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1

Quantitative analysis of complex protein mixtures using isotope-coded affinity tags  

Microsoft Academic Search

We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures. The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry. Using this strategy, we compared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source.

Steven P. Gygi; Beate Rist; Scott A. Gerber; Frantisek Turecek; Michael H. Gelb; Ruedi Aebersold

1999-01-01

2

Identification of thioredoxin target disulfides using isotope-coded affinity tags.  

PubMed

Thioredoxins (Trx) are small redox proteins that reduce disulfide bonds in various target proteins and maintain cellular thiol redox control. Here, a thiol-specific labeling and affinity enrichment approach for identification and relative quantification of Trx target disulfides in complex protein extracts is described. The procedure utilizes the isotope-coded affinity tag (ICAT) reagents containing a thiol reactive iodoacetamide group and a biotin affinity tag to target peptides containing reduced cysteine residues. The identification of substrates for Trx and the extent of target disulfide reduction is determined by LC-MS/MS-based quantification of tryptic peptides labeled with "light" ((12)C) and "heavy" ((13)C) ICAT reagents. The methodology can be adapted to monitor the effect of different reductants or oxidants on the redox status of thiol/disulfide proteomes in biological systems. PMID:24136556

Hägglund, Per; Bunkenborg, Jakob; Maeda, Kenji; Finnie, Christine; Svensson, Birte

2014-01-01

3

Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry  

Microsoft Academic Search

An approach to the systematic identification and quantification of the proteins contained in the microsomal fraction of cells is described. It consists of three steps: (1) preparation of microsomal fractions from cells or tissues representing different states; (2) covalent tagging of the proteins with isotope-coded affinity tag (ICAT) reagents followed by proteolysis of the combined labeled protein samples; and (3)

David K. Han; Jimmy Eng; Huilin Zhou; Ruedi Aebersold

2001-01-01

4

Protein Profiling with Cleavable Isotope-coded Affinity Tag (cICAT) Reagents  

Microsoft Academic Search

Protein expression profiles in yeast cells, in response to salinity stress, were determined using the cleavable iso- tope-coded affinity tag (cICAT) labeling strategy. The analysis included separation of the mixed protein samples by SDS-PAGE, followed by excision of the entire gel lane, and division of the lane into 14 gel regions. Regions were subjected to in-gel digestion, biotin affinity chromatogra-

Jiaxu Li; Hanno Steen; Steven P. Gygi

5

Mass Spectrometric Analysis of Protein Mixtures at Low Levels Using Cleavable 13 C-Isotope-coded Affinity Tag and Multidimensional Chromatography  

Microsoft Academic Search

In order to identify and compare the protein content of very low quantity samples of high complexity, a protocol has been established that combines the differential profiling strength of a new cleavable 13C isotope-coded affinity tag (cICAT) reagent with the high sequence coverage provided by multidimensional liquid chromatography and two modes of tandem mass spectrometry. Major objectives during pro- tocol

Kirk C. Hansen; Gerold Schmitt-Ulms; Robert J. Chalkley; Jan Hirsch; Michael A. Baldwin; A. L. Burlingame

6

White Spot Syndrome Virus Proteins and Differentially Expressed Host Proteins Identified in Shrimp Epithelium by Shotgun Proteomics and Cleavable Isotope-Coded Affinity Tag  

Microsoft Academic Search

Shrimp subcuticular epithelial cells are the initial and major targets of white spot syndrome virus (WSSV) infection. Proteomic studies of WSSV-infected subcuticular epithelium of Penaeus monodon were performed through two approaches, namely, subcellular fractionation coupled with shotgun proteomics to identify viral and host proteins and a quantitative time course proteomic analysis using cleavable isotope-coded affinity tags (cICATs) to identify differentially

Jinlu Wu; Qingsong Lin; Teck Kwang Lim; Tiefei Liu; Choy-Leong Hew

2007-01-01

7

Accurate Qualitative and Quantitative Proteomic Analysis of Clinical Hepatocellular Carcinoma Using Laser Capture Microdissection Coupled with Isotope-coded Affinity Tag and Two-dimensional Liquid Chromatography Mass Spectrometry  

Microsoft Academic Search

Laser capture microdissection (LCM) is a powerful tool that enables the isolation of specific cell types from tissue sections, overcoming the problem of tissue heterogeneity and contamination. This study combined the LCM with isotope-coded affinity tag (ICAT) technology and two-di- mensional liquid chromatography to investigate the qual- itative and quantitative proteomes of hepatocellular car- cinoma (HCC). The effects of three

Chen Li; Yi Hong; Ye-Xiong Tan; Hu Zhou; Jian-Hua Ai; Su-Jun Li; Lei Zhang; Qi-Chang Xia; Jia-Rui Wu; Hong-Yang Wang; Rong Zeng

2004-01-01

8

Complementary Analysis of the Mycobacterium tuberculosis Proteome by Two-dimensional Electrophoresis and Isotope-coded Affinity Tag Technology  

Microsoft Academic Search

Classical proteomics combined two-dimensional gel electrophoresis (2-DE) for the separation and quantifica- tion of proteins in a complex mixture with mass spectro- metric identification of selected proteins. More recently, the combination of liquid chromatography (LC), stable isotope tagging, and tandem mass spectrometry (MS\\/MS) has emerged as an alternative quantitative proteomics technology. We have analyzed the proteome of Mycobac- terium tuberculosis,

Frank Schmidt; Samuel Donahoe; Kristine Hagens; Jens Mattow; Ulrich E. Schaible; Stefan H. E. Kaufmann; Ruedi Aebersold; Peter R. Jungblut

2003-01-01

9

Application of isotope coded affinity tag (ICAT) analysis for the identification of differentially expressed proteins following infection of atlantic salmon (Salmo salar) with infectious hematopoietic necrosis virus (IHNV) or Renibacterium salmoninarum (BKD).  

PubMed

Aquaculture and commercial fisheries worldwide suffer from significant economic loss due to diseases of net-pen reared fish. In British Columbia, infectious hematopoietic necrosis (IHN) and bacterial kidney disease (BKD) epidemics occur because there are currently no commercially available drugs or fully licensed vaccines to treat these diseases. With a better understanding of the molecular mechanisms underlying these diseases, this circumstance might be significantly improved. In the present study, we have used a proteomics approach in an effort to identify and quantitate differentially expressed proteins in the liver and kidneys of diseased and healthy Atlantic salmon (Salmo salar). Isotope coded affinity tagging (ICAT), 2D gel electrophoresis, and multidimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC MS/MS) were used to identify hundreds of differentially expressed proteins. While the direct significance of changes in expression levels of many proteins remains to be elucidated, others appear to be more clearly related to the infectious process. Examples of the latter are discussed here and include, a natural killer cell enhancement factor (NKEF), procathepsin L, superoxide-producing NADPH oxidase and interferon-induced viral resistance protein Mx (IFI-Mx). PMID:15822907

Booy, A T; Haddow, J D; Ohlund, L B; Hardie, D B; Olafson, R W

10

Phosphoprotein Isotope-Coded Solid-Phase Tag Approach for Enrichment and Quantitative Analysis of Phosphopeptides from Complex Mixtures  

SciTech Connect

Many cellular processes are regulated by reversible protein phosphorylation and the ability to identify and quantify phosphoproteins from proteomes is essential for gaining a better understanding of these dynamic cellular processes. However, a sensitive, efficient and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a Phosphoprotein Isotope-coded Solid-phase Tag (PhIST) for isolating and measuring the relative abundance of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported Phosphoprotein Isotope-coded Affinity Tag (PhIAT)approach developed by our laboratory1-2, where the O-phosphate moiety on phosphoseryl or phosphothreonyl residues were derivatized by hydroxide ion-medated B-elimination followed by the addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either light (12C6, 14N) or heavy (13C6, 15N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary LC-MS/MS. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures was demonstrated using casein phosphoproteins. Its utility for proteomic applications is demonstrated by the labeling of soluble proteins from human breast cancer cell line.

Qian, Weijun (BATTELLE (PACIFIC NW LAB)); Goshe, Michael B. (North Carolina State University); Camp, David G. (BATTELLE (PACIFIC NW LAB)); Yu, Li-Rong (BATTELLE (PACIFIC NW LAB)); Tang, Keqi (BATTELLE (PACIFIC NW LAB)); Smith, Richard D. (BATTELLE (PACIFIC NW LAB))

2003-10-15

11

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag  

PubMed Central

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2. Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 . The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix. This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

Nilvebrant, Johan; Alm, Tove; Hober, Sophia

2012-01-01

12

Orthogonal protein purification facilitated by a small bispecific affinity tag.  

PubMed

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used. The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions. The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies. Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding, were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A, a small, bispecific molecule with affinity for both HSA and the novel target was identified. The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix. This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination. PMID:22297419

Nilvebrant, Johan; Alm, Tove; Hober, Sophia

2012-01-16

13

A Metal-coded Affinity Tag Approach to Quantitative Proteomics  

Microsoft Academic Search

The quantitative analysis of protein mixtures is pivotal for the understanding of variations in the proteome of living systems. Therefore, approaches have been recently de- vised that generally allow the relative quantitative analysis of peptides and proteins. Here we present proof of con- cept of the new metal-coded affinity tag (MeCAT) tech- nique, which allowed the quantitative determination of peptides

Robert Ahrends; Stefan Pieper; Andreas Kuhn; Hardy Weisshoff; Meike Hamester; Torsten Lindemann; Christian Scheler; Karola Lehmann; Kerstin Taubner; Michael W. Linscheid

2007-01-01

14

Affinity purification of RNA using an ARiBo tag.  

PubMed

The increased awareness of the importance of RNA in biology, illustrated by the recent attention given to RNA interference research and applications, has spurred structural and functional investigations of RNA. For these studies, the traditional purification method for in vitro transcribed RNA is denaturing polyacrylamide gel electrophoresis. However, gel-based procedures denature the RNA and can be very tedious and time-consuming. Thus, several alternative schemes have been developed for fast non-denaturing purification of RNA transcribed in vitro. In a recent report, a quick affinity purification procedure was developed for RNAs transcribed with a 3'-ARiBo tag and shown to provide RNA with exceptionally high purity and yield. The ARiBo tag contains the ?boxB RNA and the glmS ribozyme, allowing immobilization on GSH-Sepharose resin via a ?N-GST fusion protein and elution by activation of the glmS ribozyme with glucosamine-6-phosphate. This Chapter outlines the experimental details for affinity batch purification of RNAs using ARiBo tags. Although the procedure was originally developed for purification of a stable purine riboswitch mutant, it is demonstrated here for purification of the terminal loop of the let-7g precursor miRNA, an important target of the pluripotency factor Lin28. PMID:23065559

Di Tomasso, Geneviève; Dagenais, Pierre; Desjardins, Alexandre; Rompré-Brodeur, Alexis; Delfosse, Vanessa; Legault, Pascale

2012-01-01

15

An Overview of Enzymatic Reagents for the Removal of Affinity Tags  

PubMed Central

Although they are often exploited to facilitate the expression and purification of recombinant proteins, every affinity tag, whether large or small, has the potential to interfere with the structure and function of its fusion partner. For this reason, reliable methods for removing affinity tags are needed. Only enzymes have the requisite specificity to be generally useful reagents for this purpose. In this review, the advantages and disadvantages of some commonly used endo- and exoproteases are discussed in light of the latest information.

Waugh, David S.

2011-01-01

16

RNA Affinity Tags for the Rapid Purification and Investigation of RNAs and RNA-Protein Complexes  

PubMed Central

Summary Isolation of ribonucleoprotein particles from living cells and cell lysates has allowed the identification of both simple bimolecular interactions and the members of large, extended complexes. A number of different strategies have been devised to isolate these complexes by using affinity purification methods that are specific for the RNA rather than the protein components of these complexes. We describe the use of two such RNA affinity tags: small RNAs that bind with high affinity and specificity to either Sephadex beads or streptavidin affinity resins and can be eluted under mild, native conditions that retain intact complexes. The tags can be inserted into appropriate locations in genes encoding the RNA components, and ribonucleoproteins can be assembled either in vivo or in vitro before affinity isolation. Strategies toward the design and production of these tagged RNA sequences are discussed, and the purification procedure is outlined.

Walker, Scott C.; Scott, Felicia H.; Srisawat, Chatchawan; Engelke, David R.

2009-01-01

17

Histidines in affinity tags and surface clusters for immobilized metal-ion affinity chromatography of trimeric tumor necrosis factor ?  

Microsoft Academic Search

In order to achieve efficient IMAC (immobilized metal-ion affinity chromatography) purification of tumor necrosis factor ? (TNF-?) and its analogs by a common chromatographic procedure, we tested four histidine-rich affinity tags attached to the N-termini of the trimeric TNF-? molecule. Using low cultivation temperature and appropriate protease deficient E. coli strains, it was possible to obtain intact, full-length proteins with

V Gaberc-Porekar; V Menart; S Jevševar; A Videnšek; A Štalc

1999-01-01

18

Production of milligram quantities of affinity tagged-proteins using automated multistep chromatographic purification  

Microsoft Academic Search

A new chromatography system, ÄKTAxpress (GE Healthcare, Amersham Biosciences, Uppsala, Sweden) has been designed to meet the demand for high-throughput purification of proteins in structural genomics and drug discovery. The system offers a number of automated multistep purification protocols for affinity-tagged proteins. All protocols start with affinity chromatography followed by combinations of desalting, ion exchange chromatography and gel filtration. As

Rama Bhikhabhai; Anna Sjöberg; Lotta Hedkvist; Markus Galin; Pia Liljedahl; Tuomo Frigård; Niklas Pettersson; Mats Nilsson; Jill A. Sigrell-Simon; Christine Markeland-Johansson

2005-01-01

19

Dual-tagging system for the affinity purification of mammalian protein complexes  

SciTech Connect

Although affinity purification coupled with mass spectrometry (MS) provides a powerful tool to study protein-protein interactions, this strategy has encountered numerous difficulties when adapted to mammalian cells. Here we describe a Gateway{reg_sign}-compatible dual-tag affinity purification system that integrates regulatable expression, tetracysteine motifs, and various combinations of affinity tags to facilitate the cloning, detection, and purification of bait proteins and their interacting partners. Utilizing the human telomere binding protein TRF2 as a benchmark, we demonstrate bait protein recoveries upwards of approximately 16% from as little as 1-7 x 10{sup 7} cells and successfully identify known TRF2 interacting proteins, suggesting that our dual-tag affinity purification approach is a capable new tool for expanding the capacity to explore mammalian proteomic networks.

Giannone, Richard J [ORNL; McDonald, W Hayes [ORNL; Hurst, Gregory {Greg} B [ORNL; Huang, Ying [ORNL; Wu, Jun [ORNL; Liu, Yie [National Institute on Aging, Baltimore; Wang, Yisong [ORNL

2007-01-01

20

Streptavidin aptamers: affinity tags for the study of RNAs and ribonucleoproteins.  

PubMed Central

RNA affinity tags would be very useful for the study of RNAs and ribonucleoproteins (RNPs) as a means for rapid detection, immobilization, and purification. To develop a new affinity tag, streptavidin-binding RNA ligands, termed "aptamers," were identified from a random RNA library using in vitro selection. Individual aptamers were classified into two groups based on common sequences, and representative members of the groups had sufficiently low dissociation constants to suggest they would be useful affinity tools. Binding of the aptamers to streptavidin was blocked by presaturation of the streptavidin with biotin, and biotin could be used to dissociate RNA/streptavidin complexes. To investigate the practicality of using the aptamer as an affinity tag, one of the higher affinity aptamers was inserted into RPR1 RNA, the large RNA subunit of RNase P. The aptamer-tagged RNase P could be specifically isolated using commercially available streptavidin-agarose and recovered in a catalytically active form when biotin was used as an eluting agent under mild conditions. The aptamer tag was also used to demonstrate that RNase P exists in a monomeric form, and is not tightly associated with RNase MRP, a closely related ribonucleoprotein enzyme. These results show that the streptavidin aptamers are potentially powerful tools for the study of RNAs or RNPs.

Srisawat, C; Engelke, D R

2001-01-01

21

Tandem Immobilized Metal-Ion Affinity Chromatography\\/Immunoaffinity Purification of His-tagged Proteins— Evaluation of Two Anti-His-Tag Monoclonal Antibodies  

Microsoft Academic Search

A tag comprising four to six histidines genetically fused to the protein of interest (His-tag) has been widely used to purify proteins by immobilized metal-ion affinity chromatography (IMAC). Here we report the utilization of the same tag twice in series, first for IMAC and subsequently for immunoaffinity purification. Both steps are based on completely different physical principles and can therefore

Kristian M. Müller; Katja M. Arndt; Konrad Bauer; Andreas Plückthun

1998-01-01

22

Differential profiling of breast cancer plasma proteome by isotope-coded affinity tagging method reveals biotinidase as a breast cancer biomarker  

Microsoft Academic Search

BACKGROUND: Breast cancer is one of the leading causes of women's death worldwide. It is important to discover a reliable biomarker for the detection of breast cancer. Plasma is the most ideal source for cancer biomarker discovery since many cells cross-communicate through the secretion of soluble proteins into blood. METHODS: Plasma proteomes obtained from 6 breast cancer patients and 6

Un-Beom Kang; Younghee Ahn; Jong Won Lee; Yong-Hak Kim; Joon Kim; Myeong-Hee Yu; Dong-Young Noh; Cheolju Lee

2010-01-01

23

Systematic Uncovering of Multiple Pathways Underlying the Pathology of Huntington Disease by an Acid-cleavable Isotope-coded Affinity Tag Approach  

Microsoft Academic Search

Huntington disease (HD) is an autosomal dominant neuro- degenerative disease that results from a CAG (glutamine) trinucleotide expansion in exon 1 of huntingtin (Htt). The aggregation of mutant Htt has been implicated in the pro- gression of HD. The earliest degeneration occurs in the striatum. To identify proteins critical for the progression of HD, we applied acid-cleavable ICAT technology to

Ming-Chang Chiang; Chiun-Gung Juo; Hao-Hung Chang; Hui-Mei Chen; Eugene C. Yi; Yijuang Chern

2007-01-01

24

Tris-Nitrilotriacetic Acids of Sub-nanomolar Affinity Toward Hexahistidine Tagged Molecules  

PubMed Central

Nitrilotriacetic acid (NTA) has moderate affinity (10 µM) for hexahistidine (His6) and is widely used in the purification of His6-tagged proteins. The affinity can be increased significantly (10 nM) through multivalency such as using a tris-NTA. We show that the binding affinity of tris-NTA is dependent on the flexibility and length of the spacer between the mono-NTA and the scaffold: the shorter the spacer, the higher the affinity. A series of biotinylated tris-NTA having different spacers were synthesized and used to prepare tris-NTA sensor chips for surface plasmon resonance measurement of binding affinity. Sub-nanomolar affinity can be achieved with a short spacer. The new high affinity tris-NTA enables the formation of stable complexes with hexahistidine containing molecules and provides a convenient method to non-covalently attach proteins to various surfaces.

Huang, Zhaohua; Hwang, Peter; Watson, Douglas S.; Cao, Limin; Szoka, Francis C.

