protein had the same molecular weight as that isolated from the purification preparations and was specifically displaced by the same hormones.

  14. A 32,000-molecular weight protein from bovine placenta with placental lactogen-like activity in radioreceptor assays.

    PubMed

    Eakle, K A; Arima, Y; Swanson, P; Grimek, H; Bremel, R D

    1982-05-01

    Considerable discrepancies exist in the literature concerning the size and activity of bovine placental lactogen. Our bovine placental lactogen purification preparations were 20% as active as bovine PRL (bPRL) on a per weight basis when compared to bPRL in a lactogenic radioreceptor assay. To identify the active component in these preparations, the proteins were radioiodinated and bound to membrane receptors in the presence and absence of competing hormones, bPRL, and bovine GH (bGH). After centrifugation, membrane pellets were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gels were autoradiographed. Only one radioiodinated protein band was present. This protein was displaced in the presence of competing hormone and comigrated which a major component of the purification preparations. In radioreceptor assays the active component was as active as bPRL and bGH. From the migration of protein standards included with the radioiodinated purification preparations in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we estimate that the protein is 32,000 mol wt--about 10,000 mol wt larger than placental lactogens isolated in other species. The possibility that the active molecule was a precursor protein was investigated by examining proteins secreted by bovine placenta tissue cultures. The binding activity in these secretions, as well as in the purification preparations, eluted between ovalbumin (43,000 mol wt) and bPRL (22,000 mol wt) under nondenaturing conditions using high performance gel filtration chromatography. Analysis of this secreted protein, also by binding to membrane receptors, showed that the protein had the same molecular weight as that isolated from the purification preparations and was specifically displaced by the same hormones. PMID:7075536

  15. Water-soluble gold nanoclusters prepared by protein-ligand interaction as fluorescent probe for real-time assay of pyrophosphatase activity.

    PubMed

    Deng, Hao-Hua; Wang, Fei-Fei; Shi, Xiao-Qiong; Peng, Hua-Ping; Liu, Ai-Lin; Xia, Xing-Hua; Chen, Wei

    2016-09-15

    This paper reports a new and facile method for the synthesis of water-soluble thiolate-protected AuNCs via protein-ligand interaction. Using 3-mercaptopropionic acid (MPA) as a model ligand and bovine serum albumin (BSA) as a model protein, water-soluble AuNCs (BSA/MPA-AuNCs) with intense orange-yellow fluorescent emission (quantum yield=16%) are obtained. Results show that AuNCs produced with this method have hydrophobic interactions with BSA. The synthetic strategy is then successfully extended to produce water-soluble AuNCs protected by other thiolates. Moreover, a sensitive and eco-friendly sensing system is established for detection of the activity of inorganic pyrophosphatase (PPase), which relies on the selective coordination of Fe(3+)with BSA/MPA-AuNCs, the higher affinity between pyrophosphate (PPi) and Fe(3+), and the hydrolysis of PPi by PPase. A good linearity between the fluorescence intensity and PPase activity within the range from 0.1 to 3U/L is found, with a detection limit down to 0.07U/L. Additionally, the fluorescent assay developed here is utilized to assay the PPase activity in real biological samples and as well as to evaluate PPase inhibitor, illustrating the great potential for biological analysis. PMID:27093483

  16. Quantitation, with a new assay, and Theiler's virus capsid protein in the central nervous system of mice

    SciTech Connect

    Cash, E.; Chamorro, M.; Brahic, M.

    1986-11-01

    The authors developed a quantitative assay for antigens at the single-cell level. Tissue sections were reacted with (i) a primarily antibody , (ii) a biotinylated secondary antibody, or (iii) /sup 35/S-streptavidin. Binding of streptavidin to cells was quantitated by microscopic autoradiography. They showed that the number of autoradiographic grains was proportional to the amount of antigen per cell. With this assay, they studied the synthesis of Theiler's virus capsid proteins VP1, VP2, and VP3 in permissive BHK cells grown in vitro and in mouse central nervous system (CNS) cells during a persistent infection. They found that synthesis of the three capsid proteins was restricted in mouse CNS cells. Restricted virus replication could play a major role in the persistence of Theiler's virus in mouse CNS cells.

  17. Development of a Medium-Throughput Targeted LCMS Assay to Detect Endogenous Cellular Levels of Malonyl-CoA to Screen Fatty Acid Synthase Inhibitors.

    PubMed

    Hopcroft, Philip J; Fisher, David I

    2016-02-01

    The fatty acid synthase (FAS) enzyme in mammalian cells is a large multidomain protein responsible for de novo synthesis of fatty acids. The steps catalyzed by FAS involve the condensation of acetyl-CoA and malonyl-CoA moieties in the presence of NADPH until palmitate is formed. Inhibition of FAS causes an accumulation of intracellular malonyl-CoA, as this metabolite is essentially committed to fatty acid synthesis once formed. Detection of intracellular metabolites for screening can be problematic due to a lack of appropriate tools, but here we describe a targeted liquid chromatography-mass spectroscopy (LCMS) method to directly measure endogenous levels of malonyl-CoA to drive a drug development structure-activity relationship (SAR) screening cascade. Our process involves preparation of samples at 96-well scale, normalization postpermeabilization via use of a whole-well imaging platform, and the LCMS detection methodology. The assay is amenable to multiplexing cellular endpoints, has a typical Z' of >0.6, and has high reproducibility of EC50 values. PMID:26586251

  18. Isolation and immunological characterization of fatty acid binding protein isoforms from Fasciola hepatica.

    PubMed

    Espino, A M; Rodríguez Medina, J R; Hillyer, G V

    2001-10-01

    A combination of molecular sieving chromatography and 2-step preparative isoelectric focusing showed that native Fh12, a fatty acid-binding protein isolated from Fasciola hepatica adult worms, is a protein complex of at least 8 isoforms with identical molecular mass but different isoelectric points. Using enzyme-linked immunosorbent assay (ELISA) and inhibition ELISA assays, immunological differences were observed between native (nFh12) and a recombinant molecule denoted rFh15 that was obtained after screening a cDNA library from F. hepatica adult worms with an anti-Fh12 monospecific polyclonal antibody. It was confirmed that in infected rabbits, antibodies to nFh12 appear by the second week postinfection, whereas antibodies to rFh15 appear much later, by 6 wk postinfection. Four acidic forms (Fh12(1-4)) showed more immunological identity with rFh15 than with nFh12, based on the observation that they inhibited ELISA activity by nearly 50% when they were added to the anti-rFh15 polyclonal antibody at 20 microg/ml of protein concentration. Moreover, the Fh12(1-4) isoforms were poorly reactive with sera from rabbits 2-4 wk postinfection. However, the 2 acidic forms, denoted Fh12(5) and Fh12(6), and the neutral/basic forms, denoted Fh12(7) and Fh12(8), showed more immunological identity with the native nFh12 molecule than with the recombinant rFh15 because they were highly reactive with sera of rabbits with early 2-wk F. hepatica infection and inhibited ELISA activity nearly 50% when they were quantitatively added to the anti-nFh12 polyclonal antibody. These results suggest that rFh15 could be one of the acidic forms of nFh12, and that it, in fact, may be one of the less immunogenic or immunoprotective members, or both, of the nFh12 protein complex. PMID:11695360

  19. Novel molecular beacon DNA probes for protein-nucleic acid interaction studies

    NASA Astrophysics Data System (ADS)

    Li, Jianwei J.; Perlette, John; Fang, Xiaohong; Kelley, Shannon; Tan, Weihong

    2000-03-01

    We report a novel approach to study protein-nucleic acid interactions by using molecular beacons (MBs). Molecular beacons are hairpin-shaped DNA oligonucleotide probes labeled with a fluorophore and a quencher, and can report the presence of target DNA/RNA sequences. MBs can also report the existence of single-stranded DNA binding proteins (SSB) through non-sequence specific binding. The interaction between SSB and MB has resulted in significant fluorescence restoration of the MB. The fluorescence enhancement brought by SSB and by complementary DNA is very comparable. The molar ratio of the binding between SSB and the molecular beacon is 1:1 with a binding constant of 2 X 107 M-1. Using the MB-SSB binding, we are able to determine SSB at 2 X 10-10 M with a conventional spectrometer. We have also applied MB DNA probes for the analysis of an enzyme lactic dehydrogenase (LDH), and for the investigation of its binding properties with ssDNA. The biding process between MB and different isoenzymes of LDH has been studied. We also show that there are significant differences in MB binding affinity to different proteins, which will enable selective binding studies of a variety of proteins. This new approach is potentially useful for protein-DNA/RNA interaction studies that require high sensitivity, speed and convenience. The results also open the possibility of using easily obtainable, custom designed, modified DNA molecules for studies of drug interactions and targeting. Our results demonstrate that MB can be effectively used for sensitive protein quantitation and for efficient protein-DNA interaction studies. MB has the signal transduction mechanism built within the molecule, and can thus be used for quick protein assay development and for real-time measurements.

  20. Coagulant properties of Moringa oleifera protein preparations: application to humic acid removal.

    PubMed

    Santos, Andréa F S; Paiva, Patrícia M G; Teixeira, José A C; Brito, António G; Coelho, Luana C B B; Nogueira, Regina

    2012-01-01

    This work aimed to characterize the coagulant properties of protein preparations from Moringa oleifera seeds in the removal of humic acids from water. Three distinct preparations were assayed, namely extract (seeds homogenized with 0.15 M NaCl), fraction (extract precipitated with 60% w/v ammonium sulphate) and cMoL (protein purified with guar gel column chromatography). The extract showed the highest coagulant activity in a protein concentration between 1 mg/L and 180 mg/L at pH 7.0. The zeta potential of the extract (-10 mV to -15 mV) was less negative than that of the humic acid (-41 mV to -42 mV) in a pH range between 5.0 and 8.0; thus, the mechanism that might be involved in this coagulation activity is adsorption and neutralization of charges. Reduction of total organic carbon (TOC) and dissolved organic carbon (DOC) was observed in water samples containing 9 mg/L carbon as humic acid when treated with 1 mg/L of the extract. A decrease in colour and in the aromatic content of the treated water was also observed. These results suggested that the extract from M. oleifera seeds in a low concentration (1 mg/L) can be an interesting natural alternative for removing humic acid from water in developing countries. The extract dose determined in the present study does not impart odour or colour to the treated water. PMID:22519089

  1. Protein digestibility evaluations of meat and fish substrates using laboratory, avian and ileally cannulated dog assays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fish and meat protein serves as important protein sources in the human and companion animal diets: however, limited information is available on differences in protein quality. Pollock fillet, and salmon fillet, beef loin, pork loin and chicken breast, were evaluated for protein quality and amino aci...

  2. Assay for Arf GTP-binding Proteins | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Cancer Institute's Laboratory of Cellular and Molecular Biology is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize an antibody-based proteomics assay.

  3. Nucleic acid encoding a self-assembling split-fluorescent protein system

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2015-07-14

    The invention provides a protein labeling and detection system based on self-complementing fragments of fluorescent and chromophoric proteins. The system of the invention is exemplified with various combinations of self-complementing fragments derived from Aequorea victoria Green Fluorescent Protein (GFP), which are used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.

  4. Nucleic acid encoding a self-assembling split-fluorescent protein system

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2011-06-07

    The invention provides a protein labeling and detection system based on self-complementing fragments of fluorescent and chromophoric proteins. The system of the invention is exemplified with various combinations of self-complementing fragments derived from Aequorea victoria Green Fluorescent Protein (GFP), which are used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.

  5. Nucleic acid encoding a self-assembling split-fluorescent protein system

    DOEpatents

    Waldo, Geoffrey S; Cabantous, Stephanie

    2014-04-01

    The invention provides a protein labeling and detection system based on self-complementing fragments of fluorescent and chromophoric proteins. The system of the invention is exemplified with various combinations of self-complementing fragments derived from Aequorea victoria Green Fluorescent Protein (GFP), which are used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.