2009-01-01

25

SnAvi - a new tandem tag for high-affinity protein-complex purification  

PubMed Central

Systematic tandem-affinity-purification (TAP) of protein complexes was tremendously successful in yeast and has changed the general concept of how we understand protein function in eukaryotic cells. The transfer of this method to other model organisms has been difficult and may require specific adaptations. We were especially interested to establish a cell-type-specific TAP system for Caenorhabditis elegans, a model animal well suited to high-throughput analysis, proteomics and systems biology. By combining the high-affinity interaction between in vivo biotinylated target-proteins and streptavidin with the usage of a newly identified epitope of the publicly shared SB1 monoclonal antibody we created a novel in vivo fluorescent tag, the SnAvi-Tag. We show the versatile application of the SnAvi-Tag in Escherichia coli, vertebrate cells and in C. elegans for tandem affinity purification of protein complexes, western blotting and also for the in vivo sub-cellular localization of labelled proteins.

Schaffer, Ursula; Schlosser, Andreas; Muller, Kristian M.; Schafer, Angelika; Katava, Nenad; Baumeister, Ralf; Schulze, Ekkehard

2010-01-01

26

Microbial polyhydroxyalkanote synthesis repression protein PhaR as an affinity tag for recombinant protein purification  

Microsoft Academic Search

BACKGROUND: PhaR which is a repressor protein for microbial polyhydroxyalkanoates (PHA) biosynthesis, is able to attach to bacterial PHA granules in vivo, was developed as an affinity tag for in vitro protein purification. Fusion of PhaR-tagged self-cleavable Ssp DnaB intein to the N-terminus of a target protein allowed protein purification with a pH and temperature shift. During the process, the

Shuang Zhang; Zhi Hui Wang; Guo Qiang Chen

2010-01-01

27

The Monitoring and Affinity Purification of Proteins Using Dual-Tags with Tetracysteine Motifs  

SciTech Connect

Identification and characterization of protein-protein interaction networks is essential for the elucidation of biochemical mechanisms and cellular function. Affinity purification in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a very powerful tactic for the identification of specific protein-protein interactions. In this chapter we describe a comprehensive methodology that utilizes our recently developed dual-tag affinity purification system for the enrichment and identification of mammalian protein complexes. The protocol covers a series of separate but sequentially related techniques focused on the facile monitoring and purification of a dual-tagged protein of interest and its interacting partners via a system built with tetracysteine motifs and various combinations of affinity tags. Using human telomeric repeat binding factor 2 (TRF2) as an example, we have demonstrated the power of the system in terms of bait protein recovery after dual-tag affinity purification, detection of bait protein subcellular localization and expression, and successful identification of known and potentially novel TRF2 interacting proteins. Although the protocol described here has been optimized for the identification and characterization of TRF2-associated proteins, it is, in principle, applicable to the study of any other mammalian protein complexes that may be of interest to the research community.

Giannone, Richard J [ORNL; Liu, Yie [ORNL; Wang, Yisong [ORNL

2009-01-01

28

Quantification in proteomics through stable isotope coding: a review.  

PubMed

This review focuses on techniques for quantification and identification in proteomics by stable isotope coding. Methods are examined for analyzing expression, post-translational modifications, protein:protein interactions, single amino acid polymorphism, and absolute quantification. The bulk of the quantification literature in proteomics focuses on expression analysis, where a wide variety of methods targeting different features of proteins are described. Methods for the analysis of post-translational modification (PTM) focus primarily on phosphorylation and glycosylation, where quantification is achieved in two ways, either by substitution or tagging of the PTM with an isotopically coded derivatizing agent in a single process or by coding and selecting PTM modified peptides in separate operations. Absolute quantification has been achieved by age-old internal standard methods, in which an isotopically labeled isoform of an analyte is synthesized and added to a mixture at a known concentration. One of the surprises is that isotope coding can be a valuable aid in the examination of intermolecular association of proteins through stimulus:response studies. Preliminary efforts to recognize single amino acid polymorphism are also described. The review ends with the conclusion that (1) isotope ratio analysis of protein concentration between samples does not necessarily relate directly to protein expression and rate of PTM and (2) that multiple new methods must be developed and applied simultaneously to make existing stable isotope quantification methods more meaningful. Although stable isotope coding is a powerful, wonderful new technique, multiple analytical issues must be solved for the technique to reach its full potential as a tool to study biological systems. PMID:15253416

Julka, Samir; Regnier, Fred

29

A dual-tagging system for the affinity purification of mammalian protein complexes  

SciTech Connect

One popular method to elucidate protein-protein interactions involves the native co-purification of an affinity tagged protein and its interacting partners, which are subsequently identified through mass spectrometry (MS) (1). Although straightforward, reproducible, and broadly employed, this strategy is hampered by the efficacy of protein recoveries both in terms of sensitivity and specificity. This is especially pertinent to methodologies that employ a single-step of purification, where suboptimal enrichment of the bait protein and its partners over background can lead to masking of their signals. Although improvements to MS instrumentation generally increase peptide detection sensitivities, the problem of specificity, i.e. distinguishing specific from non-specific interacting proteins, remains. Thus ultimately, the limiting factor in the identification of specific interacting proteins lies with the purification itself. An effort to resolve this specificity issue has been made with the introduction of the Tandem Affinity Purification (TAP) tag. This construct consists of an IgG-binding domain and calmodulin binding peptide domain separated by a tobacco etch virus (TEV) protease cleavage site (2). The TAP method was originally developed in yeast and has best demonstrated its utility in the systematic identification of numerous multiprotein complexes in the yeast proteome (3). Although modifications to the original TAP have been successful in examining the protein networks of mammalian cells (4-7), the strategy offers a relatively low yield of bait and specific interacting proteins (8), and the success rate are usually on case-by-case basis. In addition, problems inherent to any protein tagging strategy remain, such as variable exposure of the affinity tag, disruption of the bait protein's ability to fold properly, steric exclusion of interacting partners, and/or ectopic overexpression of the fusion protein, which can lead to complications in both the purification and identification of true interactions.

Giannone, Richard J [ORNL; McDonald, W Hayes [ORNL; Hurst, Gregory {Greg} B [ORNL; Huang, Ying [ORNL; Wu, Jun [ORNL; Liu, Yie [National Institute on Aging, Baltimore; Wang, Yisong [ORNL

2007-01-01

30

Application of Ni(II)-Assisted Peptide Bond Hydrolysis to Non-Enzymatic Affinity Tag Removal  

PubMed Central

In this study, we demonstrate a non-enzymatic method for hydrolytic peptide bond cleavage, applied to the removal of an affinity tag from a recombinant fusion protein, SPI2-SRHWAP-His6. This method is based on a highly specific Ni(II) reaction with (S/T)XHZ peptide sequences. It can be applied for the protein attached to an affinity column or to the unbound protein in solution. We studied the effect of pH, temperature and Ni(II) concentration on the efficacy of cleavage and developed an analytical protocol, which provides active protein with a 90% yield and ?100% purity. The method works well in the presence of non-ionic detergents, DTT and GuHCl, therefore providing a viable alternative for currently used techniques.

Kopera, Edyta; Belczyk-Ciesielska, Agnieszka; Bal, Wojciech

2012-01-01

31

Application of Ni(II)-assisted peptide bond hydrolysis to non-enzymatic affinity tag removal.  

PubMed

In this study, we demonstrate a non-enzymatic method for hydrolytic peptide bond cleavage, applied to the removal of an affinity tag from a recombinant fusion protein, SPI2-SRHWAP-His(6). This method is based on a highly specific Ni(II) reaction with (S/T)XHZ peptide sequences. It can be applied for the protein attached to an affinity column or to the unbound protein in solution. We studied the effect of pH, temperature and Ni(II) concentration on the efficacy of cleavage and developed an analytical protocol, which provides active protein with a 90% yield and ?100% purity. The method works well in the presence of non-ionic detergents, DTT and GuHCl, therefore providing a viable alternative for currently used techniques. PMID:22574150

Kopera, Edyta; Belczyk-Ciesielska, Agnieszka; Bal, Wojciech

2012-05-04

32

The ARiBo tag: a reliable tool for affinity purification of RNAs under native conditions  

PubMed Central

Although RNA-based biological processes and therapeutics have gained increasing interest, purification of in vitro transcribed RNA generally relies on gel-based methods that are time-consuming, tedious and denature the RNA. Here, we present a reliable procedure for affinity batch purification of RNA, which exploits the high-affinity interaction between the boxB RNA and the N-peptide from bacteriophage ?. The RNA of interest is synthesized with an ARiBo tag, which consists of an activatable ribozyme (the glmS ribozyme) and the ?BoxB RNA. This ARiBo-fusion RNA is initially captured on Glutathione-Sepharose resin via a GST/?N-fusion protein, and the RNA of interest is subsequently eluted by ribozyme self-cleavage using glucosamine-6-phosphate. Several GST/?N-fusion proteins and ARiBo tags were tested to optimize RNA yield and purity. The optimized procedure enables one to quickly obtain (3?h) highly pure RNA (>99%) under native conditions and with yields comparable to standard denaturing gel-based protocols. It is widely applicable to a variety of RNAs, including riboswitches, ribozymes and microRNAs. In addition, it can be easily adapted to a wide range of applications that require RNA purification and/or immobilization, including isolation of RNA-associated complexes from living cells and high-throughput applications.

Di Tomasso, Genevieve; Lampron, Philipe; Dagenais, Pierre; Omichinski, James G.; Legault, Pascale

2011-01-01

33

MHC Class II Tetramers Made from Isolated Recombinant ? and ? Chains Refolded with Affinity-Tagged Peptides  

PubMed Central

Targeting CD4+ T cells through their unique antigen-specific, MHC class II-restricted T cell receptor makes MHC class II tetramers an attractive strategy to identify, validate and manipulate these cells at the single cell level. Currently, generating class II tetramers is a specialized undertaking effectively limiting their use and emphasizing the need for improved methods of production. Using class II chains expressed individually in E. coli as versatile recombinant reagents, we have previously generated peptide-MHC class II monomers, but failed to generate functional class II tetramers. Adding a monomer purification principle based upon affinity-tagged peptides, we here provide a robust method to produce class II tetramers and demonstrate staining of antigen-specific CD4+ T cells. We also provide evidence that both MHC class II and T cell receptor molecules largely accept affinity-tagged peptides. As a general approach to class II tetramer generation, this method should support rational CD4+ T cell epitope discovery as well as enable specific monitoring and manipulation of CD4+ T cell responses.

?sterbye, Thomas; Nielsen, Lise Lotte Bruun; Mallone, Roberto; Vindel?v, Lars; Stryhn, Anette; Buus, S?ren

2013-01-01

34

Tandem affinity purification of protein complexes from mammalian cells by the Strep/FLAG (SF)-TAP tag.  

PubMed

Isolation and dissection of native multiprotein complexes is a central theme in functional genomics. The development of the tandem affinity purification (TAP) tag has enabled efficient and large-scale purification of native protein complexes. The SF-TAP tag, a modified version of the TAP tag, allows a fast and straightforward purification of protein complexes from mammalian cells. It consists of a tandem Strep-tag II and a FLAG epitope (SF-TAP). The SF-TAP tag allows a native elution of protein complexes without proteolytic cleavage needed in the original TAP procedure. Besides the SF-TAP protocol, the principal idea of a pathway mapping by subsequent tagging of copurified proteins is demonstrated for the interactome of the MAPKKK Raf. PMID:19544034

Gloeckner, Christian Johannes; Boldt, Karsten; Schumacher, Annette; Ueffing, Marius

2009-01-01

35

Expression of the affinity tags, glutathione-S-transferase and maltose-binding protein, in tobacco chloroplasts.  

PubMed

Chloroplast transformation offers an exciting platform for the safe, inexpensive and large-scale production of recombinant proteins in plants. An important advantage for the isolation of proteins produced in the chloroplast would be the use of affinity tags for rapid purification by affinity chromatography. To date, only His-tags have been used. In this study, we have tested the feasibility of expressing two additional affinity tags: glutathione-S-transferase (GST) and a His-tagged derivative of the maltose-binding protein (His?-MBP). By using the chloroplast 16S rRNA promoter and 5' untranslated region of phage T7 gene 10, GST and His?-MBP were expressed in homoplastomic tobacco plants at approximately 7% and 37% of total soluble protein, respectively. GST could be purified by one-step-affinity purification using a glutathione column. Much better recoveries were obtained for His?-MBP by using a twin-affinity purification procedure involving first immobilised nickel followed by binding to amylose. Interestingly, expression of GST led to cytoplasmic male sterility. Overall, our work expands the tools available for purifying recombinant proteins from the chloroplast. PMID:22237946

Ahmad, Niaz; Michoux, Franck; McCarthy, James; Nixon, Peter J

2012-01-12

36

Probing the Mechanism of Electron Capture and Electron Transfer Dissociation Using Tags with Variable Electron Affinity  

PubMed Central

Electron capture dissociation (ECD) and electron transfer dissociation (ETD) of doubly protonated electron affinity (EA)-tuned peptides were studied to further illuminate the mechanism of these processes. The model peptide FQpSEEQQQTEDELQDK, containing a phosphoserine residue, was converted to EA-tuned peptides via ?-elimination and Michael addition of various thiol compounds. These include propanyl, benzyl, 4-cyanobenzyl, perfluorobenzyl, 3,5-dicyanobenzyl, 3-nitrobenzyl and 3,5-dinitrobenzyl structural moieties, having a range of EA from -1.15 to 1.65 eV, excluding the propanyl group. Typical ECD or ETD backbone fragmentations are completely inhibited in peptides with substituent tags having EA over 1.00 eV, which are referred to as electron predators in this work. Nearly identical rates of electron capture by the dications substituted by the benzyl (EA = -1.15 eV) and 3-nitrobenzyl (EA = 1.00 eV) moieties are observed, which indicates the similarity of electron capture cross sections for the two derivatized peptides. This observation leads to the inference that electron capture kinetics are governed by the long range electron-dication interaction and are not affected by side chain derivatives with positive EA. Once an electron is captured to high-n Rydberg states, however, through-space or through-bond electron transfer to the EA-tuning tags or low-n Rydberg states via potential curve crossing occurs in competition with transfer to the amide ?* orbital. The energetics of these processes are evaluated using time-dependent density functional theory with a series of reduced model systems. The intramolecular electron transfer process is modulated by structure-dependent hydrogen bonds and is heavily affected by the presence and type of electron withdrawing groups in the EA-tuning tag. The anion radicals formed by electron predators have high proton affinities (approximately 1400 kJ/mol for the 3-nitrobenzyl anion radical) in comparison to other basic sites in the model peptide dication, facilitating exothermic proton transfer from one of the two sites of protonation. This interrupts the normal sequence of events in ECD or ETD leading to backbone fragmentation by forming a stable radical intermediate. The implications which these results have for previously proposed ECD and ETD mechanisms are discussed.

Sohn, Chang Ho; Chung, Cheol K.; Yin, Sheng; Ramachandran, Prasanna; Loo, Joseph A.; Beauchamp, J. L.

2009-01-01

37

An Isotopically Coded CID-cleavable Biotinylated Cross-linker for Structural Proteomics*  

PubMed Central

Successful application of cross-linking combined with mass spectrometry for structural proteomics demands specifically designed cross-linking reagents to address challenges in the detection and assignment of cross-links. A combination of affinity enrichment, isotopic coding, and cleavage of the cross-linker is beneficial for detection and identification of the peptide cross-links. Here we describe a novel cross-linker, cyanurbiotindipropionylsuccinimide (CBDPS), that allows affinity enrichment of cross-linker-containing peptides with avidin. Affinity enrichment eliminates interfering non-cross-linked peptides and allows the researcher to focus on the analysis of the cross-linked peptides. CBDPS is also isotopically coded and CID-cleavable. The cleaved fragments still contain a portion of the isotopic label and can therefore be distinguished from unlabeled fragments by their distinct isotopic signatures in the MS/MS spectra. This cleavage information has been incorporated into a program for the automatic analysis of the MS/MS spectra of the cross-links. This allows rapid determination of cross-link type in addition to facilitating identification of the individual peptides constituting the interpeptide cross-links. Thus, affinity enrichment combined with isotopic coding and CID cleavage allows in-depth mass spectrometric analysis of the peptide cross-links. We have characterized the performance of CBDPS on the 120-kDa protein heterodimer of HIV reverse transcriptase.

Petrotchenko, Evgeniy V.; Serpa, Jason J.; Borchers, Christoph H.

2011-01-01

38

One-step metal-affinity purification of histidine-tagged proteins by temperature-triggered precipitation  

Microsoft Academic Search

The feature of elastin-like proteins (ELPs) to reversibly precipitate above their transition temperature was exploited as a general method for the purification of histidine (His)-tagged proteins. The principle of the single-step metal-affinity method is based on coordi- nated ligand-bridging between the modified ELPs and the target proteins. ELPs with repeating sequences of ((VPGVG)2(VPGKG)(VPGVG)2)21 were synthesized and the free amino groups

Hana Stiborova; Jan Kostal; Ashok Mulchandani; Wilfred Chen

2003-01-01

39

Vectors for expression of proteins with single or combinatorial fluorescent protein and tandem affinity purification tags in Dictyostelium  

PubMed Central

We constructed a series of expression vectors for purification of native proteins and protein complexes in Dictyostelium. Protein purification is achieved by either a C-terminal or N-terminal fusion of the protein of choice to the tandem affinity purification (TAP) tag. The TAP tag consists of a protein A tag and a calmodulin binding peptide (CBP) and has been successfully used for purification of native protein complexes from yeast and animal cells. Protein expression is driven by the constitutive actin 15 promoter and the vectors optionally carry additional green- or yellow fluorescent protein (GFP or YFP) tags for fusion at either a C- or N-terminal location. Tandem affinity purification of native Dictyostelium protein complexes was tested by using pArc-34, one of the members of the well characterized Dictyostelium Arp2/3 complex, as bait. After denaturation and SDS–PAGE separation of the pArc-34 associated proteins all members of the Arp2/3 complex could be identified.

Meima, Marcel E.; Weening, Karin E.; Schaap, Pauline

2007-01-01

40

Strategy for the use of Affinity Grids to prepare non-His-tagged macromolecular complexes for single-particle electron microscopy  

PubMed Central

Affinity Grids are electron microscopy (EM) grids with a pre-deposited lipid monolayer containing functionalized Nickel-nitrilotriacetic acid (Ni-NTA) lipids. Affinity Grids can be used to prepare His-tagged proteins for single-particle EM from impure solutions or even directly from cell extracts. Here we introduce the concept of His-tagged adaptor molecules, which eliminate the need for the target protein or complex to be His-tagged. The use of His-tagged protein A as adaptor molecule allows Affinity Grids to be used for the preparation of virtually any protein or complex provided that a specific antibody is available or can be raised against the target protein. The principle is that the Affinity Grid is coated with a specific antibody that is recruited to the grid by His-tagged protein A. The antibody-decorated Affinity Grid can then be used to isolate the target protein directly from a cell extract. We first established this approach by preparing negatively stained specimens of both native ribosomal complexes and ribosomal complexes carrying different purification tags directly from HEK-293T cell extract. We then used the His-tagged protein A/antibody strategy to isolate RNA polymerase II (RNAP II), still bound to native DNA, from HEK-293T cell extract, allowing us to calculate a 25-Å resolution density map by single-particle cryo-EM.