  6. Effect of Ethionine on the Ribonucleic Acid, Deoxyribonucleic Acid, and Protein Content of Escherichia coli

    PubMed Central

    Smith, Robert C.; Salmon, W. D.

    1965-01-01

    Smith, Robert C. (Auburn University, Auburn, Ala.), and W. D. Salmon. Effect of ethionine on the ribonucleic acid, deoxyribonucleic acid, and protein content of Escherichia coli. J. Bacteriol. 89:687–692. 1965.—The addition of ethionine to cultures of Escherichia coli K-12 W6, a methionine-requiring auxotroph, led to inhibition of the rate of increase in optical density when the ratio of ethionine to methionine was 200:1. When the ratio was 600:1, the increase in optical density became linear. When ethionine was substituted for methionine in the medium, the optical density of the culture increased, and there was a parallel increase in protein content. There was no cell division in these cultures. The rate of synthesis of ribonucleic acid (RNA) in a culture containing ethionine was similar to that of a culture deprived of methionine, but the synthesis of deoxyribonucleic acid in a culture with ethionine was about twice that of a culture deprived of methionine. No detectable radioactivity from ethionine-ethyl-1-C14 was incorporated into RNA. Ethionine-ethyl-1-C14 was readily incorporated into the protein fraction. PMID:14273646

  7. Fatty Acid-binding Proteins Interact with Comparative Gene Identification-58 Linking Lipolysis with Lipid Ligand Shuttling.

    PubMed

    Hofer, Peter; Boeszoermenyi, Andras; Jaeger, Doris; Feiler, Ursula; Arthanari, Haribabu; Mayer, Nicole; Zehender, Fabian; Rechberger, Gerald; Oberer, Monika; Zimmermann, Robert; Lass, Achim; Haemmerle, Guenter; Breinbauer, Rolf; Zechner, Rudolf; Preiss-Landl, Karina

    2015-07-24

    The coordinated breakdown of intracellular triglyceride (TG) stores requires the exquisitely regulated interaction of lipolytic enzymes with regulatory, accessory, and scaffolding proteins. Together they form a dynamic multiprotein network designated as the "lipolysome." Adipose triglyceride lipase (Atgl) catalyzes the initiating step of TG hydrolysis and requires comparative gene identification-58 (Cgi-58) as a potent activator of enzyme activity. Here, we identify adipocyte-type fatty acid-binding protein (A-Fabp) and other members of the fatty acid-binding protein (Fabp) family as interaction partners of Cgi-58. Co-immunoprecipitation, microscale thermophoresis, and solid phase assays proved direct protein/protein interaction between A-Fabp and Cgi-58. Using nuclear magnetic resonance titration experiments and site-directed mutagenesis, we located a potential contact region on A-Fabp. In functional terms, A-Fabp stimulates Atgl-catalyzed TG hydrolysis in a Cgi-58-dependent manner. Additionally, transcriptional transactivation assays with a luciferase reporter system revealed that Fabps enhance the ability of Atgl/Cgi-58-mediated lipolysis to induce the activity of peroxisome proliferator-activated receptors. Our studies identify Fabps as crucial structural and functional components of the lipolysome. PMID:25953897

  8. Fatty Acid-binding Proteins Interact with Comparative Gene Identification-58 Linking Lipolysis with Lipid Ligand Shuttling*

    PubMed Central

    Hofer, Peter; Boeszoermenyi, Andras; Jaeger, Doris; Feiler, Ursula; Arthanari, Haribabu; Mayer, Nicole; Zehender, Fabian; Rechberger, Gerald; Oberer, Monika; Zimmermann, Robert; Lass, Achim; Haemmerle, Guenter; Breinbauer, Rolf; Zechner, Rudolf; Preiss-Landl, Karina

    2015-01-01

    The coordinated breakdown of intracellular triglyceride (TG) stores requires the exquisitely regulated interaction of lipolytic enzymes with regulatory, accessory, and scaffolding proteins. Together they form a dynamic multiprotein network designated as the “lipolysome.” Adipose triglyceride lipase (Atgl) catalyzes the initiating step of TG hydrolysis and requires comparative gene identification-58 (Cgi-58) as a potent activator of enzyme activity. Here, we identify adipocyte-type fatty acid-binding protein (A-Fabp) and other members of the fatty acid-binding protein (Fabp) family as interaction partners of Cgi-58. Co-immunoprecipitation, microscale thermophoresis, and solid phase assays proved direct protein/protein interaction between A-Fabp and Cgi-58. Using nuclear magnetic resonance titration experiments and site-directed mutagenesis, we located a potential contact region on A-Fabp. In functional terms, A-Fabp stimulates Atgl-catalyzed TG hydrolysis in a Cgi-58-dependent manner. Additionally, transcriptional transactivation assays with a luciferase reporter system revealed that Fabps enhance the ability of Atgl/Cgi-58-mediated lipolysis to induce the activity of peroxisome proliferator-activated receptors. Our studies identify Fabps as crucial structural and functional components of the lipolysome. PMID:25953897

  9. The requirements of protein & amino acid during acute & chronic infections.

    PubMed

    Kurpad, Anura V

    2006-08-01

    Nutrition and infection interact with each other in a synergistic vicious cycle, leading to an adverse nutritional status and increased susceptibility to infection. Infectious episodes result in hypermetabolism and a negative nitrogen balance which is modulated by hormones, cytokines and other pro-inflammatory mediators, and is compounded by a reduced food intake. The extent of the negative nitrogen balance varies with the type of infection and its duration; however, it is reasonable to suggest that the loss of body protein could be minimized by the provision of dietary nitrogen, although anorexia will limit this. Further, distinctions need to be made about the provision of nutrients or protein during the catabolic and anabolic or recovery phase of the infection, since the capacity of the body to retain protein is enhanced in the anabolic recovery phase. Meeting the increased requirement for protein (and other nutrients) in infection does not imply a complete therapeutic strategy. Infections need to be treated appropriately, with nutrition as an adjunct to the treatment. Prior undernutrition could also impair the body's response to infection, although the weight of the evidence would suggest that this happens more particularly in oedematous undernutrition. In general, the amount of extra protein that would appear to be needed is of the order of 20-25 per cent of the recommended intake, for most infections. In acute infections, this is particularly relevant during the convalescence period. Community trials have suggested that lysine supplementation to the level required for normal daily nutriture, in predominantly wheat eating or potentially lysine deficient communities, improves immune function among other functional nutritional parameters; however, there is as yet insufficient evidence to suggest a specific requirement for amino acids in infections over and above the normal daily requirement as based on recent evidence. Some clinical studies that have showed

  10. Retinoic acid binding protein in normal and neopolastic rat prostate.

    PubMed

    Gesell, M S; Brandes, M J; Arnold, E A; Isaacs, J T; Ueda, H; Millan, J C; Brandes, D

    1982-01-01

    Sucrose density gradient analysis of cytosol from normal and neoplastic rat prostatic tissues exhibited a peak of (3H) retinoic acid binding in the 2S region, corresponding to the cytoplasmic retinoic acid binding protein (cRABP). In the Fisher-Copenhagen F1 rat, cRABP was present in the lateral lobe, but could not be detected in the ventral nor in the dorsal prostatic lobes. Four sublines of the R-3327 rat prostatic tumor contained similar levels of this binding protein. The absence of cRABP in the normal tissue of origin of the R-3327 tumor, the rat dorsal prostate, and reappearance in the neoplastic tissues follows a pattern described in other human and animal tumors. The occurrence of cRABP in the well-differentiated as well as in the anaplastic R-3327 tumors in which markers which reflect a state of differentiation and hormonal regulation, such as androgen receptor, 5 alpha reductase, and secretory acid phosphatase are either markedly reduced or absent, points to cRABP as a marker of malignant transformation. PMID:6283503

  11. Nanodisc-Tm: Rapid functional assessment of nanodisc reconstituted membrane proteins by CPM assay

    PubMed Central

    Ashok, Yashwanth; Jaakola, Veli-Pekka

    2016-01-01

    Membrane proteins are generally unstable in detergents. Therefore, biochemical and biophysical studies of membrane proteins in lipidic environments provides a near native-like environment suitable for membrane proteins. However, manipulation of proteins embedded in lipid bilayer has remained difficult. Methods such as nanodiscs and lipid cubic phase have been developed for easy manipulation of membrane proteins and have yielded significant insights into membrane proteins. Traditionally functional reconstitution of receptors in nanodiscs has been studied with radioligands. We present a simple and faster method for studying the functionality of reconstituted membrane proteins for routine characterization of protein batches after initial optimization of suitable conditions using radioligands. The benefits of the method are • Faster and generic method to assess functional reconstitution of membrane proteins. • Adaptable in high throughput format (≥96 well format). • Stability measurement in near-native lipid environment and lipid dependent melting temperatures. PMID:27054097

  12. Foamy Virus Protein-Nucleic Acid Interactions during Particle Morphogenesis.

    PubMed

    Hamann, Martin V; Lindemann, Dirk

    2016-01-01

    Compared with orthoretroviruses, our understanding of the molecular and cellular replication mechanism of foamy viruses (FVs), a subfamily of retroviruses, is less advanced. The FV replication cycle differs in several key aspects from orthoretroviruses, which leaves established retroviral models debatable for FVs. Here, we review the general aspect of the FV protein-nucleic acid interactions during virus morphogenesis. We provide a summary of the current knowledge of the FV genome structure and essential sequence motifs required for RNA encapsidation as well as Gag and Pol binding in combination with details about the Gag and Pol biosynthesis. This leads us to address open questions in FV RNA engagement, binding and packaging. Based on recent findings, we propose to shift the point of view from individual glycine-arginine-rich motifs having functions in RNA interactions towards envisioning the FV Gag C-terminus as a general RNA binding protein module. We encourage further investigating a potential new retroviral RNA packaging mechanism, which seems more complex in terms of the components that need to be gathered to form an infectious particle. Additional molecular insights into retroviral protein-nucleic acid interactions help us to develop safer, more specific and more efficient vectors in an era of booming genome engineering and gene therapy approaches. PMID:27589786

  13. Human Skeletal Muscle Protein Metabolism Responses to Amino Acid Nutrition.

    PubMed

    Mitchell, W Kyle; Wilkinson, Daniel J; Phillips, Bethan E; Lund, Jonathan N; Smith, Kenneth; Atherton, Philip J

    2016-07-01

    Healthy individuals maintain remarkably constant skeletal muscle mass across much of adult life, suggesting the existence of robust homeostatic mechanisms. Muscle exists in dynamic equilibrium whereby the influx of amino acids (AAs) and the resulting increases in muscle protein synthesis (MPS) associated with the intake of dietary proteins cancel out the efflux of AAs from muscle protein breakdown that occurs between meals. Dysregulated proteostasis is evident with aging, especially beyond the sixth decade of life. Women and men aged 75 y lose muscle mass at a rate of ∼0.7% and 1%/y, respectively (sarcopenia), and lose strength 2- to 5-fold faster (dynapenia) as muscle "quality" decreases. Factors contributing to the disruption of an otherwise robust proteostatic system represent targets for potential therapies that promote healthy aging. Understanding age-related impairments in anabolic responses to AAs and identifying strategies to mitigate these factors constitute major areas of interest. Numerous studies have aimed to identify 1) the influence of distinct protein sources on absorption kinetics and muscle anabolism, 2) the latency and time course of MPS responses to protein/AAs, 3) the impacts of protein/AA intake on muscle microvascular recruitment, and 4) the role of certain AAs (e.g., leucine) as signaling molecules, which are able to trigger anabolic pathways in tissues. This review aims to discuss these 4 issues listed, to provide historical and modern perspectives of AAs as modulators of human skeletal muscle protein metabolism, to describe how advances in stable isotope/mass spectrometric approaches and instrumentation have underpinned these advances, and to highlight relevant differences between young adults and older individuals. Whenever possible, observations are based on human studies, with additional consideration of relevant nonhuman studies. PMID:27422520

  14. Amino acid profiles and digestible indispensable amino acid scores of proteins from the prioritized key foods in Bangladesh.