Kelly, Deborah F.; Dukovski, Danijela; Walz, Thomas

2010-01-01

41

Immuno-qPCR detection of the tandem affinity purification (TAP)-tag as a sensitive and accurate tool suitable for large-scale protein quantification  

Microsoft Academic Search

The tandem affinity purification (TAP)-tag has rapidly gained a wide popularity, mostly in studies on protein interactions, but lately also in large-scale protein quantification studies. We have developed an immuno-quantitative real-time PCR (qPCR) method to achieve rapid, sensitive and accurate quantification of TAP-tagged (and protein A-tagged) proteins in yeast with a detection range between 10 7 and 10 10 molecules.

Kristina Lind; Joakim Norbeck

2007-01-01

42

In-house preparation of hydrogels for batch affinity purification of glutathione S-transferase tagged recombinant proteins  

PubMed Central

Background Many branches of biomedical research find use for pure recombinant proteins for direct application or to study other molecules and pathways. Glutathione affinity purification is commonly used to isolate and purify glutathione S-transferase (GST)-tagged fusion proteins from total cellular proteins in lysates. Although GST affinity materials are commercially available as glutathione immobilized on beaded agarose resins, few simple options for in-house production of those systems exist. Herein, we describe a novel method for the purification of GST-tagged recombinant proteins. Results Glutathione was conjugated to low molecular weight poly(ethylene glycol) diacrylate (PEGDA) via thiol-ene “click” chemistry. With our in-house prepared PEGDA:glutathione (PEGDA:GSH) homogenates, we were able to purify a glutathione S-transferase (GST) green fluorescent protein (GFP) fusion protein (GST-GFP) from the soluble fraction of E. coli lysate. Further, microspheres were formed from the PEGDA:GSH hydrogels and improved protein binding to a level comparable to purchased GSH-agarose beads. Conclusions GSH containing polymers might find use as in-house methods of protein purification. They exhibited similar ability to purify GST tagged proteins as purchased GSH agarose beads.

2012-01-01

43

Separation and identification of four distinct serine-phosphorylation states of ovalbumin by Phos-tag affinity electrophoresis.  

PubMed

Ovalbumin (OVA) derived from egg white contains two residues that can be phosphorylated: Ser-68 and Ser-344. Native polyacrylamide gel electrophoresis(PAGE) shows the presence of three distinct migration bands corresponding to phosphorylation states with two, one, or no phosphate groups, respectively. Phosphate-affinity sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Zn(2+)-Phos-tag SDS-PAGE), on the other hand, showed the presence of four distinct phosphorylated states in intact OVA. In addition to the diphosphorylated and nonphosphorylated forms, two distinct species, one with a phosphate group at Ser-68 and one with a phosphate group at Ser-344, were separately visualized. The content of the OVA monophosphorylated at Ser-68 was greater than that of OVA monophosphorylated at Ser-344. Zn(2+)-Phos-tag SDS-PAGE is therefore a useful method for the quantitative analysis of the detailed phosphorylation status of food proteins. PMID:22522539

Kinoshita-Kikuta, Emiko; Kinoshita, Eiji; Koike, Tohru

2012-03-01

44

Metal affinity engineering of proinsulin carrying genetically attached (His)10-X-Met affinity tail and removal of the tag by cyanogen bromide.  

PubMed

An E. coli expression clone coding for human proinsulin, which was fused to NH2-terminal beta-galactosidase, was engineered for the separation from host proteins by introducing peptide devices, and for the sequential removal of the fused polypeptide by cyanogen bromide in front of the NH2 terminal residue (methionine) of the human proinsulin gene. Short synthetic genes encoding oligopeptide residues including (Glu)n, (His)n, (Trp)n, and (Ser)n (n = 10 or 11), which have certain characteristic physical properties such as metal-affinity, polarity, hydrophobicity, and hydrophilicity, respectively, were inserted at the junction region of the gene fusion. Interestingly, it was found that among the oligopeptides, the oligohistidine residue as an affinity-tag has greatly facilitated the procedures for FPI purification, particularly in the manner of selective metal-affinity precipitation. The chelating peptide covering the NH2-terminal beta-galactosidase portion could then be removed simply after purification to generate a protein with the natural amino acid sequence of proinsulin by cyanogen bromide. PMID:7765485

Ko, J H; Chung, W J; Koh, S; Park, B C; Kwon, S T; Kim, C H; Lee, D S

1994-09-01

45

Natural poly-histidine affinity tag for purification of recombinant proteins on cobalt(II)-carboxymethylaspartate crosslinked agarose.  

PubMed

A natural 19-amino-acid poly-histidine affinity tag was cloned at the N-terminus of three recombinant proteins. The vectors containing the DNA of the fusion proteins were used for transformation of Escherichia coli DH5alpha cells. Each protein was expressed, extracted and purified in one chromatographic step. The purification procedure for each protein can be accomplished in less than 1 h. A new type of immobilized metal ion affinity chromatography adsorbent--Co2+-carboxymethylaspartate agarose Superflow--was utilized at linear flow-rates as high as 5 cm/min. The final preparation of each protein is with purity greater than 95% as ascertained by sodium dodecyl sulfate-electrophoresis. Recovery for each purified protein was higher than 77% of the initial loaded amount as judged by biological activity. The operational capacity of Co2+-carboxymethylaspartate agarose for each protein was determined. PMID:10669292

Chaga, G; Bochkariov, D E; Jokhadze, G G; Hopp, J; Nelson, P

1999-12-24

46

Purification of histidine-tagged nucleocapsid protein of Nipah virus using immobilized metal affinity chromatography  

Microsoft Academic Search

Nucleocapsid (N) protein of Nipah virus (NiV) is a potential serological marker used in the diagnosis of NiV infections. In this study, a rapid and efficient purification system, HisTrap™ 6 Fast Flow packed bed column was applied to purify recombinant histidine-tagged N protein of NiV from clarified feedstock. The optimizations of binding and elution conditions of N protein of NiV

Fui Chin Chong; Wen Siang Tan; Dayang Radiah Awang Biak; Tau Chuan Ling; Beng Ti Tey

2009-01-01

47

A novel recombinant system for functional expression of myonecrotic snake phospholipase A 2 in Escherichia coli using a new fusion affinity tag  

Microsoft Academic Search

A novel recombinant expression system in Escherichia coli was developed using conger eel galectin, namely, congerin II, as an affinity tag. This system was applied for the functional expression of myotoxic lysine-49-phospholipase A2 ([Lys49]PLA2), termed BPII and obtained from Protobothrops flavoviridis (Pf) venom. Recombinant Pf BPII fused with a congerin tag has been successfully expressed as a soluble fraction and

Minae Seto; Tomohisa Ogawa; Kyousuke Kodama; Koji Muramoto; Yoshitaka Kanayama; Yasuo Sakai; Takahito Chijiwa; Motonori Ohno

2008-01-01

48

Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element  

Microsoft Academic Search

A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce

Shaorong Chong; Fana B Mersha; Donald G Comb; Melissa E Scott; David Landry; Luis M Vence; Francine B Perler; Jack Benner; Rebecca B Kucera; Christine A Hirvonen; John J Pelletier; Henry Paulus; Ming-Qun Xu

1997-01-01

49

Application of metal-coded affinity tags (MeCAT): absolute protein quantification with top-down and bottom-up workflows by metal-coded tagging.  

PubMed

As the quantification of peptides and proteins extends from comparative analyses to the determination of actual amounts, methodologies for absolute protein quantification are desirable. Metal-coded affinity tags (MeCAT) are chemical labels for peptides and proteins with a lanthanide-bearing chelator as a core. This modification of analytes with non-naturally occurring heteroelements adds the analytical possibilities of inductively coupled plasma mass spectrometry (ICPMS) to quantitative proteomics. We here present the absolute quantification of recombinantly expressed aprotinin out of its host cell protein background using two independent MeCAT methodologies. A bottom-up strategy employs labeling of primary amino groups on peptide level. Synthetic peptides with a MeCAT label which are externally quantified by flow injection analysis (FIA)-ICPMS serve as internal standard in nanoHPLC-ESI-MS/MS. In the top-down approach, protein is labeled on cysteine residues and separated by two-dimensional gel electrophoresis. Flow injection analysis of dissolved gel spots by ICPMS yields the individual protein amount via its lanthanide label content. The enzymatic determination of the fusion protein via its ?-galactosidase activity found 8.3 and 9.8 ng/?g (nanogram fusion protein per microgram sample) for batches 1 and 2, respectively. Using MeCAT values of 4.0 and 5.4 ng/?g are obtained for top-down analysis, while 14.5 and 15.9 ng/?g were found in the bottom-up analysis. PMID:22659083

Bergmann, U; Ahrends, R; Neumann, B; Scheler, C; Linscheid, M W

2012-05-31

50

Binuclear Ni(II)-DpaTyr complex as a high affinity probe for an oligo-aspartate Tag tethered to proteins.  

PubMed

A complementary recognition pair of a short-peptide tag and a small molecular probe is a versatile molecular tool for protein detection, handling, and purification, and so forth. In this manuscript, we report that the binuclear Ni(II)-DpaTyr (DpaTyr=bis((dipicolylamino)methyl)tyrosine) complex serves as a strong binding probe for an oligo-aspartate tag tethered to a protein. Among various binuclear metal complexes of M-DpaTyr (M=Zn(II), Ni(II), Mn(II), Cu(II), Cd(II), Co(III), and Fe(III)), we have found that Ni(II)-DpaTyr (1-2Ni(II)) displays a strong-binding affinity (apparent binding constant: K(app) approximately 10(5) M(-1)) for an oligo-aspartate peptide under neutral aqueous conditions (50 mM HEPES, 100 mM NaCl, pH 7.2). Detailed isothermal-titration calorimetry (ITC) studies reveal that the tri-aspartate D3-tag (DDD) is an optimal sequence recognized by 1-2Ni(II) in a 1:1 binding stoichiometry. On the other hand, other metal complexes of DpaTyr, except for Ni(II)- and Zn(II)-DpaTyr, show a negligible binding affinity for the oligo-aspartate peptide. The binding affinity was greatly enhanced in the pair between the dimer of Ni(II)-DpaTyr and the repeated D3 tag peptide (D3x2), such as DDDXXDDD, on the basis of the multivalent coordination interaction between them. Most notably, a remarkably high-binding affinity (K(app)=2x10(9) M(-1)) was achieved between the Ni(II)-DpaTyr dimer 4-4Ni(II) and the D3x2 tag peptide (DDDNGDDD). This affinity is approximately 100-fold stronger than that observed in the binding pair of the Zn(II)-DpaTyr (4-4Zn(II)) and the D4x2 tag (DDDDGDDDD), a useful tag-probe pair previously reported by us. The recognition pair of the Ni(II)-DpaTyr probe and the D3x2 tag can also work effectively on a protein surface, that is, 4-4Ni(II) is strongly bound to the FKBP12 protein tethered with the D3x2 tag (DDDNGDDD) with a large K(app) value of 5x10(8) M(-1). Taking advantage of the strong-binding affinity, this pair was successfully applied to the selective inactivation of the tag-fused beta-galactosidase by using the chromophore-assisted light inactivation (CALI) technique under crude conditions, such as cell lysate. PMID:20143369

Ojida, Akio; Fujishima, Sho-Hei; Honda, Kei; Nonaka, Hiroshi; Uchinomiya, Sho-Hei; Hamachi, Itaru

2010-04-01

51

A Modified Metal-Ion Affinity Chromatography Procedure for the Purification of Histidine-Tagged Recombinant Proteins Expressed in Drosophila S2 Cells  

Microsoft Academic Search

We have developed a modified method of immobilized metal-ion affinity chromatography (IMAC) that can be used for the purification of histidine-tagged proteins from conditioned medium containing free copper ions. Classical methods of IMAC purification, using resins such as Ni–NTA, have proven inefficient for this type of purification and require multiple steps due to the interference of divalent copper ions with

Ruth V. Lehr; Louis C. Elefante; Kristine K. Kikly; Shawn P. O'Brien; Robert B. Kirkpatrick

2000-01-01

52

Affinity binding via Zinc(II) for controlled orientation and electrochemistry of Histidine-tagged nitrate reductase in self-assembled monolayers.  

PubMed

Monolayers of Cu(II)-complexes on electrode surfaces are frequently applied for the immobilization and controlled orientation of His-tagged redox proteins. However, affinity binding is limited to applications that require potentials less negative than the reduction potential of the metal complexes. In order to overcome this limitation, we used Zn(2+) cations on nitrilotriacetic acid (NTA) modified carbon electrodes for the coordination of His-tagged nitrate reductase (NaR). The NTA modified electrodes were prepared upon diazotation and electrochemical reduction of an aniline functionalized NTA ligand. After coordination of Zn(2+) to the bound NTA ligand, self-assembly of NaR is achieved via coordination of the imidazole groups from the His-tag to the NTA-Zn(II) complex. The electrochemical investigations of the NaR monolayer on NTA-Zn(II) films demonstrate the catalytic activity for reduction of nitrate to nitrite in the presence of methyl viologen. The catalytic current density correlates with the one expected for a fully active enzyme monolayer. Moreover, the reduction of Zn(2+) is not observed at the potential necessary for the reduction of methyl viologen. Therefore, affinity binding based on Zn(2+) may be used for the immobilization and electrochemical applications of His-tagged NaR. PMID:22894913

Campbell, Wilbur H; Henig, Jörg; Plumeré, Nicolas

2012-07-23

53

A novel recombinant system for functional expression of myonecrotic snake phospholipase A(2) in Escherichia coli using a new fusion affinity tag.  

PubMed

A novel recombinant expression system in Escherichia coli was developed using conger eel galectin, namely, congerin II, as an affinity tag. This system was applied for the functional expression of myotoxic lysine-49-phospholipase A(2) ([Lys(49)]PLA(2)), termed BPII and obtained from Protobothrops flavoviridis (Pf) venom. Recombinant Pf BPII fused with a congerin tag has been successfully expressed as a soluble fraction and showed better quantitative yield when folded correctly. The solubility of the recombinant congerin II-tagged BPII increased up to >90% in E. coli strain JM109 when coexpressed with the molecular chaperones GroEL, GroES, and trigger factor (Tf). The tag protein was cleaved by digestion with restriction protease, such as alpha-thrombin or Microbacterium liquefaciens protease (MLP), to obtain completely active recombinant BPII. Thus, the congerin-tagged fusion systems containing the cleavage recognition site for alpha-thrombin or MLP were demonstrated to be highly efficient and useful for producing proteins of desired solubility and activity. PMID:18207418

Seto, Minae; Ogawa, Tomohisa; Kodama, Kyousuke; Muramoto, Koji; Kanayama, Yoshitaka; Sakai, Yasuo; Chijiwa, Takahito; Ohno, Motonori

2007-12-08

54

Tandem Affinity Tags for the Purification of Bivalent Anti-DNA Single-Chain Fv Expressed in Escherichia coli  

Microsoft Academic Search

Antibodies to DNA define an important autospecificity that arises in systemic lupus erythematosus (SLE). To elucidate the molecular features that may explain the pathogenesis of SLE, a heterologous system for expression of cloned V genes is often desirable. Here, a single-chain Fv coding domain was constructed by using the heavy- and light-chain V genes of a high-affinity site-directed mutant of

Brian A. Cocca; Samarendra N. Seal; Marko Z. Radic

1999-01-01

55

Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications.  

PubMed

Identifying sites of post-translational modifications on proteins is a major challenge in proteomics. O-Linked beta-N-acetylglucosamine (O-GlcNAc) is a dynamic nucleocytoplasmic modification more analogous to phosphorylation than to classical complex O-glycosylation. We describe a mass spectrometry-based method for the identification of sites modified by O-GlcNAc that relies on mild beta-elimination followed by Michael addition with dithiothreitol (BEMAD). Using synthetic peptides, we also show that biotin pentylamine can replace dithiothreitol as the nucleophile. The modified peptides can be efficiently enriched by affinity chromatography, and the sites can be mapped using tandem mass spectrometry. This same methodology can be applied to mapping sites of serine and threonine phosphorylation, and we provide a strategy that uses modification-specific antibodies and enzymes to discriminate between the two post-translational modifications. The BEMAD methodology was validated by mapping three previously identified O-GlcNAc sites, as well as three novel sites, on Synapsin I purified from rat brain. BEMAD was then used on a purified nuclear pore complex preparation to map novel sites of O-GlcNAc modification on the Lamin B receptor and the nucleoporin Nup155. This method is amenable for performing quantitative mass spectrometry and can also be adapted to quantify cysteine residues. In addition, our studies emphasize the importance of distinguishing between O-phosphate versus O-GlcNAc when mapping sites of serine and threonine post-translational modification using beta-elimination/Michael addition methods. PMID:12438562

Wells, Lance; Vosseller, Keith; Cole, Robert N; Cronshaw, Janet M; Matunis, Michael J; Hart, Gerald W

2002-10-01

56

Observed octameric assembly of a Plasmodium yoelii peroxiredoxin can be explained by the replacement of native "ball-and-socket" interacting residues by an affinity tag.  

PubMed

Peroxiredoxins (Prxs) are ubiquitous and efficient antioxidant enzymes crucial for redox homeostasis in most organisms, and are of special importance for disease-causing parasites that must protect themselves against the oxidative weapons of the human immune system. Here, we describe reanalyses of crystal structures of two Prxs from malaria parasites. In addition to producing improved structures, we provide normalizing explanations for features that had been noted as unusual in the original report of these structures (Qiu et al., BMC Struct Biol 2012;12:2). Most importantly, we provide evidence that the unusual octameric assembly seen for Plasmodium yoelii Prx1a is not physiologically relevant, but arises because the structure is not of authentic P. yoelii Prx1a, but a variant we designate PyPrx1a(N*) that has seven native N-terminal residues replaced by an affinity tag. This N-terminal modification disrupts a previously unrecognized, hydrophobic "ball-and-socket" interaction conserved at the B-type dimer interface of Prx1 subfamily enzymes, and is accommodated by a fascinating two-residue "?-slip" type register shift in the ?-strand association at a dimer interface. The resulting change in the geometry of the dimer provides a simple explanation for octamer formation. This study illustrates how substantive impacts can occur in protein variants in which native residues have been altered. PMID:23934758

Gretes, Michael C; Karplus, P Andrew

2013-10-01

57

A Method to Site-Specifically Identify and Quantitate Carbonyl End Products of Protein Oxidation Using Oxidation-Dependent Element Coded Affinity Tags (O-ECAT) and NanoLiquid Chromatography Fourier Transform Mass Spectrometry  

SciTech Connect

Protein oxidation is linked to cellular stress, aging, and disease. Protein oxidations that result in reactive species are of particular interest, since these reactive oxidation products may react with other proteins or biomolecules in an unmediated and irreversible fashion, providing a potential marker for a variety of disease mechanisms. We have developed a novel system to identify and quantitate, relative to other states, the sites of oxidation on a given protein. A specially designed Oxidation-dependent carbonyl-specific Element-Coded Affinity Mass Tag (O-ECAT), AOD, ((S)-2-(4-(2-aminooxy)-acetamido)-benzyl)-1, 4, 7, 10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid, is used to covalently tag the residues of a protein oxidized to aldehyde or keto end products. After proteolysis, the resulting AOD-tagged peptides are affinity purified, and analyzed by nanoLC-FTICR-MS, which provides high specificity in extracting co-eluting AOD mass pairs with a unique mass difference and affords relative quantitation based on isotopic ratios. Using this methodology, we have mapped the surface oxidation sites on a model protein, recombinant human serum albumin (rHSA) in its native form (as purchased) and after FeEDTA oxidation. A variety of modified amino acid residues including lysine, arginine, proline, histidine, threonine, aspartic and glutamic acids, were found to be oxidized to aldehyde and keto end products. The sensitivity of this methodology is shown by the number of peptides identified, twenty peptides on the native protein and twenty-nine after surface oxidation using FeEDTA and ascorbate. All identified peptides map to the surface of the HSA crystal structure validating this method for identifying oxidized amino acids on protein surfaces. In relative quantitation experiments between FeEDTA oxidation and native protein oxidation, identified sites showed different relative propensities towards oxidation independent of amino acid residue. We expect to extend this methodology to study disease-related oxidation systems.

Lee, S; Young, N L; Whetstone, P A; Cheal, S M; Benner, W H; Lebrilla, C B; Meares, C F

2005-08-25

58

In situ activation of caspases and serine proteases during apoptosis detected by affinity labeling their enzyme active centers with fluorochrome-tagged inhibitors  

Microsoft Academic Search

Activation of caspases is the key event of apoptosis. To detect this event in situ we applied flu- orochrome-labeled inhibitors of caspases (FLICA) as affinity labels of active centers of these enzymes. The FLICA are fluorescein- or sulforhodamine-conjugated peptide-fluoromethyl ketones that covalently, with 1:1 stoichiometry, bind to enzymatic centers of caspases; the specificity is provided by the peptide sequence of

Jerzy Grabarek; Zbigniew Darzynkiewicz

2002-01-01

59

Variability in the immunodetection of His-tagged recombinant proteins  

Microsoft Academic Search

Labeling of recombinant proteins with polypeptide fusion partners, or affinity tagging, is a useful method to facilitate subsequent protein purification and detection. Poly-histidine tags (His-tags) are among the most commonly used affinity tags. We report strikingly variable immunodetection of two His-tagged recombinant human erythropoietins (Epo): wild type Epo (Epowt) and Epo containing an R103A mutation (EpoR103A). Both were engineered to

Nataša Debeljak; Laurie Feldman; Kerry L. Davis; Radovan Komel; Arthur J. Sytkowski

2006-01-01

60

Isotope Coded Protein Labeling analysis of plasma specimens from acute severe dengue fever patients  

PubMed Central

Background Dengue fever is the most important arthropod born viral disease of public health significance. Although most patients suffer only from flu-like symptoms, a small group of patient experiences more severe forms of the disease. To contribute to a better understanding of its pathogenesis this study aims to identify proteins differentially expressed in a pool of five viremic plasma from severe dengue patients relative to a pool of five non-severe dengue patients. Results The use of Isotope Coded Protein Labeling (ICPLTM) to analyze plasma depleted of twenty high-abundance proteins allowed for the identification of 51 differentially expressed proteins, which were characterized by mass spectrometry. Using quantitative ELISA, three of these proteins (Leucine-rich glycoprotein 1, Vitamin D binding-protein and Ferritin) were confirmed as having an increased expression in a panel of severe dengue plasma. The proteins identified as overexpressed by ICPLTM in severe dengue plasma involve in clear up action after cell injury, tissue coherence and immune defense. Conclusion This ICPLTM study evaluating differences between acute severe dengue plasmas and acute non-severe dengue plasmas suggests that the three proteins identified are overexpressed early in the course of the disease. Their possible use as biomarkers for the prognostic of disease severity is discussed.

2012-01-01

61

Protein Cross-Linking Analysis Using Mass Spectrometry, Isotope-Coded Cross-Linkers, and Integrated Computational Data Processing  

Microsoft Academic Search

Distance constraints in proteins and protein complexes provide invaluable information for calculation of 3D structures, identification of protein binding partners and localization of protein-protein contact sites. We have developed an integrative approach to identify and characterize such sites through the analysis of proteolytic products derived from proteins chemically cross-linked by isotopically coded cross-linkers using LC-MALDI tandem mass spectrometry and computer

Jan Seebacher; Parag Mallick; Ning Zhang; James S. Eddes; Ruedi Aebersold; Michael H. Gelb

2006-01-01

62

Stable isotope coded derivatizing reagents as internal standards in metabolite profiling.  

PubMed

Gas chromatography (GC) and liquid chromatography (LC) coupled to mass spectrometric (MS) detection have become the two main techniques for the analysis of metabolite pools (i.e. Metabolomics). These technologies are especially suited for Metabolite Profiling analysis of various metabolite groups due to high separation capabilities of the chromatographs and high sensitivity of the mass analysers. The trend in quantitative Metabolite Profiling is to add more metabolites and metabolite groups in a single method. This should not be done by compromising the analytical precision. Mass spectrometric detection comes with certain limitations, especially in the quantitative aspects as standards are needed for conversion of ion abundance to concentration and ionization efficiencies are directly dependent on eluent conditions. This calls for novel strategies to counteract all variables that can influence the quantitative precision. Usually, internal standards are used to correct any technical variation. For quantitation of single or just a few analytes this can be executed with spiking isotopically labeled standards. However, for more comprehensive analytical tasks, e.g. profiling tens or hundreds of analytes simultaneously, this strategy becomes expensive and in many cases isotopically labeled standards are not available. An alternative is to introduce a derivatizing step where the sample is derivatized with naturally labeled reagent, while a standard solution is separately derivatized with isotopically labeled reagent and spiked into the sample solution prior to analysis. This strategy, named isotope coded derivatization - ICD, is attractive in the emerging field of quantitative Metabolite Profiling where current protocols can easily comprise over hundred metabolites. This review provides an overview of isotopically labeled derivatizing reagents that have been developed for important metabolite groups with the aim to improve analytical performance and precision. PMID:23628173

Bruheim, Per; Kvitvang, Hans Fredrik Nyvold; Villas-Boas, Silas G

2013-04-09

63

Purification of proteins containing zinc finger domains using immobilized metal ion affinity chromatography  

Microsoft Academic Search

Heterologous proteins are frequently purified by immobilized metal ion affinity chromatography (IMAC) based on their modification with a hexa-histidine affinity tag (His-tag). The terminal His-tag can, however, alter functional properties of the tagged protein. Numerous strategies for the tag removal have been developed including chemical treatment and insertion of protease target sequences in the protein sequence. Instead of using these

Irena Vorá?ková; Šárka Suchanová; Pavel Ulbrich; William E. Diehl; Tomáš Ruml

2011-01-01

64

Deciphering systemic wound responses of the pumpkin extrafascicular phloem by metabolomics and stable isotope-coded protein labeling.  

PubMed

In cucurbits, phloem latex exudes from cut sieve tubes of the extrafascicular phloem (EFP), serving in defense against herbivores. We analyzed inducible defense mechanisms in the EFP of pumpkin (Cucurbita maxima) after leaf damage. As an early systemic response, wounding elicited transient accumulation of jasmonates and a decrease in exudation probably due to partial sieve tube occlusion by callose. The energy status of the EFP was enhanced as indicated by increased levels of ATP, phosphate, and intermediates of the citric acid cycle. Gas chromatography coupled to mass spectrometry also revealed that sucrose transport, gluconeogenesis/glycolysis, and amino acid metabolism were up-regulated after wounding. Combining ProteoMiner technology for the enrichment of low-abundance proteins with stable isotope-coded protein labeling, we identified 51 wound-regulated phloem proteins. Two Sucrose-Nonfermenting1-related protein kinases and a 32-kD 14-3-3 protein are candidate central regulators of stress metabolism in the EFP. Other proteins, such as the Silverleaf Whitefly-Induced Protein1, Mitogen Activated Protein Kinase6, and Heat Shock Protein81, have known defensive functions. Isotope-coded protein labeling and western-blot analyses indicated that Cyclophilin18 is a reliable marker for stress responses of the EFP. As a hint toward the induction of redox signaling, we have observed delayed oxidation-triggered polymerization of the major Phloem Protein1 (PP1) and PP2, which correlated with a decline in carbonylation of PP2. In sum, wounding triggered transient sieve tube occlusion, enhanced energy metabolism, and accumulation of defense-related proteins in the pumpkin EFP. The systemic wound response was mediated by jasmonate and redox signaling. PMID:23085839

Gaupels, Frank; Sarioglu, Hakan; Beckmann, Manfred; Hause, Bettina; Spannagl, Manuel; Draper, John; Lindermayr, Christian; Durner, Jörg

2012-10-19

65

tagging, communities, vocabulary, evolution  

Microsoft Academic Search

A tagging community's vocabulary of tags forms the basis for social navigation and shared expression. We present a user-centric model of vocabulary evolution in tagging com- munities based on community influence and personal ten- dency. We evaluate our model in an emergent tagging sys- tem by introducing tagging features into the MovieLens rec- ommender system. We explore four tag selection

Shilad Sen; Shyong K. Lam; Al Mamunur Rashid; Dan Cosley; Dan Frankowski; Jeremy Osterhouse; F. Maxwell Harper; John Riedl

2006-01-01

66

Sense Tagging: Semantic Tagging with a Lexicon  

Microsoft Academic Search

Sense tagging, the automatic assignment of the appropriate sense from some lexicon to each of the words in a text, is a specialised instance of the general problem of seman- tic tagging by category or type. We discuss which recent word sense disambignation al- gorithms are appropriate for sense tagging. It is our belief that sense tagging can be carried

Yorick Wilks; Mark Stevenson

1997-01-01

67

Improved methodology for the affinity isolation of human protein complexes expressed at near endogenous levels  

PubMed Central

An efficient and reliable procedure for the capture of affinity-tagged proteins and associated complexes from human cell lines is reported. Through multiple optimizations, high yield and low background affinity-purifications are achieved from modest quantities of human cells expressing endogenous-level tagged proteins. Isolations of triple-FLAG and GFP-tagged fusion proteins involved in RNA metabolism are presented.

Domanski, Michal; Molloy, Kelly; Jiang, Hua; Chait, Brian T.; Rout, Michael P.; Jensen, Torben Heick; LaCava, John

2013-01-01

68

Germ Tag  

NSDL National Science Digital Library

In this version of tag, a large group of learners model how the body fights infection. Learners act as germs, as lymphocytes, and as the body's cells threatened by germs. After playing one round, subsequent rounds can use different numbers of germs and/or lymphocytes to see how the infection rate is changed. When learners set up a free account at Kinetic City, they can answer bonus questions at the end of the activity as a quick assessment. They can also keep track of their progress in all of the Kinetic City activities, and compare their progress to other participants worldwide.

Science, American A.

2009-01-01

69

Analysis of proteins copurifying with the cd4\\/lck complex using one-dimensional polyacrylamide gel electrophoresis and mass spectrometry: comparison with affinity-tag based protein detection and evaluation of different solubilization methods  

Microsoft Academic Search

Mass spectrometry-based identification of the components of affinity purified protein complexes after polyacrylamide gel electrophoresis\\u000a (PAGE) and in-gel digest has become very popular for the detection of novel protein interactions. As an alternative, the entire\\u000a protein complex can be subjected to proteolytic cleavage followed by chromatographic separation of the peptides. Based on\\u000a our earlier report of a method using affinity

Oliver K. Bernhard; Anthony L. Cunningham; Margaret M. Sheil

2004-01-01

70

Affinity Chromatography  

NSDL National Science Digital Library

This is an experiment showing the application of affinity chromatography to the separation of albumin from horse serum. A brief introduction of affinity chromatography and how it is being used in this specific experiment is given. This appears to be a good experiment to show the advantages of affinity chromatography in separating specific proteins from a complex matrix and would be useful in a biochemistry course or a course that is specifically looking at differing types of chromatography.

Diresta, Dan

2011-05-23

71

Affinity Chromatography.  

ERIC Educational Resources Information Center

|Presents selected recent advances in immobilization chemistry which have important connections to affinity chromatography. Discusses ligand immobilization and support modification. Cites 51 references. (CS)|

Gray, Gary R.

1980-01-01

72

Detection and Quantitation of Hexa-Histidine-Tagged Recombinant Proteins on Western Blots and by a Surface Plasmon Resonance Biosensor Technique  

Microsoft Academic Search

The use of short peptide affinity tag sequences has become commonplace for the expression and purification of recombinant proteins. Many of these tags are antibody epitopes and detection of tagged proteins via Western blots is straightforward. However, the most common affinity tag used at present for the expression of recombinant proteins is a hexa-histidine, or like sequence, which exhibits strong

D. J. Oshannessy; K. C. Odonnell; J. Martin; M. Brighamburke

1995-01-01

73

A tag recommendation algorithm based on SCOT tag ontology  

Microsoft Academic Search

Responding to the problem of tag ambiguity and lack of tag semantics, we propose a new recommendation algorithm based on SCOT tag ontology. The algorithm utilizes rich semantics provided by SCOT ontology to expand tag co-occurrence and eliminate tag disambiguation based on tag semantic similarity for candidate tag sets, aiming to eliminate ambiguity tags and recommend semantic related tags, thereby

Gang-gang Hui; Yi-dan Su; Hua Qin; Xin-lun Zhang

2010-01-01

74

Shark Tagging Activities.  

ERIC Educational Resources Information Center

|In this group activity, children learn about the purpose of tagging and how scientists tag a shark. Using a cut-out of a shark, students identify, measure, record data, read coordinates, and tag a shark. Includes introductory information about the purpose of tagging and the procedure, a data sheet showing original tagging data from Tampa Bay, and…

Current: The Journal of Marine Education, 1998

1998-01-01

75

Isotope-Coded Dimethyl Tagging for Differential Quantification of Posttranslational Protein Carbonylation by 4-Hydroxy-2-nonenal, an End-Product of Lipid Peroxidation  

PubMed Central

Peroxidation of cellular membrane lipids, rich in polyunsaturated fatty acids, generates electrophilic, ?,?-unsaturated aldehydes such as 4-hydroxy-2-nonenal (HNE). HNE is a highly reactive and cytotoxic molecule that can react with the nucleophilic sites in proteins causing posttranslational modification. The identification of protein targets is an important first step; however, quantitative profiling of site-specific modifications is necessary to understand the biological impact of HNE-induced carbonylation. We report a method that uses light (H12CHO) and heavy (D13CDO) isotopic variant of formaldehyde to differentially label primary amines (N-termini and ?-amino group of lysines) in peptides through reductive methylation and, combined with selective enrichment of modified peptides, permits comparison of the extent of carbonylation in two samples after mixing for simultaneous liquid chromatography–mass spectrometry. Specifically, dimethyl-labeled peptide carbonyls were fractionated from unmodified peptides using solid-phase hydrazide chemistry to immobilize them to porous glass beads and, after removing the unmodified peptides by thoroughly washing the beads, subsequently recover them by acid-catalyzed hydrolysis. The method was developed using HNE-modified synthetic peptides and also showing enrichment from a complex matrix of digested human plasma proteins. Applicability was confirmed using apomyoglobin as an analyte, implicating thereby its potential value to proteome-wide identification and relative quantification of posttranslational protein carbonylation with residue-specific information. Because HNE attachment may not necessarily cause change in protein abundance, this modification-focused quantification should facilitate the characterization of accompanied changes in protein function and, also, provide important insights into molecular signaling mechanisms and a better understanding of cellular processes associated with oxidative stress.

Rauniyar, Navin; Prokai, Laszlo

2011-01-01

76

Tags, micro-tags and tag editing: improving internet search  

NASA Astrophysics Data System (ADS)

Social tagging is an emerging methodology that allows individual users to assign semantic keywords to content on the web. Popular web services allow the community of users to search for content based on these user-defined tags. Tags are typically attached to a whole entity such as a web page (e.g., del.icio.us), a video (e.g., YouTube), a product description (e.g., Amazon) or a photograph (e.g., Flickr). However, finding specific information within a whole entity can be a difficult, time-intensive process. This is especially true for content such as video, where the information sought may be a small segment within a very long presentation. Moreover, the tags provided by a community of users may be incorrect, conflicting, or incomplete when used as search terms. In this paper we introduce a system that allows users to create "micro-tags," that is, semantic markers that are attached to subsets of information. These micro-tags give the user the ability to direct attention to specific subsets within a larger and more complex entity, and the set of micro-tags provides a more nuanced description of the full content. Also, when these micro-tags are used as search terms, there is no need to do a serial search of the content, since micro-tags draw attention to the semantic content of interest. This system also provides a mechanism that allows users in the community to edit and delete each others' tags, using the community to refine and improve tag quality. We will also report on empirical studies that demonstrate the value of micro-tagging and tag editing and explore the role micro-tags and tag editing will play in future applications.

Rogowitz, Bernice E.; Topkara, Mercan

2009-02-01

77

Oil Tagging System Study.  

National Technical Information Service (NTIS)

Several methods of identifying the source of oil pollution are critically examined. These methods are grouped into two categories: passive tagging and active tagging. Passive tagging assumes that oils are so chemically diverse that their contents constitu...

1970-01-01

78

Evaluation of Alternate Isotope-Coded Derivatization Assay (AIDA) in the LC–MS\\/MS analysis of aldehydes in exhaled breath condensate  

Microsoft Academic Search

We present the application of a novel isotope dilution method, named Alternate Isotope-Coded Derivatization Assay (AIDA), to the quantitative analysis of hydrazone derivatives of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) in exhaled breath condensate (EBC) samples using liquid chromatography–tandem mass spectrometry. AIDA is based on the alternate derivatization of the analyte(s) with reagents that are available in two pure isotopic forms,

Paola Manini; Roberta Andreoli; Stefano Sforza; Chiara Dall’Asta; Gianni Galaverna; Antonio Mutti; Wilfried M. A. Niessen

2010-01-01

79

Affinity Chromatography  

NSDL National Science Digital Library

Using exposition, graphics, and commercial videos, this module teaches the theory and application of affinity chromatography in the characterization of proteins, nucleic acids, and other biochemical/biomedical systems. Problems and application examples support the tutorial material.

2011-05-25

80

Influence of histidine tag attachment on picosecond protein dynamics.  