    PubMed

    Shaheen, Nazma; Islam, Saiful; Munmun, Sarah; Mohiduzzaman, Md; Longvah, Thingnganing

    2016-12-15

    Concentrations of standard amino acids were determined in the composite samples (representing 30 agro-ecological zones of Bangladesh) of six prioritized key dietary protein sources: Oryza sativa (rice), Triticum aestivum (wheat flour), Lens culinaris (lentils), Pangusius pangusius (pangas), Labeo rohita (rohu) and Oreochromis mossambicus (tilapia). Digestible indispensable amino acid scores (DIAAS) was calculated using published data on amino acids' digestibility to evaluate the protein quality of these foods. Indispensable amino acid (IAA) contents (mg IAA/g protein), found to be highest in pangas (430) and lowest in wheat (336), of all these analyzed foods exceeded the FAO recommended daily allowance (277mg IAA/g protein) and contributed on average 40% to total amino acid contents. Untruncated DIAAS values ranged from 51% (lysine) in wheat to 106% (histidine) in pangas and distinguished pangas, rohu, and tilapia containing 'excellent quality' protein (DIAAS>100%) with potential to complement lower quality protein of cereals, fruits, and vegetables. PMID:27451158

  15. Studies on fatty acid-binding proteins. The detection and quantification of the protein from rat liver by using a fluorescent fatty acid analogue.

    PubMed Central

    Wilkinson, T C; Wilton, D C

    1986-01-01

    Fatty acid-binding protein from rat liver is shown to bind the fluorescent fatty acid probe dansyl undecanoic acid. Binding is accompanied by a shift in the fluorescence emission maximum from 550 nm to 500 nm and a 60-fold fluorescence enhancement at 500 nm. These spectral properties have allowed the use of this probe to detect and quantify microgram amounts of liver fatty acid-binding protein during purification procedures. In conjunction with h.p.l.c. the method allows the rapid estimation of liver fatty acid-binding protein in biological samples. The validity of the method is demonstrated by measuring the concentration of fatty acid-binding protein in livers from control and hypolipidaemic-drug-treated rats. The dramatic diurnal rhythm previously reported for this protein [Dempsey (1984) Curr. Top. Cell. Regul. 24, 63-86] was not observed with this method. Images Fig. 1. PMID:3800946

  16. Roles of nucleic acid substrates and cofactors in the vhs protein activity of pseudorabies virus.

    PubMed

    Liu, Ya-Fen; Tsai, Pei-Yun; Lin, Fong-Yuan; Lin, Kuan-Hsun; Chang, Tien-Jye; Lin, Hui-Wen; Chulakasian, Songkhla; Hsu, Wei-Li

    2015-01-01

    Pseudorabies virus (PrV) belongs to the α-herpesvirinae of which human simplex virus (HSV) is the prototype virus. One of the hallmarks of HSV infection is shutoff of protein synthesis that is mediated by various viral proteins including vhs (virion host shutoff), which is encoded by the UL41 gene. However, the function of PrV vhs is poorly understood. Due to the low sequence similarity (39.3%) between the HSV and PrV UL41 proteins, vhs might not share the same biochemistry characteristics. The purpose of this study was to characterize the nuclease activity of the PrV vhs protein with respect to substrate specificity, its requirements in terms of cofactors, and the protein regions, as well as key amino acids, which contribute to vhs activity. Our results indicated that, similar to HSV vhs, PrV vhs is able to degrade ssRNA and mRNA. However, PrV vhs also targeted rRNA for degradation, which is novel compared to the HSV-1 vhs. Activity assays indicated that Mg(2+) alone enhances RNA degradation mediated by PrV vhs, while K(+) and ATP are not sufficient to induce activity. Finally, we demonstrated that each of the four highly conserved functional boxes of PrV vhs contributes to RNA degradation and that, in particular, residues 152, 169, 171, 172, 173 343, 345, 352 and 356, which are conserved among α-herpesviruses, are key amino acids needed for PrV vhs ribonuclease activity. PMID:26704628

  17. Correlation of secreted protein acidic and rich in cysteine with diabetic nephropathy

    PubMed Central

    Li, Lei; Song, Hai-Yan; Liu, Kai; An, Meng-Meng

    2015-01-01

    To detect the serum concentrations of secreted protein acidic and rich in cysteine (SPARC) in patients with diabetic nephropathy and SPARC mRNA and protein expressions in renal tissue of db/db mice (C57BL/KsJ, diabetic nephropathy mice), thus preliminary exploration on the role of secreted protein acidic riches in cysteine in the development of diabetic nephropathy were carried out. Serum SPARC levels in normal subjects, patients with type 2 diabetes mellitus (without diabetic nephropathy), chronic renal failure (without diabetes mellitus), and diabetic nephropathy were determined with enzyme-linked immunosorbent assay. 12-week-old db/db mice (db/db group) and its littermate wild-type control mice (NC group) were selected with 6 from each group, and the kidney tissue were taken. RT-PCR, Western blot, and immunofluorescence were used to detect the mRNA, targeted protein expressions of SPARC and the staining of renal tissue. The serum level of SPARC in diabetic nephropathy group was significantly higher than those in normal group, type 2 diabetes mellitus, and chronic renal failure group (P < 0.05 or P < 0.01). The SPARC level in the type 2 diabetes mellitus group was higher than that in normal group (P < 0.05), but there was no difference between normal group and chronic renal failure. SPARC mRNA and protein levels in renal tissue of db/db mice were higher compared with the normal control group (P < 0.05). The long term hyperglycemic state in patients with diabetic nephropathy causes pathological change of renal tissue. Simultaneously, increased secretion of SPARC from renal tissue results in elevation of serum SPARC level. SPARC correlates with the occurrence and progression of diabetes, and it may play a role in pathological change of diabetic nephropathy. PMID:26550188

  18. Detection of inhibition of 5-aminoimidazole-4-carboxamide ribotide transformylase by thioinosinic acid and azathioprine by a new colorimetric assay.