PubMed

Polyhistidine affinity tags are routinely employed as a convenient means of purifying recombinantly expressed proteins. A tacit assumption is commonly made that His tags have little influence on protein structure and function. Attachment of a His tag to the N-terminus of the robust globular protein myoglobin leads to only minor changes to the electrostatic environment of the heme pocket, as evinced by the nearly unchanged Fourier transform infrared spectrum of CO bound to the heme of His-tagged myoglobin. Experiments employing two-dimensional infrared vibrational echo spectroscopy of the heme-bound CO, however, find that significant changes occur to the short time scale (picoseconds) dynamics of myoglobin as a result of His tag incorporation. The His tag mainly reduces the dynamics on the 1.4 ps time scale and also alters protein motions of myoglobin on the slower, >100 ps time scale, as demonstrated by the His tag's influence on the fluctuations of the CO vibrational frequency, which reports on protein structural dynamics. The results suggest that affinity tags may have effects on protein function and indicate that investigators of affinity-tagged proteins should take this into consideration when investigating the dynamics and other properties of such proteins. PMID:21619030

Thielges, Megan C; Chung, Jean K; Axup, Jun Y; Fayer, Michael D

2011-06-06

81

The Influence of Histidine Tag Attachment on Picosecond Protein Dynamics†  

PubMed Central

Poly-Histidine (His) affinity tags are routinely employed as a convenient means of purifying recombinantly expressed proteins. A tacit assumption is commonly made that His tags have little influence on protein structure and function. Attachment of a His tag to the N terminus of the robust globular protein myoglobin leads to only minor changes to the electrostatic environment of the heme pocket, as evinced by the nearly unchanged FT-IR spectrum of CO bound to the heme of His-tagged myoglobin. Experiments employing 2D IR vibrational echo spectroscopy of the heme bound CO, however, find that significant changes occur to the short time scale (ps) dynamics of myoglobin as a result of His tag incorporation. The His tag mainly reduces the dynamics on the 1.4 ps timescale and also alters protein motions of myoglobin on the slower, >100s ps timescale, as demonstrated by the His tag's influence on the fluctuations of the CO vibrational frequency, which reports on protein structural dynamics. The results suggest that affinity tags may have effects on protein function and indicate that investigators of affinity tagged proteins should take this into consideration when investigating the dynamics and other properties of such proteins.

Thielges, Megan C; Chung, Jean K.; Axup, Jun Y.; Fayer, Michael D.

2011-01-01

82

Ftr Tag Burnup.  

National Technical Information Service (NTIS)

The gas tag burnup changes investigated were limited to the three tags (Kr-78/Kr-80, Xe-126/Xe-129 and Kr-82/Kr-80) currently accepted as being the most desirable. Control rod tag burnup was significantly greater than fuel rod tag burnup. This occurs beca...

R. B. Kidman

1976-01-01

83

Tag switching architecture overview  

Microsoft Academic Search

Abstract Tag switching is a way to combine the label-swapping forwarding paradigm with network layer routing. This has several advantages. Tags can have a wide spectrum of forwarding granularities, so at one end of the spectrum a tag could be associated with a group of destinations, while at the other a tag could be associated with a single application flow.

B. Davm; D. Katz; G. Swallow; D. Famnacci

1996-01-01

84

Tag switching architecture overview  

Microsoft Academic Search

Tag switching is a way to combine the label-swapping forwarding paradigm with network-layer routing with particular application to the Internet. This has several advantages. Tags can have a wide spectrum of forwarding granularities, so at one end of the spectrum a tag could be associated with a group of destinations, while at the other end, a tag could be associated

YAKOV REKHTER; BRUCE DAVIE; ERIC ROSEN; GEORGE SWALLOW; DINO FARINACCI; DAVE KATZ

1997-01-01

85

Conversational tagging in twitter  

Microsoft Academic Search

Users on Twitter, a microblogging service, started the phenomenon of adding tags to their messages sometime around February 2008. These tags are distinct from those in other Web 2.0 systems because users are less likely to index messages for later retrieval. We compare tagging patterns in Twitter with those in Delicious to show that tagging behavior in Twitter is different

Jeff Huang; Katherine M. Thornton; Efthimis N. Efthimiadis

2010-01-01

86

Variability in the Immunodetection of His-tagged Recombinant Proteins  

PubMed Central

Labeling of recombinant proteins with polypeptide fusion partners, or affinity tagging, is a useful method to facilitate subsequent protein purification and detection. Poly-histidine tags (His-tags) are among the most commonly used affinity tags. We report strikingly variable immunodetection of two His-tagged recombinant human erythropoietins (Epo), wild type Epo (Epowt) and Epo containing an R103A mutation (EpoR103A). Both were engineered to contain a C-terminal six residue His-tag. The cDNA constructs were stably transfected into CHO cells and COS-7 cells. Clones from the CHO cell transfections were selected for further characterization and larger-scale protein expression. Three chromatographic steps were utilized to achieve pharmacologically pure Epo. Conditioned media from the Epo-expressing cell lines and protein-containing samples from each step of purification were analyzed by SDS-PAGE and dot blot, using both monoclonal anti-human Epo antibody (AE7A5) and anti-His antibodies. While the successful incorporation of the His-tag into our constructs was confirmed by Epo binding to Ni2+-NTA resin and by ?LC/MS/MS amino acid sequencing, the levels of immunodetection of His-tagged protein varied markedly depending on the particular anti His-tag antibody used. Such variability in His-tag immunorecognition can lead to critical adverse effects on several analytical methods.

Debeljak, Natasa; Feldman, Laurie; Davis, Kerry L.; Komel, Radovan; Sytkowski, Arthur J.

2006-01-01

87

Myocardial tagging with SSFP.  

PubMed

This work presents the first implementation of myocardial tagging with refocused steady-state free precession (SSFP) and magnetization preparation. The combination of myocardial tagging (a noninvasive method for quantitative measurement of regional and global cardiac function) with the high tissue signal-to-noise ratio (SNR) obtained with SSFP is shown to yield improvements in terms of the myocardium-tag contrast-to-noise ratio (CNR) and tag persistence when compared to the current standard fast gradient-echo (FGRE) tagging protocol. Myocardium-tag CNR and tag persistence were studied using numerical simulations as well as phantom and human experiments. Both quantities were found to decrease with increasing imaging flip angle (alpha) due to an increased tag decay rate and a decrease in myocardial steady-state signal. However, higher alpha yielded better blood-myocardium contrast, indicating that optimal alpha is dependent on the application: higher alpha for better blood-myocardium boundary visualization, and lower alpha for better tag persistence. SSFP tagging provided the same myocardium-tag CNR as FGRE tagging when acquired at four times the bandwidth and better tag- and blood-myocardium CNRs than FGRE tagging when acquired at equal or twice the receiver bandwidth (RBW). The increased acquisition efficiency of SSFP allowed decreases in breath-hold duration, or increases in temporal resolution, as compared to FGRE. PMID:12541254

Herzka, Daniel A; Guttman, Michael A; McVeigh, Elliot R

2003-02-01

88

Myocardial Tagging With SSFP  

PubMed Central

This work presents the first implementation of myocardial tagging with refocused steady-state free precession (SSFP) and magnetization preparation. The combination of myocardial tagging (a noninvasive method for quantitative measurement of regional and global cardiac function) with the high tissue signal-to-noise ratio (SNR) obtained with SSFP is shown to yield improvements in terms of the myocardium–tag contrast-to-noise ratio (CNR) and tag persistence when compared to the current standard fast gradient-echo (FGRE) tagging protocol. Myocardium–tag CNR and tag persistence were studied using numerical simulations as well as phantom and human experiments. Both quantities were found to decrease with increasing imaging flip angle (?) due to an increased tag decay rate and a decrease in myocardial steady-state signal. However, higher ? yielded better blood–myocardium contrast, indicating that optimal ? is dependent on the application: higher ? for better blood–myocardium boundary visualization, and lower ? for better tag persistence. SSFP tagging provided the same myocardium–tag CNR as FGRE tagging when acquired at four times the bandwidth and better tag– and blood–myocardium CNRs than FGRE tagging when acquired at equal or twice the receiver bandwidth (RBW). The increased acquisition efficiency of SSFP allowed decreases in breath-hold duration, or increases in temporal resolution, as compared to FGRE.

Herzka, Daniel A.; Guttman, Michael A.; McVeigh, Elliot R.

2007-01-01

89

New cobalt resin for optimal purification of His-tagged proteins  

Microsoft Academic Search

The expression and purification of recombinant proteins is central to protein regulation, structure and function studies. The majority of recombinant proteins are expressed as fusions with short affinity tags, the most popular being the polyhistidine (6xHis) tag. The method used to purify these recombinant His-tagged proteins is immobilized metal affinity chromatography (IMAC) consisting of chelating resins charged with either nickel

Michael Major; Marty Wilkes; Martin Bremmer; Navid Haghdoost; Barbara Kaboord; Thermo Fisher

90

Combined hydrophobic-metal binding fusion tags for applications in aqueous two-phase partitioning  

Microsoft Academic Search

In this work, we studied the influence of fusion affinity tags containing both hydrophobic and histidines residues on the partitioning of the green fluorescent protein, GFPuv, in aqueous two-phase system. The tags were fused to the N-terminal of GFPuv and tested by immobilized metal affinity partitioning, in a PEG\\/salt system. The presence of both types of residues in the tag

Florent Bernaudat; Leif Bülow

2006-01-01

91

Donor Tag Game  

MedlinePLUS

... Games > Donor Tag Game Printable Version Donor Tag Game This feature requires version 6 or later of ... Needles Blood Donor Community Donor Stories Recipient Stories Games Facebook Fanbox Avatars and Badges Banners eCards Twitter ...

92

Design, synthesis, and application of the trimethoprim-based chemical tag for live-cell imaging.  

PubMed

Over the past decade, chemical tags have been developed to complement the use of fluorescent proteins in live-cell imaging. Chemical tags retain the specificity of protein labeling achieved with fluorescent proteins through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon output and tailored functionalities. The trimethoprim-based chemical tag (TMP-tag) was initially developed based on the high affinity interaction between E. coli dihydrofolate reductase and the antibiotic trimethoprim and was subsequently rendered covalent and fluorogenic via proximity-induced protein labeling reactions. To date, the TMP-tag is one of the few chemical tags that enable intracellular protein labeling and high-resolution live-cell imaging. Here we describe the general design, chemical synthesis, and application of TMP-tag for live-cell imaging. Alternate protocols for synthesizing and using the covalent and the fluorogenic TMP-tags are also included. PMID:23839994

Jing, Chaoran; Cornish, Virginia W

2013-06-01

93

TAG-1 and TAG-2 Proteins and Uses Thereof.  

National Technical Information Service (NTIS)

The present invention is directed to a newly discovered gene family with multiple isoforms, designated TAG-1, TAG-2a, TAG-2b, TAG-2c, and TAG-3, nucleic acid sequences encoding those proteins, and antibodies generated against said proteins. The genes, pro...

C. L. Slingluff K. T. Hogan

2004-01-01

94

Relative position of the hexahistidine tag effects binding properties of a tumor-associated single-chain Fv construct  

Microsoft Academic Search

Hexahistidine tag (His-tag) is the most widely used tag for affinity purification of recombinant proteins for their structural and functional analysis. In the present study, single chain Fv (scFv) constructs were engineered form the monoclonal antibody (MAb) CC49 which is among the most extensively studied MAb for cancer therapy. For achieving efficient purification of scFvs by immobilized metal–ion affinity chromatography

Apollina Goel; David Colcher; Ja-Seok Koo; Barbara J. M. Booth; Gabriela Pavlinkova; Surinder K. Batra

2000-01-01

95

Classifying XML tags through \\  

Microsoft Academic Search

Some tags used in XML documents create arbitrary breaks in the natural flow of the text. This may constitute an impediment to the application of some methods of document engineering. This article introduces the concept of ``reading contexts'', and gives clues to handle it theorically and in practice. This work should notably allow to recognize emphasis tags in a text,

Xavier Tannier; Jean-Jacques Girardot; Mihaela Mathieu

2005-01-01

96

Dynamic optical tags  

NASA Astrophysics Data System (ADS)

The goal of the DARPA Dynamic Optical Tags (DOTs) program is to develop a small, robust, persistent, 2-way tagging, tracking and locating device that also supports communications at data rates greater than 100 kbps and can be interrogated at significant range. These tags will allow for two-way data exchange and tagging operations in friendly and denied areas. The DOTs will be passive and non-RF. To accomplish this, the DOTs program will develop small, thin, retro-reflecting modulators. The tags will operate for long periods of time (greater than two months) in real-world environmental conditions (-40° to +70° C) and allow for a wide interrogation angle (+/-60°). The tags will be passive (in the sleep mode) for most of the time and only become active when interrogated by a laser with the correct code. Once correctly interrogated, the tags will begin to modulate and retro-reflect the incoming beam. The program will also develop two tag specific transceiver systems that are eye-safe, employ automated scanning algorithms, and are capable of short search and interrogate times.

Griggs, Steven P.; Mark, Martin B.; Feldman, Barry J.

2004-07-01

97

Cutaneous skin tag  

MedlinePLUS

Skin tag; Acrochordon; Fibroepithelial polyp ... have diabetes. They are thought to occur from skin rubbing against skin. ... The tag sticks out of the skin and may have a short, narrow stalk connecting it to the surface of the skin. It is usually small, but may be ...

98

Personalization of tagging systems  

Microsoft Academic Search

Social media systems have encouraged end user participation in the Inter- net, for the purpose of storing and distributing Internet content, sharing opinions and maintaining relationships. Collaborative tagging allows users to annotate the resulting user-generated content, and enables effective re- trieval of otherwise uncategorised data. However, compared to professional web content production, collaborative tagging systems face the challenge that end-users

Jun Wang; Maarten Clements; Jie Yang; Arjen P. De Vries; Marcel J. T. Reinders

2010-01-01

99

Preparation and immunogenicity of tag-free recombinant human eppin  

PubMed Central

Human epididymal protease inhibitor (eppin) may be effective as a male contraceptive vaccine. In a number of studies, eppin with an engineered His6-tag has been produced using prokaryotic expression systems. For production of pharmaceutical-grade proteins for human use, however, the His6-tag must be removed. This study describes a method for producing recombinant human eppin without a His6-tag. We constructed plasmid pET28a (+)-His6-tobacco etch virus (TEV)-eppin for expression in Escherichia coli. After purification and refolding, the fusion protein His6-TEV-eppin was digested with TEV protease to remove the His6-tag and was further purified by NTA-Ni2+ affinity chromatography. Using this procedure, 2 mg of eppin without a His6-tag was isolated from 1 l of culture with a purity of >95%. The immunogenicity of the eppin was characterized using male Balb/c mice.

Zhang, Jie; Ding, Xin-Liang; Bian, Zeng-Hui; Xia, Yan-Kai; Wang, Shou-Lin; Song, Ling; Wang, Xin-Ru

2011-01-01

100

Passive optoelectronic tag  

NASA Astrophysics Data System (ADS)

In response to a pressing demand for tagging systems and technologies developing, Physical Optics Corporation (POC) proposes a novel Passive Optoelectronical (POET) Tag system. The POET tag is an omnidirectional (360° in azimuth), with up to 180° field-of-view in elevation, retroreflection optical system with a high frequency multiple quantum well (MQW) light intensity modulator for free space IR optical communication. The POET tag optical scheme is a compact, high quality generalized fish-eye lens with telecentric arrangement in image space. The telecentric arrangement in image space provides perfect omnidirectional retroreflection of a recall beam and an optimum divergent of light at the MQW providing maximum modulation contrast ratios. The important POET tag features are low power consumption, zero probability of jamming and intercepting (high security of communication,) because it operates in a passive retroreflection mode with a highly-directed optical beam.

Agurok, Il'ya P.; Jannson, Tomasz P.; Savant, Gajendra D.

2003-09-01

101

Report: Affinity Chromatography.  

ERIC Educational Resources Information Center

|Supports, affinity ligands, immobilization, elution methods, and a number of applications are among the topics considered in this discussion of affinity chromatography. An outline of the basic principles of affinity chromatography is included. (JN)|

Walters, Rodney R.

1985-01-01

102

Analysis of the eukaryotic prenylome by isoprenoid affinity tagging  

Microsoft Academic Search

Protein prenylation is a widespread phenomenon in eukaryotic cells that affects many important signaling molecules. We describe the structure-guided design of engineered protein prenyltransferases and their universal synthetic substrate, biotin-geranylpyrophosphate. These new tools allowed us to detect femtomolar amounts of prenylatable proteins in cells and organs and to identify their cognate protein prenyltransferases. Using this approach, we analyzed the in

Uyen T T Nguyen; Zhong Guo; Christine Delon; Yaowen Wu; Celine Deraeve; Benjamin Fränzel; Robin S Bon; Wulf Blankenfeldt; Roger S Goody; Herbert Waldmann; Dirk Wolters; Kirill Alexandrov

2009-01-01

103

Engineering a Novel Multifunctional Green Fluorescent Protein Tag for a Wide Variety of Protein Research  

PubMed Central

Background Genetically encoded tag is a powerful tool for protein research. Various kinds of tags have been developed: fluorescent proteins for live-cell imaging, affinity tags for protein isolation, and epitope tags for immunological detections. One of the major problems concerning the protein tagging is that many constructs with different tags have to be made for different applications, which is time- and resource-consuming. Methodology/Principal Findings Here we report a novel multifunctional green fluorescent protein (mfGFP) tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8×His), streptavidin-binding peptide (SBP), and c-Myc tag, in tandem into a loop of GFP. When fused to various proteins, mfGFP monitored their localization in living cells. Streptavidin agarose column chromatography with the SBP tag successfully isolated the protein complexes in a native form with a high purity. Tandem affinity purification (TAP) with 8×His and SBP tags in mfGFP further purified the protein complexes. mfGFP was clearly detected by c-Myc-specific antibody both in immunofluorescence and immuno-electron microscopy (EM). These findings indicate that mfGFP works well as a multifunctional tag in mammalian cells. The tag insertion was also successful in other fluorescent protein, mCherry. Conclusions and Significance The multifunctional fluorescent protein tag is a useful tool for a wide variety of protein research, and may have the advantage over other multiple tag systems in its higher expandability and compatibility with existing and future tag technologies.

Kobayashi, Takuya; Morone, Nobuhiro; Kashiyama, Taku; Oyamada, Hideto; Kurebayashi, Nagomi; Murayama, Takashi

2008-01-01

104

Improved native affinity purification of RNA.  

PubMed

RNA biochemical or structural studies often require an RNA sample that is chemically pure, and most protocols for its in vitro production use denaturing polyacrylamide gel electrophoresis to achieve this. Unfortunately, many RNAs do not quantitatively refold into an active conformation after denaturation, creating significant problems for downstream characterization or use. In addition, this traditional purification method is not amenable to studies demanding high-throughput RNA production. Recently, we presented the first general method for producing almost any RNA sequence that employs an affinity tag that is removed during the purification process. Because technical difficulties prevented application of this method to many RNAs, we have developed an improved version that utilizes a different activatable ribozyme and affinity tag that are considerably more robust, rapid, and broadly applicable. PMID:17548432

Batey, Robert T; Kieft, Jeffrey S

2007-06-04

105

Use of Streamline chelating for capture and purification of poly-His-tagged recombinant proteins  

Microsoft Academic Search

Expression of recombinant proteins with poly-histidine tags enables their convenient capture and purification using immobilized metal affinity chromatography (IMAC). The 6×His-tagged protein binds to a chelating resin charged with metal ions such as Ni2+, Cu2+ or Zn2+, and can therefore be separated from proteins which have lower, or no, affinity for the resin. Two recombinant proteins, a malaria transmission-blocking vaccine

Santosh Noronha; Jeanne Kaufman; Joseph Shiloach

1999-01-01

106

Method for protein tagging in Schizosaccharomyces pombe.  