    PubMed Central

    Ha, T; Morgan, S L; Vaughn, W H; Eto, I; Baggott, J E

    1990-01-01

    The colorimetric assay for 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase (phosphoribosylamino-imidazolecarboxamide formyltransferase; EC 2.1.2.3) has been extensively modified. The modified assay is based upon the short-term permanganate oxidation of the folate product, tetrahydrofolate (H4folate) to p-aminobenzoyl glutamate (pABG). The modified assay was used to detect the transformylase activity in crude extracts of peripheral-blood mononuclear cells (PBMCs). Azathioprine and its metabolite, thioinosinic acid (tIMP), are competitive inhibitors (with respect to AICAR) of the chicken liver transformylase and the transformylase from PBMCs of the MRL/lpr mouse, an animal model of systemic autoimmune disease. The Ki values of tIMP and azathioprine for the chicken liver enzyme are 39 +/- 4 microM and 120 +/- 10 microM, whereas the Ki values for the enzyme from PBMCs of the MRL/lpr mouse are 110 +/- 20 microM and 90 +/- 14 microM respectively. The anti-inflammatory drugs ibuprofen and naproxen are also inhibitors of the transformylase. PMID:2268263

  19. Amino acid sequence of the Amur tiger prion protein.

    PubMed

    Wu, Changde; Pang, Wanyong; Zhao, Deming

    2006-10-01

    Prion diseases are fatal neurodegenerative disorders in human and animal associated with conformational conversion of a cellular prion protein (PrP(C)) into the pathologic isoform (PrP(Sc)). Various data indicate that the polymorphisms within the open reading frame (ORF) of PrP are associated with the susceptibility and control the species barrier in prion diseases. In the present study, partial Prnp from 25 Amur tigers (tPrnp) were cloned and screened for polymorphisms. Four single nucleotide polymorphisms (T423C, A501G, C511A, A610G) were found; the C511A and A610G nucleotide substitutions resulted in the amino acid changes Lysine171Glutamine and Alanine204Threoine, respectively. The tPrnp amino acid sequence is similar to house cat (Felis catus ) and sheep, but differs significantly from other two cat Prnp sequences that were previously deposited in GenBank. PMID:16780982

  20. Evaluation of Surfactants-Assisted Folic Acid-Loaded Pectin Submicrospheres: Characterization and Hemocompatibility Assay.

    PubMed

    Varuna Kumara, J B; Ravikumara, N R; Madhusudhan, Basavaraj

    2016-10-01

    Folic acid is used for preventing and treating multiple diseases and disorders, administered in the form of oral supplements. The present research work was aimed to study the influence of two non-ionic surfactants Poloxamer and Tween 80 (Polysorbate 80) on pectin submicrospheres formulations. Typical natural polymer pectin was used to encapsulate folic acid by cross linking method. The resultant submicrospheres contributed to improve the aqueous solubility to enhance the bioavailability of folic acid. During investigation, it was observed that pectin polymers influenced kinetics of the rate of reaction more intensively than the surfactants. The physical phenomenon caused the change in their size, shape and chemistry of pectin polymers transforming into submicrospheres in aqueous condition. The characteristic differences of submicrospheres were assessed by scanning electron microscopy, differential scanning calorimetry and Fourier-transform infrared spectroscopy. The average diameters of the submicrospheres ranged between 250 and 500 nm. The encapsulation efficiency of submicrospheres ranged between 80 and 96 %. The characteristic swelling behavior of lyophilized submicrospheres was influenced by the ratio of pectin polymers and folic acid used in the formulations. The submicrospheres systems exhibited controlled release of folic acid due to the pH-dependent solubility of pectin polymers in aqueous medium. The submicrospheres showed good haemocompatibility suggesting them to be promising candidates for oral delivery. PMID:27605736

  1. Simplified protein design biased for prebiotic amino acids yields a foldable, halophilic protein.

    PubMed

    Longo, Liam M; Lee, Jihun; Blaber, Michael

    2013-02-01

    A compendium of different types of abiotic chemical syntheses identifies a consensus set of 10 "prebiotic" α-amino acids. Before the emergence of biosynthetic pathways, this set is the most plausible resource for protein formation (i.e., proteogenesis) within the overall process of abiogenesis. An essential unsolved question regarding this prebiotic set is whether it defines a "foldable set"--that is, does it contain sufficient chemical information to permit cooperatively folding polypeptides? If so, what (if any) characteristic properties might such polypeptides exhibit? To investigate these questions, two "primitive" versions of an extant protein fold (the β-trefoil) were produced by top-down symmetric deconstruction, resulting in a reduced alphabet size of 12 or 13 amino acids and a percentage of prebiotic amino acids approaching 80%. These proteins show a substantial acidification of pI and require high salt concentrations for cooperative folding. The results suggest that the prebiotic amino acids do comprise a foldable set within the halophile environment. PMID:23341608

  2. Simplified protein design biased for prebiotic amino acids yields a foldable, halophilic protein

    PubMed Central

    Longo, Liam M.; Lee, Jihun; Blaber, Michael

    2013-01-01

    A compendium of different types of abiotic chemical syntheses identifies a consensus set of 10 “prebiotic” α-amino acids. Before the emergence of biosynthetic pathways, this set is the most plausible resource for protein formation (i.e., proteogenesis) within the overall process of abiogenesis. An essential unsolved question regarding this prebiotic set is whether it defines a “foldable set”—that is, does it contain sufficient chemical information to permit cooperatively folding polypeptides? If so, what (if any) characteristic properties might such polypeptides exhibit? To investigate these questions, two “primitive” versions of an extant protein fold (the β-trefoil) were produced by top-down symmetric deconstruction, resulting in a reduced alphabet size of 12 or 13 amino acids and a percentage of prebiotic amino acids approaching 80%. These proteins show a substantial acidification of pI and require high salt concentrations for cooperative folding. The results suggest that the prebiotic amino acids do comprise a foldable set within the halophile environment. PMID:23341608