PubMed

Tagging is a useful method for the investigation of proteins. It allows the localization of the proteins in the cell, their purification in order to investigate their function and the determination of their expression. The aim of the present study was to tag the Rad32 protein of fission yeast (which is the homologue of Mre11 protein from humans) at its N-terminus. Rad32p as well as Mre11p are involved in the repair of DNA double strand breaks and in the DNA damage checkpoint. We carried out this tagging using the Cre-loxp recombination system. In a first step, a 2 kb DNA fragment was integrated upstream of the initiating codon of rad32 gene. This fragment encoded the TAP-tag (tandem affinity purification), a loxp site, a selectable marker (sup3-5), an exogenous promoter (nmt1) and a second loxp site, in this sequence. Following transformation of this DNA fragment into S. pombe cells, rad32 was under the control of the artificial promotor, which allows a controlled expression of the gene by thiamine. In a second step, the cells were transformed with a plasmid coding for Cre recombinase, which catalyses the excision of the DNA sequence between the two loxp sites, removing the marker and the artificial promotor. Thus the tag became attached to the rad32 gene upstream of the ATG, placing the gene under the control of its native promotor. The strain thus obtained will be subsequently used for evidencing the tagged protein by Western blotting and then for its purification in order to investigate its function. PMID:17802953

Petrescu-D?nil?, Elena; Voicu, Pia-Manuela; Poi?elea, M; Stoica, B; St?nescu, Raluca; Rusu, M

107

A generalized tagging method  

NASA Astrophysics Data System (ADS)

The understanding of causes of changes in climate-chemistry simulations is an important, but often challenging task. In atmospheric chemistry, one approach is to tag species according to their origin (e.g. emission categories) and to inherit these tags to other species during subsequent reactions. This concept was recently employed to calculate the contribution of atmospheric processes to temperature. Here a new concept for tagging any state variable is presented. This generalized tagging method results from a sensitivity analysis of the individual forcing terms of the right hand side of the governing differential equations. In a couple of examples, the consistency with previous approaches and the synergy by using different analysis techniques are shown. Since the method is based on a ratio describing relative sensitivities, singularities occur where the method is not applicable. For some applications, such as in atmospheric chemistry, these singularities can easily be removed. However, one theoretical example is given, where this method is not applicable at all.

Grewe, V.

2013-02-01

108

Automatically Identifying Tag Types  

Microsoft Academic Search

Web 2.0 applications such as delicious, flickr or lastfm have recently become extremely popular and as a result, a large amount of semantically rich metadata produced by users becomes\\u000a available and exploitable. Tag information can be used for many purposes (e.g. user profiling, recommendations, clustering etc), though the benefit of tags for search is by far the most discussed usage.

Kerstin Bischoff; Claudiu S. Firan; Cristina Kadar; Wolfgang Nejdl; Raluca Paiu

2009-01-01

109

Neuron Chain Tag  

NSDL National Science Digital Library

In this outdoor activity, learners play a game of Tag to discover how neurons attach themselves to each other to form a chain. The game starts with one learner who is "it" and represents the first neuron. When "it" tags another player, the tagger player must hold the hand of "it" and work together to form a long a chain. The game ends when all the players are part of the neuron chain.

Yoshioka, Melissa

2009-01-01

110

Tag-switching architecture: overview  

Microsoft Academic Search

Tag switching is a way to combine the label-swapping forwarding paradigm with network layer routing. This has several advantages. Tags can have a wide spectrum of forwarding granularities, so at one end of the spectrum a tag could be associated with a group of desti-nations, while at the other a tag could be associated with a single application flow. At

Yakov Rekhter; B. Davie; E. Rosen; G. Swallow; D. Farinacci; D. Katz

1997-01-01

111

Distributed RFID Tag Storage Infrastructures  

Microsoft Academic Search

We leverage increasing passive RFID tag memory to pro- pose distributed RFID tag storage infrastructures (D-RFID stores). A D-RFID store is a large set of tags with signicantly sized re-writeable storage. Interrogators interact with D-RFID stores by reading from and writing to tags, providing a wide range of possible applications that are otherwise resource-inecien t. Examples include tagging trees in

Victor K. Y. Wu; Mirko Montanari; Nitin H. Vaidya; Roy H. Campbell

112

Expression and fast-flow purification of a polyhistidine-tagged myoglobin-like aerotaxis transducer  

Microsoft Academic Search

A Co2+-affinity, fast-flow perfusion chromatography method to purify a polyhistidine-tagged myoglobin-like aerotaxis transducer HemAT-Hs has been developed. The method relies upon a six-histidine affinity tag fused to the C-terminus and N-terminus of HemAT-Hs for expression in the native host, an extremely halophilic Archaeon Halobacterium salinarum, and in the heterologous host Escherichia coli, respectively. The His-tagged HemAT-Hs can be purified rapidly

Mikhail Piatibratov; Shaobin Hou; Alexei Brooun; Jinsheng Yang; Huaiyang Chen; Maqsudul Alam

2000-01-01

113

Affinity recovery of lentivirus by diaminopelargonic acid mediated desthiobiotin labelling  

Microsoft Academic Search

Desthiobiotin-tagged lentiviral vectors have been metabolically produced by DBL producer cells in a 7,8-diaminopelargonic acid (7-DAPA) dependent manner for envelope independent, single-step affinity purification. 7-DAPA, which has little or no affinity for avidin\\/streptavidin, was synthesised and verified by NMR spectroscopy and mass spectrometry. By expressing the biotin acceptor, biotin ligase and desthiobiotin synthase bioD, DBL cells converted exogenous 7-DAPA into

Rongjun Chen; Najeem Folarin; Vincent H. B. Ho; David McNally; David Darling; Farzin Farzaneh; Nigel K. H. Slater

2010-01-01

114

Cell Surface Glypicans Are Low-Affinity Endostatin Receptors  

Microsoft Academic Search

Endostatin, a collagen XVIII fragment, is a potent anti-angiogenic protein. We sought to identify its endothelial cell surface receptor(s). Alkaline phosphatase– tagged endostatin bound endothelial cells revealing two binding affinities. Expression cloning identified glypican, a cell surface proteoglycan as the lower-affinity receptor. Biochemical and genetic studies indicated that glypicans' heparan sulfate glycosaminoglycans were critical for endostatin binding. Furthermore, endostatin selected

S. Ananth Karumanchi; Vivekanand Jha; Ramani Ramchandran; Anil Karihaloo; Leonidas Tsiokas; Barden Chan; Mohanraj Dhanabal; Jun-ichi Hanai; Ganesh Venkataraman; Zachary Shriver; Nishla Keiser; Raghu Kalluri; Huiyan Zeng; Debabrata Mukhopadhyay; Robert L Chen; Arthur D Lander; Kazuki Hagihara; Yu Yamaguchi; Ram Sasisekharan; Lloyd Cantley; Vikas P Sukhatme

2001-01-01

115

A Reliable Tag Anti-Collision Algorithm for Mobile Tags  

NASA Astrophysics Data System (ADS)

As RFID technology is being more widely adopted, it is fairly common to read mobile tags using RFID systems, such as packages on conveyer belt and unit loads on pallet jack or forklift truck. In RFID systems, multiple tags use a shared medium for communicating with a reader. It is quite possible that tags will exit the reading area without being read, which results in tag leaking. In this letter, a reliable tag anti-collision algorithm for mobile tags is proposed. It reliably estimates the expectation of the number of tags arriving during a time slot when new tags continually enter the reader's reading area and no tag leaves without being read. In addition, it gives priority to tags that arrived early among read cycles and applies the expectation of the number of tags arriving during a time slot to the determination of the number of slots in the initial inventory round of the next read cycle. Simulation results show that the reliability of the proposed algorithm is close to that of DFSA algorithm when the expectation of the number of tags entering the reading area during a time slot is a given, and is better than that of DFSA algorithm when the number of time slots in the initial inventory round of next read cycle is set to 1 assuming that the number of tags arriving during a time slot follows Poisson distribution.

Deng, Xiaodong; Rong, Mengtian; Liu, Tao

116

Highly Efficient Tandem Affinity Purification of Trypanosome Protein Complexes Based on a Novel Epitope Combination  

Microsoft Academic Search

Received 13 July 2005\\/Accepted 22 August 2005 Tandem affinity purification (TAP) allows for rapid and efficient purification of epitope-tagged protein complexes from crude extracts under native conditions. The method was established in yeast and has been successfully applied to other organisms, including mammals and trypanosomes. However, we found that the original method, which is based on the TAP tag, consisting

Bernd Schimanski; Tu N. Nguyen; Arthur Gunzl

2005-01-01

117

Comparing tagging vocabularies among four enterprise tag-based services  

Microsoft Academic Search

We compare four tagging-based enterprise services, which respectively stored bookmarks to webpages and documents, to people, to blog entries, and to hierarchically-structured activity records. Analysis of user data and tag data showed relatively small overlaps in tags used. Conventional normalization strategies produced only modest improvement. These results suggest difficulties in combining exploratory searches across multiple social-tagging services. We recommend strategies

Michael J. Muller

2007-01-01

118

Discourse Tagging Tool and Discourse-Tagged Multilingual Corpora  

Microsoft Academic Search

As a part of our on-going research on multilingual anaphora resolution (cf. Aone and McKee [3],Aone [1]), we have built a graphical tool to tag texts with antecedent-anaphor relations and havecreated corpora tagged with such relations. The tool, called the Discourse Tagging Tool (DTTool),has been used to manually tag Japanese, Spanish and English texts with anaphora, their types (suchas pronouns

Chinatsu Aone; Scott W. Bennett

1994-01-01

119

Project 8 Tags  

ERIC Educational Resources Information Center

|In this article, the author describes 8 Tags, a project that she used with her eighth-grade studio art students to encourage them to come up with original and creative solutions to an assignment. She also wanted to incorporate their knowledge of the elements of art and principles of design. In this project, students were challenged to create an…

Ramos-Burrows, Michele

2010-01-01

120

Imperfect Tagging Revisited  

Microsoft Academic Search

Revisiting Parsons' 1996 article about disability insurance with imperfect tagging in a two type-economy -- individuals are either able or disabled. Here Parsons' analysis is extended in several directions. The model is generalized to allow for different utility functions over work status. The analysis extends to three different cases of a two-type economy. Finally Parsons' model is extended to three

Eric Rehn

2007-01-01

121

Tags are not Metadata, but \\  

Microsoft Academic Search

The authoring of tags - unlike the authoring of traditional metadata - is highly popular among users. This harbours un- precedented opportunities for organizing content. However, tags are still poorly understood. What do they \\

Bettina Berendt; Christoph Hanser

2007-01-01

122

The HaloTag: Improving Soluble Expression and Applications in Protein Functional Analysis  

PubMed Central

Technological and methodological advances have been critical for the rapidly evolving field of proteomics. The development of fusion tag systems is essential for purification and analysis of recombinant proteins. The HaloTag is a 34 KDa monomeric protein derived from a bacterial haloalkane dehalogenase. The majority of fusion tags in use today utilize a reversible binding interaction with a specific ligand. The HaloTag system is unique in that it forms a covalent linkage to its chloroalkane ligand. This linkage permits attachment of the HaloTag to a variety of functional reporters, which can be used to label and immobilize recombinant proteins. The success rate for HaloTag expression of soluble proteins is very high and comparable to maltose binding protein (MBP) tag. Furthermore, cleavage of the HaloTag does not result in protein insolubility that often is observed with the MBP tag. In the present report, we describe applications of the HaloTag system in our ongoing investigation of protein-protein interactions of the Y. pestis Type 3 secretion system on a custom protein microarray. We also describe the utilization of affinity purification/mass spectroscopy (AP/MS) to evaluate the utility of the Halo Tag system to characterize DNA binding activity and protein specificity.

N Peterson, Scott; Kwon, Keehwan

2012-01-01

123

Cultural difference in image tagging  

Microsoft Academic Search

Do people from different cultures tag digital images differently? The current study compared the content of tags for digital images created by two cultural groups: European Americans and Chinese. In line with previous findings on cultural differences in attentional patterns, we found similar cultural differences in the order of the image parts (e.g., foreground or background objects) that people tag.

Wei Dong; Wai-Tat Fu

2010-01-01

124

Tagsplanations: explaining recommendations using tags  

Microsoft Academic Search

While recommender systems tell users what items they might like, explanations of recommendations reveal why they might like them. Explanations provide many benefits, from im- proving user satisfaction to helping users make better deci- sions. This paper introduces tagsplanations, which are ex- planations based on community tags. Tagsplanations have two key components: tag relevance, the degree to which a tag

Jesse Vig; Shilad Sen; John Riedl

2009-01-01

125

Development of the Twin-Strep-tag® and its application for purification of recombinant proteins from cell culture supernatants.  

PubMed

Short peptide affinity tags have become indispensable in protein research. They cannot only be used for affinity purification but also downstream for detection and assay of an arbitrary fused recombinant protein without the need for any prior knowledge of its biochemical properties. Strep-tag®II is particularly popular for providing recombinant proteins at high purity and functionality by using physiological conditions within a rapid one-step protocol. The affinity receptor for Strep-tag®II is affinity engineered streptavidin, named Strep-Tactin®. Strep-tag®II binds to the biotin binding pocket enabling mild competitive elution with biotin derivatives, preferably desthiobiotin, for repeated use of the Strep-Tactin® affinity resins. Fast binding and dissociation kinetics allow comparatively high flow rates throughout column chromatography including elution. Fast dissociation kinetics may be, however, limiting for using Strep-tag®II for direct purification of target proteins from large volumes of diluted extracts like mammalian cell culture supernatants or in assay formats requiring extended washing like ELISA. For this reason, binding characteristics were improved by development of the Twin-Strep-tag® consisting of two Strep-tag®II moieties connected by a short linker. The resulting avidity effect, i.e., the combined synergistic binding of two Strep-tag®II moieties to tetrameric Strep-Tactin®, reduces the off-rate for more steady binding under non-competitive conditions. The addition of a competitor, however, reverses the synergistic avidity effect and, hence, efficient elution capability is preserved. In fact, the Twin-Strep-tag® features all beneficial properties of Strep-tag®II, including efficient elution under gentle competitive conditions, but, due to its higher affinity, additionally enables a more universal use in applications requiring stable binding. PMID:24012791

Schmidt, Thomas G M; Batz, Lilia; Bonet, Lidia; Carl, Uwe; Holzapfel, Gerd; Kiem, Klaus; Matulewicz, Kamila; Niermeier, Dennis; Schuchardt, Isabel; Stanar, Kristian

2013-09-06

126

Activity assay of His-tagged E. coli DNA photolyase by RP-HPLC and SE-HPLC  

Microsoft Academic Search

Escherichia coli DNA photolyase was expressed as C-terminal 6× histidine-fused protein. Purification of His-tagged E. coli DNA photolyase was developed using immobilized metal affinity chromatography with Chelating Sepharose Fast Flow. By one-step affinity chromatography, approximate 4.6 mg DNA photolyase was obtained from 400 ml E. coli culture. The purified His-tagged enzyme was combined with two chromophors, FADH and MTHF. Using

Wanmeng Mu; Dongfang Zhang; Lei Xu; Zhaofeng Luo; Yuzhen Wang

2005-01-01

127

Phosphate-binding tag, a new tool to visualize phosphorylated proteins.  

PubMed

We introduce two methods for the visualization of phosphorylated proteins using alkoxide-bridged dinuclear metal (i.e. Zn(2+) or Mn(2+)) complexes as novel phosphate-binding tag (Phos-tag) molecules. Both Zn(2+)- and Mn(2+)-Phos-tag molecules preferentially capture phosphomonoester dianions bound to Ser, Thr, and Tyr residues. One method is based on an ECL system using biotin-pendant Zn(2+)-Phos-tag and horseradish peroxidase-conjugated streptavidin. We demonstrate the electroblotting analyses of protein phosphorylation status by the phosphate-selective ECL signals. Another method is based on the mobility shift of phosphorylated proteins in SDS-PAGE with polyacrylamide-bound Mn(2+)-Phos-tag. Phosphorylated proteins in the gel are visualized as slower migration bands compared with corresponding dephosphorylated proteins. We demonstrate the kinase and phosphatase assays by phosphate affinity electrophoresis (Mn(2+)-Phos-tag SDS-PAGE). PMID:16340016

Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Takiyama, Kei; Koike, Tohru

2005-12-11

128

TagReel: A Visualization of Tag Relations among User Interests in the Social Tagging System  

Microsoft Academic Search

Social tagging systems provide users with the ability to share information and extend their field of knowledge. The purpose of this paper is to explore the tag relations of user interest in these systems and examine the semantic relations of tag usage in terms of user interests. To do this, a classifying method was used to characterize words into seven

Joohee Bae; Kyungwon Lee

2009-01-01

129

TagMyDoc  

NSDL National Science Digital Library

Do you need to share documents quickly with a number of different users? You may want to give TagMyDoc a look. Visitors can choose their documents, and upload them so they can be scanned and retrieved as virtual copies. Additionally, users can sign up for free accounts for enhanced functionality and there's an explanatory video here as well. This version is compatible with all operating systems.

2012-01-01

130

Evaluation of chromatographic recycling for imidazole used in the chromatographic purification of His-tag recombinant proteins  

Microsoft Academic Search

The aim of this work was to test a recycling method for imidazole used in immobilized metal affinity chromatography (IMAC) as eluent for recombinant histidine-tag (His-tag) protein. After evaluating two supports, the method was optimized with a mixture of bovine serum albumin, sodium chloride and imidazole. Recycling was performed with an eluate fraction from IMAC of His-tag enhanced green fluorescent

A. M. Noubhani; W. Dieryck; N. Bakalara; L. Latxague; X. Santarelli

2003-01-01

131

Quantitative evaluation of his-tag purification and immunoprecipitation of tristetraprolin and its mutant proteins from transfected human cells  

Technology Transfer Automated Retrieval System (TEKTRAN)

Histidine (His)-tag is widely used for affinity purification of recombinant proteins, but the yield and purity of expressed proteins are quite different. Little information is available about quantitative evaluation of this procedure. The objective of the current study was to evaluate the His-tag pr...

132

Shark tagging: a brief history of methods  

Microsoft Academic Search

Thefirst large-scale shark tagging programmes were initiated during the 1940s using Petersen disc tags (or similar variants) wired through the relatively rigid first dorsal fin. During the same period in Australia, school sharks were tagged with internal Nesbit tags inserted into the body cavity. These tags, developed because of concerns over high shedding rates of early fin tags, still hold

John Stevens

133

The Comprehensive Native Interactome of a Fully Functional Tagged Prion Protein  

PubMed Central

The enumeration of the interaction partners of the cellular prion protein, PrPC, may help clarifying its elusive molecular function. Here we added a carboxy proximal myc epitope tag to PrPC. When expressed in transgenic mice, PrPmyc carried a GPI anchor, was targeted to lipid rafts, and was glycosylated similarly to PrPC. PrPmyc antagonized the toxicity of truncated PrP, restored prion infectibility of PrPC-deficient mice, and was physically incorporated into PrPSc aggregates, indicating that it possessed all functional characteristics of genuine PrPC. We then immunopurified myc epitope-containing protein complexes from PrPmyc transgenic mouse brains. Gentle differential elution with epitope-mimetic decapeptides, or a scrambled version thereof, yielded 96 specifically released proteins. Quantitative mass spectrometry with isotope-coded tags identified seven proteins which co-eluted equimolarly with PrPC and may represent component of a multiprotein complex. Selected PrPC interactors were validated using independent methods. Several of these proteins appear to exert functions in axomyelinic maintenance.

Moos, Rita; Brunner, Erich; Rulicke, Thomas; Calella, Anna Maria; Aguzzi, Adriano

2009-01-01

134

Fluorescence: Molecular Tagging  

NSDL National Science Digital Library

In this activity, by the Concord Consortium's Molecular Literacy project, students will be introduced to the concept of fluorescence and how this method allows scientists to better analyze objects at a molecular level. The site is interactive and allows students to interact with virtual cells and tag them just as a scientist would. The focus of the topic can be applied to many different fields, such as: mineralogy, biochemistry, medicine, forensics and biotechnology. The activity itself is a java-based interactive resource built upon the free, open source Molecular Workbench software. In addition, visitors will find an overview of the activity, assessments, and concepts and their correlation to AAAS and NSES standards.

2008-10-17

135

Review on SAW RFID tags.  

PubMed

SAW tags were invented more than 30 years ago, but only today are the conditions united for mass application of this technology. The devices in the 2.4-GHz ISM band can be routinely produced with optical lithography, high-resolution radar systems can be built up using highly sophisticated, but low-cost RF-chips, and the Internet is available for global access to the tag databases. The "Internet of Things," or I-o-T, will demand trillions of cheap tags and sensors. The SAW tags can overcome semiconductor-based analogs in many aspects: they can be read at a distance of a few meters with readers radiating power levels 2 to 3 orders lower, they are cheap, and they can operate in robust environments. Passive SAW tags are easily combined with sensors. Even the "anti-collision" problem (i.e., the simultaneous reading of many nearby tags) has adequate solutions for many practical applications. In this paper, we discuss the state-of-the-art in the development of SAW tags. The design approaches will be reviewed and optimal tag designs, as well as encoding methods, will be demonstrated. We discuss ways to reduce the size and cost of these devices. A few practical examples of tags using a time-position coding with 10(6) different codes will be demonstrated. Phase-coded devices can additionally increase the number of codes at the expense of a reduction of reading distance. We also discuss new and exciting perspectives of using ultra wide band (UWB) technology for SAW-tag systems. The wide frequency band available for this standard provides a great opportunity for SAW tags to be radically reduced in size to about 1 x 1 mm(2) while keeping a practically infinite number of possible different codes. Finally, the reader technology will be discussed, as well as detailed comparison made between SAW tags and IC-based semiconductor device. PMID:20211785

Plessky, Victor P; Reindl, Leonhard M

2010-03-01

136

Single step surface modification of highly stable magnetic nanoparticles for purification of His-tag proteins  

Microsoft Academic Search

The aim of this study was to develop a simple, cheap, and rapid method for purification of His-tag recombinant proteins with\\u000a high yields. The new immobilized metal ion affinity adsorbent containing superparamagnetic nanoparticles and hydrophilic resins\\u000a are proposed here to improve the purification of His-tagged recombinant proteins. In this report, we have described the preparation\\u000a of nanosized superparamagnetic nanoparticles (Fe3O4)

Sumanta Kumar Sahu; Arindam Chakrabarty; Dipsikha Bhattacharya; Sudip K. Ghosh; Panchanan Pramanik

2011-01-01

137

A Novel Purification Method for Histidine-Tagged Proteins Containing a Thrombin Cleavage Site  

Microsoft Academic Search

A general procedure for the purification of histidine-tagged proteins has been developed using immobilized metal-ion affinity chromatography. This two-step purification method can be used for proteins containing a hexahistidine tag and a thrombin cleavage site, yielding high amounts of purified protein. The advantage of this method is that thrombin is used instead of imidazole in the final purification step. Imidazole

Marco H. Hefti; Caroline J. G. Van Vugt-Van der Toorn; Ray Dixon; Jacques Vervoort

2001-01-01

138

Rapid High-Yield Purification and Liposome Reconstitution of Polyhistidine-Tagged Sensory Rhodopsin I  

Microsoft Academic Search

We have used Ni2+-affinity chromatography as a rapid and efficient method to purify a sensory rhodopsin I (SR-I) derivative containing six consecutive histidine residues at its C-terminus (His-tagged SR-I). The protein was expressed inHalobacterium salinariumby integrating the corresponding gene at the chromosomal bacterioopsin locus under the control of the bacterioopsin promoter. His-tagged SR-I retains native SR-I photochemical reactions in purified

Mark P. Krebs; Elena N. Spudich; John L. Spudich

1995-01-01

139

Protein-protein interactions of tandem affinity purified protein kinases from rice.  

PubMed

Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex. PMID:19690613

Rohila, Jai S; Chen, Mei; Chen, Shuo; Chen, Johann; Cerny, Ronald L; Dardick, Christopher; Canlas, Patrick; Fujii, Hiroaki; Gribskov, Michael; Kanrar, Siddhartha; Knoflicek, Lucas; Stevenson, Becky; Xie, Mingtang; Xu, Xia; Zheng, Xianwu; Zhu, Jian-Kang; Ronald, Pamela; Fromm, Michael E

2009-08-19

140

Stop thinking, start tagging: tag semantics emerge from collaborative verbosity  

Microsoft Academic Search

Recent research provides evidence for the presence of emergent semantics in collaborative tagging systems. While several methods have been proposed, little is known about the factors that influence the evolution of semantic structures in these systems. A natural hypothesis is that the quality of the emergent semantics depends on the pragmatics of tagging: Users with certain usage patterns might contribute

Christian Körner; Dominik Benz; Andreas Hotho; Markus Strohmaier; Gerd Stumme

2010-01-01

141

Extended Social Tags: Identity Tags Meet Social Networks  

Microsoft Academic Search

This paper proposes a new approach that uses social networks and common sense deduction rules to adapt the description tags of the photos for the current viewer. We exploit social graphs to enrich the tags associated to the concerned persons in the photo by following the different links between people (i.e. viewer and captured people in the photos). The main

Sonia Lajmi; Johann Stan; Hakim Hacid; Elöd Egyed-zsigmond; Pierre Maret

2009-01-01

142

Isocratic Method for Affinity Enrichment of Covalently-linked Peptides in Cyanogen Bromide Cleavage of Proteins.  

PubMed

The low resolution three-dimensional structure of a protein can be inferred from existing disulfide bridges or experimentally introduced chemical crosslinks. The general procedure involves enzymatic digestion of a protein followed by mass spectrometry-based identification of covalently-linked peptides, native disulfide-linked peptides and chemically cross-linked peptides. To facilitate unambiguous identification of these peptides, an isocratic purification method was developed for selective enrichment of covalently-linked cyanogen bromide (CNBr) fragments. This method capitalizes on the ability of homoserine lactone moieties at the C-termini of CNBr cleavage products for selective conjugation of primary-amine containing affinity tag. The availability of two C-termini within covalently-linked peptides allows for the conjugation of two affinity tags, whereas the other peptides have only one affinity tag at the C-terminus, which enables selective enrichment of covalently-linked peptides by utilization of affinity tag with moderate dissociation constant. Here we demonstrate successful implementation of this method with tetrahistidine as the affinity tag for enrichment of covalently-linked CNBr fragments of test peptides and proteins. PMID:21751378

Shi, Tujin; Liu, Jing; Yan, Chen; Wang, Xiaocong

2011-07-01

143

A dielectric affinity microbiosensor  

NASA Astrophysics Data System (ADS)

We present an affinity biosensing approach that exploits changes in dielectric properties of a polymer due to its specific, reversible binding with an analyte. The approach is demonstrated using a microsensor comprising a pair of thin-film capacitive electrodes sandwiching a solution of poly(acrylamide-ran-3-acrylamidophenylboronic acid), a synthetic polymer with specific affinity to glucose. Binding with glucose induces changes in the permittivity of the polymer, which can be measured capacitively for specific glucose detection, as confirmed by experimental results at physiologically relevant concentrations. The dielectric affinity biosensing approach holds the potential for practical applications such as long-term continuous glucose monitoring.

Huang, Xian; Li, Siqi; Schultz, Jerome S.; Wang, Qian; Lin, Qiao

2010-01-01

144

Investigation of UHF RFID tag backscatter  

Microsoft Academic Search

The results of this paper show that the backscatter from an RFID tag can be strongly influenced by the presence of other tags. This occurs even if the tag spacing is not very small. In fact, for tag spacings approaching normal antenna array spacings, the group of tags appears to behave like a parasitically coupled antenna array. The change in

Justin Johnson; Robert Sainati

2007-01-01

145

An Overview of Social Tagging and Applications  

NASA Astrophysics Data System (ADS)

Social tagging on online portals has become a trend now. It has emerged as one of the best ways of associating metadata with web objects. With the increase in the kinds of web objects becoming available, collaborative tagging of such objects is also developing along new dimensions. This popularity has led to a vast literature on social tagging. In this survey paper, we would like to summarize different techniques employed to study various aspects of tagging. Broadly, we would discuss about properties of tag streams, tagging models, tag semantics, generating recommendations using tags, visualizations of tags, applications of tags, integration of different tagging systems and problems associated with tagging usage. We would discuss topics like why people tag, what influences the choice of tags, how to model the tagging process, kinds of tags, different power laws observed in tagging domain, how tags are created and how to choose the right tags for recommendation. Metadata generated in the form of tags can be efficiently used to improve web search, for web object classification, for generating ontologies, for enhanced browsing etc. We would discuss these applications and conclude with thoughts on future work in the area.

Gupta, Manish; Li, Rui; Yin, Zhijun; Han, Jiawei

146

Anti-Histidine Antibodies as Tools for Reversible Capturing of His-Tagged Fusion Proteins for Subsequent Binding Analysis  

Microsoft Academic Search

\\u000a The hexahistidine tag is one of most commonly used fusion tags in affinity purification of recombinantly expressed proteins.\\u000a Real-time binding analysis using Biacore technology allows in-depth characterization of respective association and dissociation\\u000a patterns of potential binders. Here we tested four commercially available anti-His antibodies for reversible capturing of\\u000a His-tagged proteins as a basis for a subsequent interaction analysis with non-His-tagged

H.-M. Zenn; S. Hutschenreiter; F. W. Herberg

147

Expedient synthesis of a modular phosphate affinity reagent.  

PubMed

Isolation and identification of phosphorylated macromolecules is essential for the deconvolution of most biological regulatory networks. Koike and co-workers recently reported the application of a dinuclear zinc-(pyridylmethyl)amine complex to phosphate-specific affinity purifications and gave it the shorthand name "phos-tag". This complex is valuable for studying phosphorylation because it binds selectively to phosphate dianion in the presence of acidic functional groups at physiological pH, and because the binding is largely independent of molecular context. These properties of phos-tag recommend it for applications in phosphoproteomics, metabolomics, and nucleic acid biology. The catch has been that the molecule is difficult to make and prohibitively expensive to buy. Here, we describe an efficient and inexpensive synthesis of a phos-tag derivative with a versatile alkyne handle. The alkyne handle allows for attachment of phos-tag to alkyl azides via the copper(I)-catalyzed azide-alkyne cycloaddition reaction ("click chemistry"). We characterize the phosphate binding behavior of the new phos-tag derivative in a variety of experimental assays, including its conjugation to a fluorescent reporter, to acrylamide gels, and to sepharose chromatography resin. The synthesis we report should enable a broader use of phos-tag for phosphate-related biochemistry, as both an analytical and a preparative reagent. PMID:20491467

Tilmans, Nicolas P; Krusemark, Casey J; Harbury, Pehr A B

2010-06-16

148

Special Report: Affinity Chromatography.  

ERIC Educational Resources Information Center

Describes the nature of affinity chromatography and its use in purifying enzymes, studying cell interactions, exploring hormone receptors, and other areas. The potential the technique may have in treating disease is also considered. (JN)

Parikh, Indu; Cuatrecasas, Pedro

1985-01-01

149

Affine processes are regular  

Microsoft Academic Search

We show that stochastically continuous, time-homogeneous affine processes on the canonical state space $${\\\\mathbb{R}_{\\\\geq 0}^m \\\\times \\\\mathbb{R}^n}$$ are always regular. In the paper of Duffie et al. (Ann Appl Probab 13(3):984–1053, 2003) regularity was used as a crucial\\u000a basic assumption. It was left open whether this regularity condition is automatically satisfied for stochastically continuous\\u000a affine processes. We now show that the

Martin Keller-Ressel; Walter Schachermayer; Josef Teichmann

2009-01-01

150

Sitab: Combating Spam in Tagging Systems via Users' Implicit Tagging Behavior  

Microsoft Academic Search

Resisting spam in tagging system is very chal- lenging. This paper presents Sitab, a novel spam-resistant tagging system which can significantly diminish spam in tag search results based on users' implicit tagging behavior. Sitab is trained to obtain the weights of the client's each type of implicit tagging behavior. For each tag search, Sitab ranks each resource in the results

Longzhi Du; Yonggang Wang; Jianbin Hun; Zhong Chen

2011-01-01

151

Enhancement of ionization efficiency and selective enrichment of phosphorylated peptides from complex protein mixtures using a reversible poly-histidine tag  

Microsoft Academic Search

To improve the detection of phosphorylated peptides\\/proteins, a combination of optimized MS-based strategies were used involving\\u000a chemical derivatization with a polyhistidine-tag (His-tag) and affinity enrichment of the resulting His-tag peptides on a\\u000a nanoscale Ni2+-IMAC column. The phosphoserine and phosphothreonine peptides were derivatized using a one-pot ?-elimination\\/Michael addition\\u000a reaction with a reversible His-tag possessing a thiol-containing Cys residue. The His-tag peptides

Pegah R. Jalili; Deepti Sharma; Haydn L. Ball

2007-01-01

152

Optimal use of tandem biotin and V5 tags in ChIP assays  

PubMed Central

Background Chromatin immunoprecipitation (ChIP) assays coupled to genome arrays (Chip-on-chip) or massive parallel sequencing (ChIP-seq) lead to the genome wide identification of binding sites of chromatin associated proteins. However, the highly variable quality of antibodies and the availability of epitopes in crosslinked chromatin can compromise genomic ChIP outcomes. Epitope tags have often been used as more reliable alternatives. In addition, we have employed protein in vivo biotinylation tagging as a very high affinity alternative to antibodies. In this paper we describe the optimization of biotinylation tagging for ChIP and its coupling to a known epitope tag in providing a reliable and efficient alternative to antibodies. Results Using the biotin tagged erythroid transcription factor GATA-1 as example, we describe several optimization steps for the application of the high affinity biotin streptavidin system in ChIP. We find that the omission of SDS during sonication, the use of fish skin gelatin as blocking agent and choice of streptavidin beads can lead to significantly improved ChIP enrichments and lower background compared to antibodies. We also show that the V5 epitope tag performs equally well under the conditions worked out for streptavidin ChIP and that it may suffer less from the effects of formaldehyde crosslinking. Conclusion The combined use of the very high affinity biotin tag with the less sensitive to crosslinking V5 tag provides for a flexible ChIP platform with potential implications in ChIP sequencing outcomes.

Kolodziej, Katarzyna E; Pourfarzad, Farzin; de Boer, Ernie; Krpic, Sanja; Grosveld, Frank; Strouboulis, John

2009-01-01

153

D-TAG: erasing the tag of gang membership.  

PubMed

Gangs are noted for establishing their territory, flaunting gang affiliation, intimidating nonmembers, and documenting their "services performed." These examples are a few reasons for the practice of "tagging," the labeling of an area, person, or object with gang-related graffiti or markings, such as tattoos. This article describes a school nurse's response to gang "tagging" and her efforts to assist former gang members who request removal of their tattoos, to get them removed-in essence to D-TAG themselves from their gang affiliation. D-TAG is a volunteer rehabilitation program utilizing family and community interaction to support gang tattoo removal and direct activities away from gang affiliations toward alternative educational programs and life styles. PMID:9146217

Gurke, B; Armstrong, M L

1997-04-01

154

His6 tag-assisted chemical protein synthesis  

PubMed Central

To make more practical the total chemical synthesis of proteins by the ligation of unprotected peptide building blocks, we have developed a method to facilitate the isolation and handling of intermediate products. The synthetic technique makes use of a His6 tag at the C terminus of the target polypeptide chain, introduced during the synthesis of the C-terminal peptide segment building block. The presence of a His6 tag enables the isolation of peptide or protein products directly from ligation reaction mixtures by Ni-NTA affinity column purification. This simple approach enables facile buffer exchange to alternate reaction conditions and is compatible with direct analytical control by protein MS of the multiple ligation steps involved in protein synthesis. We used syntheses of crambin and a modular tetratricopeptide repeat protein of 17 kDa as models to examine the utility of this affinity purification approach. The results show that His6 tag-assisted chemical protein synthesis is a useful method that substantially reduces handling losses and provides for rapid chemical protein syntheses.

Bang, Duhee; Kent, Stephen B. H.

2005-01-01

155

High-throughput T7 LIC vector for introducing C-terminal poly-histidine tags with variable lengths without extra sequences  

Microsoft Academic Search

Immobilized metal ion affinity chromatography (IMAC) has become one of the most popular protein purification methods for recombinant proteins with a hexa-histidine tag (His-tag) placed at the C- or N-terminus of proteins. Nevertheless, there are always difficult proteins that show weak binding to the metal chelating resin and thus low purity. These difficulties are often overcome by increasing the His-tag

Jonas Lee; Sung-Hou Kim

2009-01-01

156

Development of a Dehalogenase-Based Protein Fusion Tag Capable of Rapid, Selective and Covalent Attachment to Customizable Ligands  

PubMed Central

Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenable to modular synthetic design. Taken together, the design features and molecular evolution of HT7 have resulted in a superior alternative to common tags for the overexpression, detection, and isolation of target proteins.

Encell, Lance P; Friedman Ohana, Rachel; Zimmerman, Kris; Otto, Paul; Vidugiris, Gediminas; Wood, Monika G; Los, Georgyi V; McDougall, Mark G; Zimprich, Chad; Karassina, Natasha; Learish, Randall D; Hurst, Robin; Hartnett, James; Wheeler, Sarah; Stecha, Pete; English, Jami; Zhao, Kate; Mendez, Jacqui; Benink, Helene A; Murphy, Nancy; Daniels, Danette L; Slater, Michael R; Urh, Marjeta; Darzins, Aldis; Klaubert, Dieter H; Bulleit, Robert F; Wood, Keith V

2012-01-01

157

Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems  

Microsoft Academic Search

. In response to the rapidly growing field of proteomics, the use of recombinant proteins has increased greatly in recent years. Recombinant hybrids containing a polypeptide fusion partner, termed affinity tag, to facilitate the purification of the target polypeptides are widely used. Many different proteins, domains, or peptides can be fused with the target protein. The advantages of using fusion

K. Terpe

2003-01-01

158

Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins.  