  3. Acid-base chemistry of frustrated water at protein interfaces.

    PubMed

    Fernández, Ariel

    2016-01-01

    Water molecules at a protein interface are often frustrated in hydrogen-bonding opportunities due to subnanoscale confinement. As shown, this condition makes them behave as a general base that may titrate side-chain ammonium and guanidinium cations. Frustration-based chemistry is captured by a quantum mechanical treatment of proton transference and shown to remove same-charge uncompensated anticontacts at the interface found in the crystallographic record and in other spectroscopic information on the aqueous interface. Such observations are untenable within classical arguments, as hydronium is a stronger acid than ammonium or guanidinium. Frustration enables a directed Grotthuss mechanism for proton transference stabilizing same-charge anticontacts. PMID:26762189

  4. Nucleic Acid Sandwich Hybridization Assay with Quantum Dot-Induced Fluorescence Resonance Energy Transfer for Pathogen Detection

    PubMed Central

    Chou, Cheng-Chung; Huang, Yi-Han

    2012-01-01

    This paper reports a nucleic acid sandwich hybridization assay with a quantum dot (QD)-induced fluorescence resonance energy transfer (FRET) reporter system. Two label-free hemagglutinin H5 sequences (60-mer DNA and 630-nt cDNA fragment) of avian influenza viruses were used as the targets in this work. Two oligonucleotides (16 mers and 18 mers) that specifically recognize two separate but neighboring regions of the H5 sequences were served as the capturing and reporter probes, respectively. The capturing probe was conjugated to QD655 (donor) in a molar ratio of 10:1 (probe-to-QD), and the reporter probe was labeled with Alexa Fluor 660 dye (acceptor) during synthesis. The sandwich hybridization assay was done in a 20 μL transparent, adhesive frame-confined microchamber on a disposable, temperature-adjustable indium tin oxide (ITO) glass slide. The FRET signal in response to the sandwich hybridization was monitored by a homemade optical sensor comprising a single 400 nm UV light-emitting diode (LED), optical fibers, and a miniature 16-bit spectrophotometer. The target with a concentration ranging from 0.5 nM to 1 μM was successfully correlated with both QD emission decrease at 653 nm and dye emission increase at 690 nm. To sum up, this work is beneficial for developing a portable QD-based nucleic acid sensor for on-site pathogen detection. PMID:23211753

  5. Use of hydrophilic extra-viral domain of canine distemper virus H protein for enzyme-linked immunosorbent assay development

    PubMed Central

    Cho, Ki-hyun; Kim, Jeongmi; Yoo, Hyun-ah; Kim, Dae-hee; Park, Seung-yong; Song, Chang-seon; Choi, In-soo

    2014-01-01

    Simple methods for measuring the levels of serum antibody against canine distemper virus (CDV) would assist in the effective vaccination of dogs. To develop an enzyme-linked immunosorbent assay (ELISA) specific for CDV, we expressed hydrophilic extra-viral domain (HEVD) protein of the A75/17-CDV H gene in a pET 28a plasmid-based Escherichia (E.) coli vector system. Expression was confirmed by dot and Western blotting. We proposed that detection of E. coli-expressed H protein might be conformation-dependent because intensities of the reactions observed with these two methods varied. The H gene HEVD protein was further purified and used as an antigen for an ELISA. Samples from dogs with undetectable to high anti-CDV antibody titers were analyzed using this HEVD-specific ELISA and a commercial CDV antibody detection kit (ImmunoComb). Levels of HEVD antigenicity measured with the assays and immunochromatography correlated. These data indicated that the HEDV protein may be used as antigen to develop techniques for detecting antibodies against CDV. PMID:25234325

  6. Ultrasensitive enzyme-linked immunosorbent assay (ELISA) of proteins by combination with the thio-NAD cycling method

    PubMed Central

    Watabe, Satoshi; Kodama, Hiromi; Kaneda, Mugiho; Morikawa, Mika; Nakaishi, Kazunari; Yoshimura, Teruki; Iwai, Atsushi; Miura, Toshiaki; Ito, Etsuro

    2014-01-01

    An ultrasensitive method for the determination of proteins is described that combines an enzyme-linked immunosorbent assay (ELISA) and a thionicotinamide-adenine dinucleotide (thio-NAD) cycling method. A sandwich method using a primary and a secondary antibody for antigens is employed in an ELISA. An androsterone derivative, 3α-hydroxysteroid, is produced by the hydrolysis of 3α-hydroxysteroid 3-phosphate with alkaline phosphatase linked to the secondary antibody. This 3α-hydroxysteroid is oxidized to a 3-ketosteroid by 3α- hydroxysteroid dehydrogenase (3α-HSD) with a cofactor thio-NAD. By the opposite reaction, the 3-ketosteroid is reduced to a 3α-hydroxysteroid by 3α-HSD with a cofactor NADH. During this cycling reaction, thio-NADH accumulates in a quadratic function-like fashion. Accumulated thio-NADH can be measured directly at an absorbance of 400 nm without any interference from other cofactors. These features enable us to detect a target protein with ultrasensitivity (10−19 mol/assay) by measuring the cumulative quantity of thio-NADH. Our ultrasensitive determination of proteins thus allows for the detection of small amounts of proteins only by the application of thio-NAD cycling reagents to the usual ELISA system. PMID:27493498

  7. Use of hydrophilic extra-viral domain of canine distemper virus H protein for enzyme-linked immunosorbent assay development.

    PubMed

    Cho, Ki-hyun; Kim, Jeongmi; Yoo, Hyun-ah; Kim, Dae-hee; Park, Seung-yong; Song, Chang-seon; Choi, In-soo; Lee, Joong-bok

    2014-12-01

    Simple methods for measuring the levels of serum antibody against canine distemper virus (CDV) would assist in the effective vaccination of dogs. To develop an enzyme-linked immunosorbent assay (ELISA) specific for CDV, we expressed hydrophilic extra-viral domain (HEVD) protein of the A75/17-CDV H gene in a pET 28a plasmid-based Escherichia (E.) coli vector system. Expression was confirmed by dot and Western blotting. We proposed that detection of E. coli-expressed H protein might be conformation- dependent because intensities of the reactions observed with these two methods varied. The H gene HEVD protein was further purified and used as an antigen for an ELISA. Samples from dogs with undetectable to high anti-CDV antibody titers were analyzed using this HEVD-specific ELISA and a commercial CDV antibody detection kit (ImmunoComb). Levels of HEVD antigenicity measured with the assays and immunochromatography correlated. These data indicated that the HEDV protein may be used as antigen to develop techniques for detecting antibodies against CDV. PMID:25234325

  8. Protein digestibility of the same protein preparations by human and rat assays and by in vitro enzymic digestion methods.

    PubMed

    Bodwell, C E; Satterlee, L D; Hackler, L R

    1980-03-01

    The apparent and true digestibilities of the same preparations of six proteins (spray dried whole egg, cottage cheese, canned tuna, peanut flour, soy isolate, and wheat gluten) were estimated in four to five men and in rats and compared to estimates of digestibility from three different in vitro enzymic digestion procedures. For all six proteins, the correlation coefficient was 0.46 between true digestibility in humans and in rats; with values for tuna excluded, r = 0.96. With all six proteins, none of the in vitro values was significantly correlated with values from humans or rats. However, with either the three animal proteins alone or the three plant proteins alone, correlations were high (r greater than 0.90) between one or more of the in vitro estimates and the observed true or apparent human and rat digestibilities. The differences in the relationship between enzymic digestion estimates and the human digestibility estimates for plant or animal proteins suggest that for accurate prediction of protein digestibility in humans by these enzymic methods, different equations would have to be used for plant and animal proteins. For protein sources containing both plant and animal protein, use of the in vitro enzymic procedures would give only an approximate estimate of digestibility in humans. PMID:6986763

  9. Effect of benzene and lead on relationship between. delta. -aminolevulinic acid and brain myelin proteins

    SciTech Connect

    Muzyka, V.I.; Bogovskii, L.A.

    1986-03-01

    The aim of this investigation was to study binding of ALA by brain myelin under normal conditions and under the influence of lead and benzene. Rabbits of three different groups were given an intercranial injection of /sup 14/C-ALA (50 microCi) in physiological saline in a volume of 0.25 ml. The myelin fraction was obtained by differential centrifugation and the /sup 14/C-ALA in it was assayed on an LKB liquid scintillation counter. By determining the quantity of exogenous /sup 14/C-ALA bound with myelin, the authors found that myelin of white matter in the brain contains more of the acid than myelin of the gray matter. Data on binding of /sup 14/C-ALA calculated per milligram of each group of myelin proteins isolated is given.

  10. Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis

    NASA Astrophysics Data System (ADS)

    Hong, Seok Hoon; Kwon, Yong-Chan; Jewett, Michael

    2014-06-01

    Incorporating non-standard amino acids (NSAAs) into proteins enables new chemical properties, new structures, and new functions. In recent years, improvements in cell-free protein synthesis (CFPS) systems have opened the way to accurate and efficient incorporation of NSAAs into proteins. The driving force behind this development has been three-fold. First, a technical renaissance has enabled high-yielding (>1 g/L) and long-lasting (>10 h in batch operation) CFPS in systems derived from Escherichia coli. Second, the efficiency of orthogonal translation systems has improved. Third, the open nature of the CFPS platform has brought about an unprecedented level of control and freedom of design. Here, we review recent developments in CFPS platforms designed to precisely incorporate NSAAs. In the coming years, we anticipate that CFPS systems will impact efforts to elucidate structure/function relationships of proteins and to make biomaterials and sequence-defined biopolymers for medical and industrial applications.

  11. The Citrus transcription factor, CitERF13, regulates citric acid accumulation via a protein-protein interaction with the vacuolar proton pump, CitVHA-c4

    PubMed Central

    Li, Shao-jia; Yin, Xue-ren; Xie, Xiu-lan; Allan, Andrew C.; Ge, Hang; Shen, Shu-ling; Chen, Kun-song

    2016-01-01

    Organic acids are essential to fruit flavor. The vacuolar H+ transporting adenosine triphosphatase (V-ATPase) plays an important role in organic acid transport and accumulation. However, less is known of V-ATPase interacting proteins and their relationship with organic acid accumulation. The relationship between V-ATPase and citric acid was investigated, using the citrus tangerine varieties ‘Ordinary Ponkan (OPK)’ and an early maturing mutant ‘Zaoshu Ponkan (ZPK)’. Five V-ATPase genes (CitVHA) were predicted as important to citric acid accumulation. Among the genes, CitVHA-c4 was observed, using a yeast two-hybrid screen, to interact at the protein level with an ethylene response factor, CitERF13. This was verified using bimolecular fluorescence complementation assays. A similar interaction was also observed between Arabidopsis AtERF017 (a CitERF13 homolog) and AtVHA-c4 (a CitVHA-c4 homolog). A synergistic effect on citric acid levels was observed between V-ATPase proteins and interacting ERFs when analyzed using transient over-expression in tobacco and Arabidopsis mutants. Furthermore, the transcript abundance of CitERF13 was concomitant with CitVHA-c4. CitERF13 or AtERF017 over-expression leads to significant citric acid accumulation. This accumulation was abolished in an AtVHA-c4 mutant background. ERF-VHA interactions appear to be involved in citric acid accumulation, which was observed in both citrus and Arabidopsis. PMID:26837571

  12. DNA binding proteins that alter nucleic acid flexibility