PubMed

Immobilized metal ion affinity chromatography (IMAC) is now a widely accepted technique for the separation of natural or artificial products that is beginning to find industrial applications. Here, we introduce a novel procedure for the separation of phosphopeptides and phosphorylated proteins by immobilized zinc(II) affinity chromatography. The phosphate-binding site of the affinity gel is an alkoxide-bridged dinuclear zinc(II) complex, the 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex (Phos-tag), which is linked to a highly cross-linked 4% (w/v) agarose. The affinity gel (Phos-tag agarose) was prepared by the quantitative reaction of N-hydroxysuccinimide-activated Sepharose and a Phos-tag derivative having a 2-aminoethylcarbamoyl group in dry CH3CN. Phosphopeptides were retrieved in a quantitative and highly selective manner by a spin column method using Phos-tag agarose at room temperature. Furthermore, in this study, we demonstrate a simple, rapid, and reusable affinity column chromatography for the separation of phosphorylated proteins such as ovalbumin, alpha(s1)-casein, and beta-casein at physiological pH. PMID:15754823

Kinoshita, Eiji; Yamada, Atsushi; Takeda, Hironori; Kinoshita-Kikuta, Emiko; Koike, Tohru

2005-02-01

159

MEASUREMENT OF BACKSCATTERING FROM RFID TAGS  

Microsoft Academic Search

This paper presents a method for measuring signal backscattering from an RFID tag and calculating tag radar cross-section (RCS), which depends on the chip input impedance. We present a derivation of a theoretical formula for RFID tag radar cross-section and an experimental RCS measurement method using a network analyzer connected to an antenna in an anechoic chamber where the tag

Pavel V. Nikitin; K. V. S. Rao

160

Latent dirichlet allocation for tag recommendation  

Microsoft Academic Search

Tagging systems have become major infrastructures on the Web. They allow users to create tags that annotate and cat- egorize content and share them with other users, very helpful in particular for searching multimedia content. However, as tagging is not constrained by a controlled vocabulary and annotation guidelines, tags tend to be noisy and sparse. Es- pecially new resources annotated

Ralf Krestel; Peter Fankhauser; Wolfgang Nejdl

2009-01-01

161

Extracting Representative Tags for Flickr Users  

Microsoft Academic Search

Tags are very popular in online social communities (like You tube, Flickr) and provide valuable and crucial information for these communities. But at the same time, there exist a lot of noisy tags, which leads many researches to tag suggestion, tag recommendation for the items, such as to the websites, photos, books, movies, and so on. Most of them used

Xian Chen; Hyoseop Shin

2010-01-01

162

Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification.  

PubMed

His-tag affinity purification is one of the most commonly used methods to purify recombinant proteins expressed in E. coli. One drawback of using the His-tag is the co-purification of contaminating histidine-rich E. coli proteins. We engineered a new E. coli expression strain, LOBSTR (low background strain), which eliminates the most abundant contaminants. LOBSTR is derived from the E. coli BL21(DE3) strain and carries genomically modified copies of arnA and slyD, whose protein products exhibit reduced affinities to Ni and Co resins, resulting in a much higher purity of the target protein. The use of LOBSTR enables the pursuit of challenging low-expressing protein targets by reducing background contamination with no additional purification steps, materials, or costs, and thus pushes the limits of standard His-tag purifications. Proteins 2013; 81:1857-1861. © 2013 Wiley Periodicals, Inc. PMID:23852738

Andersen, Kasper R; Leksa, Nina C; Schwartz, Thomas U

2013-08-23

163

Circular UHF RFID tag antenna and relationship between reading range and RCS of the tag antenna  

Microsoft Academic Search

The different sizes of circular tag antennas have been designed. The different sizes of circular tags will be fit to any circular object such as bottle caps of cosmetics. The outer radius from 8 mm to 40 mm of tags have been fabricated and tested. The 12 mm circular tag has about 1 m reading distance, and 35 mm tag

Goojo Kim; You Chung Chung

2007-01-01

164

Using Weighted Tagging to Facilitate Enterprise Search  

Microsoft Academic Search

\\u000a Motivated by the success of social tagging in web communities, this paper proposes a novel document tagging method more suitable\\u000a for the enterprise environment, named weighted tagging. The method allows users to tag a document with weighted tags which\\u000a are then used as an additional source for the query matching and relevance scoring to improve the search results. The method

Shengwen Yang; Jianming Jin; Yuhong Xiong

2010-01-01

165

Learning to Tag Multilingual Texts Through Observation  

Microsoft Academic Search

This paper describes RoboTag, an ad- vanced prototype for a machine learning- based multilingual information extraction system. First, we describe a general client\\/server architecture used in learning from observation. Then we give a detailed description of our novel decision-tree tag- ging approach. RoboTag performance for the proper noun tagging task in English and Japanese is compared against human- tagged keys

Scott W. Bennett; Chinatsu Aone; Craig Lovell

1997-01-01

166

Purification of phage display-modified bacteriophage T4 by affinity chromatography  

PubMed Central

Background Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system. Results Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml. Conclusions Affinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be considered as a new phage purification method, appropriate for further investigations and development.

2011-01-01

167

Cost-effective isobaric tagging for quantitative phosphoproteomics using DiART reagents.  

PubMed

We describe the use of an isobaric tagging reagent, Deuterium isobaric Amine Reactive Tag (DiART), for quantitative phosphoproteomic experiments. Using DiART tagged custom mixtures of two phosphorylated peptides from alpha casein and their non-phosphorylated counterparts, we demonstrate the compatibility of DiART with TiO2 affinity purification of phosphorylated peptides. Comparison of theoretical vs. experimental reporter ion ratios reveals accurate quantification of phosphorylated peptides over a dynamic range of more than 15-fold. Using DiART labelling and TiO2 enrichment (DiART-TiO2) with large quantities of proteins (8 mg) from the cell lysate of model fungus Aspergillus nidulans, we quantified 744 unique phosphopeptides. Overlap of median values of TiO2 enriched phosphopeptides with theoretical values indicates accurate trends. Altogether these findings confirm the feasibility of performing quantitative phosphoproteomic experiments in a cost-effective manner using isobaric tagging reagents, DiART. PMID:24129742

Ramsubramaniam, Nikhil; Tao, Feng; Li, Shuwei; Marten, Mark R

2013-10-16

168

The modification of quantum dot probes used for the targeted imaging of his-tagged fusion proteins  

Microsoft Academic Search

In molecular biology and protein detection the immobilized metal ion clusters using a NTA-chelator is a powerful technique in identification and isolation of histidine-tagged fusion proteins. The Oligo-histidine tag should serve as a high affinity binding sequence for the purification of any fusion protein via metal chelating adsorbents. We described the preparation and characterization of bioinorganic conjugates made with highly

Pan K. Bae; Kyung N. Kim; Seung J. Lee; Hyun J. Chang; Chong K. Lee; Joung K. Park

2009-01-01

169

Characterization of native and histidine-tagged deoxyxylulose 5-phosphate reductoisomerase from the cyanobacterium Synechocystis sp. PCC6803  

Microsoft Academic Search

The dxr gene encoding the 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) from the cyanobacterium Synechocystis sp. PCC6803 was expressed in Escherichia coli to produce both the native and N-terminal histidine-tagged forms of DXR. The enzymes were purified from the cell extracts using either anion exchange chromatography or metal affinity chromatography and gel filtration. The purified recombinant native and histidine-tagged enzymes each displayed

Xihou Yin; Philip J Proteau

2003-01-01

170

Stereoscopic flow-tagging velocimetry  

NASA Astrophysics Data System (ADS)

A laser-based method for measuring the three components of the velocity in a plane simultaneously and instantaneously without seed particles is presented. This is achieved by combining a laser flow-tagging technique with stereoscopic detection, in which the tagged flow is viewed from two different directions. A single CCD camera is employed for this purpose by using a new optical detection system. The flow tagging is performed by two consecutive laser pulses, i.e., ``write'' and ``read'' laser pulses. The write laser creates a grid of tracer molecules (NO) by inducing a photodissociation process. The three-dimensional motion of the tracer molecules is measured by a thick read laser sheet.

Krüger, S.; Grünefeld, G.

171

Tagging the European eel Anguilla anguilla (L.) with coded wire tags  

Microsoft Academic Search

The coded wire tag (CWT) system was examined as a possible tool for tagging European eels (Anguilla anguilla). Two size groups of eels (3.8 and 10.2 g) were tagged with CWTs in the dorsal musculature. Tag loss 28 days after tagging was 3.1% for the small and 0.7% for the large groups of eels. Of the tag loss in the

Søren Thomassen; Michael Ingemann Pedersen; Gert Holdensgaard

2000-01-01

172

WebTag: Web Browsing into Sensor Tags over NFC  

PubMed Central

Information and Communication Technologies (ICTs) continue to overcome many of the challenges related to wireless sensor monitoring, such as for example the design of smarter embedded processors, the improvement of the network architectures, the development of efficient communication protocols or the maximization of the life cycle autonomy. This work tries to improve the communication link of the data transmission in wireless sensor monitoring. The upstream communication link is usually based on standard IP technologies, but the downstream side is always masked with the proprietary protocols used for the wireless link (like ZigBee, Bluetooth, RFID, etc.). This work presents a novel solution (WebTag) for a direct IP based access to a sensor tag over the Near Field Communication (NFC) technology for secure applications. WebTag allows a direct web access to the sensor tag by means of a standard web browser, it reads the sensor data, configures the sampling rate and implements IP based security policies. It is, definitely, a new step towards the evolution of the Internet of Things paradigm.

Echevarria, Juan Jose; Ruiz-de-Garibay, Jonathan; Legarda, Jon; Alvarez, Maite; Ayerbe, Ana; Vazquez, Juan Ignacio

2012-01-01

173

Deciphering Protein Complexes and Protein Interaction Networks by Tandem Affinity Purification and Mass Spectrometry  

Microsoft Academic Search

We employed a combination of tandem affinity purifica- tion and mass spectrometry for deciphering protein com- plexes and the protein interaction network in budding yeast. 53 genes were epitope-tagged, and their interac- tion partners were isolated by two-step immunoaffinity chromatography from whole cell lysates. 38 baits pulled down a total of 220 interaction partners, which are mem- bers of 19

Anna Shevchenko; Daniel Schaft; Assen Roguev; W. W. M. Pim; A. Francis Stewart; Andrej Shevchenko

174

Conjugation of DNA with protein using His-tag chemistry and its application to the aptamer-based detection system  

Microsoft Academic Search

We propose a novel method to prepare a DNA–protein conjugate using histidine-tag (His-tag) chemistry. Oligo-DNA was modified\\u000a with nitrilotriacetate (NTA), which has high affinity to a His-tag on recombinant protein via the complexation of Ni2+. Investigations using a microplate which displayed a complementary DNA-strand revealed that a NTA-modified DNA–protein conjugate\\u000a was formed and immobilized in the presence of Ni2+ on

Josui Shimada; Tatsuo Maruyama; Takuya Hosogi; Jo Tominaga; Noriho Kamiya; Masahiro Goto

2008-01-01

175

Click Chemistry Facilitates Formation of Reporter Ions and Simplified Synthesis of Amine-Reactive Multiplexed Isobaric Tags for Protein Quantification  

PubMed Central

We report the development of novel reagents for cell-level protein quantification, referred to as Caltech Isobaric Tags (CITs), which offer several advantages in comparison with other isobaric tags (e.g., iTRAQ and TMT). Click chemistry, copper-catalyzed azide-alkyne cycloaddition (CuAAC), is applied to generate a gas-phase cleavable linker suitable for the formation of reporter ions. Upon collisional activation, the 1,2,3-triazole ring constructed by CuAAC participates in a nucleophilic displacement reaction forming a six-membered ring and releasing a stable cationic reporter ion. To investigate its utility in peptide mass spectrometry, the energetics of the observed fragmentation pathway are examined by density functional theory. When this functional group is covalently attached to a target peptide, it is found that the nucleophilic displacement occurs in competition with formation of b- and y-type backbone fragment ions regardless of the amino acid side-chains present in the parent bioconjugate, confirming that calculated reaction energetics of reporter ion formation are similar to those of backbone fragmentations. Based on these results, we apply this selective fragmentation pathway for the development of CIT reagents. For demonstration purposes, duplex CIT reagent is prepared using a single isotope-coded precursor, allyl-d5-bromide, with reporter ions appearing at m/z 164 and 169. Isotope-coded allyl azides for the construction of the reporter ion group can be prepared from halogenated alkyl groups which are also employed for the mass balance group via N-alkylation, reducing the cost and effort for synthesis of isobaric pairs. Owing to their modular designs, an unlimited number of isobaric combinations of CIT reagents are, in principle, possible. The reporter ion mass can be easily tuned to avoid overlapping with common peptide MS/MS fragments as well as the low mass cut-off problems inherent in ion trap mass spectrometers. The applicability of the CIT reagent is tested with several model systems involving protein mixtures and cellular systems.

Ho Sohn, Chang; Lee, J. Eugene; Sweredoski, Michael J.; Graham, Robert L.J.; Smith, Geoffrey T.; Hess, Sonja; Czerwieniec, Gregg; Loo, Joseph A.; Deshaies, Raymond J.; Beauchamp, J. L.

2012-01-01

176

Comparing Social Tags to Microblogs  

Microsoft Academic Search

As Internet usage and e-commerce grow, online social media serve as popular outlets for consumers to express sentiments about products. On Amazon, users can tag an album with a keyword, while tweets on Twitter represent a more natural conversation. The differing natures of these media make them difficult to compare. This project collects and analyzes social media data for newly

Victoria Lai; Christopher Rajashekar; William Rand

2011-01-01

177

Named entity tagged language models  

Microsoft Academic Search

We introduce named entity (NE) language modelling, a stochastic finite state machine approach to identifying both words and NE categories from a stream of spoken data. We provide an overview of our approach to NE tagged language model (LM) generation together with results of the application of such a LM to the task of out-of-vocabulary (OOV) word reduction in large

Y. Gotoh; S. Renals; G. Williams

1999-01-01

178

Approximation properties of haplotype tagging  

Microsoft Academic Search

Background: Single nucleotide polymorphisms (SNPs) are locations at which the genomic sequences of population members differ. Since these differences are known to follow patterns, disease association studies are facilitated by identifying SNPs that allow the unique identification of such patterns. This process, known as haplotype tagging, is formulated as a combinatorial optimization problem and analyzed in terms of complexity and

Staal A. Vinterbo; Stephan Dreiseitl; Lucila Ohno-machado

2006-01-01

179

What Do Tag Games Teach?  

ERIC Educational Resources Information Center

Tag games have been described as "Chasing, fleeing, and dodging" type activities. Most "fleeing" activities involve dramatic play, use of movement concepts (such as quick and light), or movement changes without a partner, while many of the chasing and dodging activities utilize dodging concepts between partners or within small groups and are…

Belka, David

2006-01-01

180

The EIPeptiDi tool: enhancing peptide discovery in ICAT-based LC MS\\/MS experiments  

Microsoft Academic Search

BACKGROUND: Isotope-coded affinity tags (ICAT) is a method for quantitative proteomics based on differential isotopic labeling, sample digestion and mass spectrometry (MS). The method allows the identification and relative quantification of proteins present in two samples and consists of the following phases. First, cysteine residues are either labeled using the ICAT Light or ICAT Heavy reagent (having identical chemical properties

Mario Cannataro; Giovanni Cuda; Marco Gaspari; Sergio Greco; Giuseppe Tradigo; Pierangelo Veltri

2007-01-01

181

The Pseudomonas aeruginosa Proteome during Anaerobic Growth  

Microsoft Academic Search

Isotope-coded affinity tag analysis and two-dimensional gel electrophoresis followed by tandem mass spec- trometry were used to identify Pseudomonas aeruginosa proteins expressed during anaerobic growth. Out of the 617 proteins identified, 158 were changed in abundance during anaerobic growth compared to during aerobic growth, including proteins whose increased expression was expected based on their role in anaerobic metab- olism. These

Manhong Wu; Tina Guina; Mitchell Brittnacher; Hai Nguyen; Jimmy Eng; Samuel I. Miller

2005-01-01

182

Oligo-Asp tag/Zn(II) complex probe as a new pair for labeling and fluorescence imaging of proteins.  

PubMed

To accomplish the selective labeling of a specific protein in complicated biological systems, a peptide tag incorporated into the protein and a complementary small molecular probe are required. Although a variety of peptide tag/probe pairs have been developed as molecular tools for protein analyses, the availability of pairs suitable for real-time imaging of proteins is still limited. We now report a new peptide tag/artificial probe pair composed of a genetically encodable oligo-aspartate sequence (D4 tag, (D4)n, n = 1-3) and the corresponding multinuclear Zn(II) complexes (Zn(II)-DpaTyrs). The strong binding affinity of the Zn(II)-DpaTyr probes with the D4 tag was a result of the multiple coordination bonds and the multivalent effect. It was measured quantitatively by isothermal titration calorimetry. The high affinity between the tag and the probe, indispensable for the selective protein labeling, enabled the pair to be used for the labeling and fluorescence imaging of a membrane-bound receptor protein tethering a triply repeated D4 tag ((D4)3) in an intact cell configuration without significantly affecting the receptor signal transduction. PMID:16895410

Ojida, Akio; Honda, Kei; Shinmi, Daisuke; Kiyonaka, Shigeki; Mori, Yasuo; Hamachi, Itaru

2006-08-16

183

Characterization of polystyrene-binding peptides (PS-tags) for site-specific immobilization of proteins.  

PubMed

In this study, we characterized polystyrene-binding peptides (PS-tags) that possess a specific binding affinity for hydrophilic polystyrene (phi-PS) plates. Both the FITC-labeled PS19-1 (RAFIASRRIKRP) and PS19-6 (RIIIRRIRR) peptides showed strong binding affinity for commercially available hydrophilic, but not hydrophobic, PS plates in the presence of the non-ionic surfactant Tween 20. The dissociation constants (K(d)) of the PS19-1 and PS19-6 peptides for the hydrophilic PS-A plate were 169 and 86 nM, respectively, and the K(d) of both peptides increased with the concentration of NaCl or urea. Based on adsorption yield and residual activity of glutathione S-transferase (GST) after fusion with the PS19-6 peptide or its variants, it was found that the basic amino acid in the PS-tags, i.e., Arg was essential for the strong binding affinity of PS-tags in both the peptide and peptide-fused protein forms The aliphatic amino acids in PS19-6 and PS19-6L, such as Ile or Leu, were also effective. Thus, a series of PS-tags that possess this unusual feature, especially the peptides PS19-6 (RIIIRRIRR) and PS19-6L (RLLLRRLRR), are potential candidate affinity peptide tags for site-specific immobilization of proteins onto hydrophilic PS plates, which show potential as solid supports for protein-based biochips. PMID:20471598

Kumada, Yoichi; Kuroki, Daisuke; Yasui, Hidefumi; Ohse, Takuhito; Kishimoto, Michimasa

2009-12-14

184

Buddy Tag's motion sensing and analysis subsystem  

SciTech Connect

Buddy Tag is one of several types of tags being developed as a means of verifying arms control limitations on numbers of treaty limited items (TLIs). The TLIs being focused on for now are missile systems. Buddy Tag has the attractive feature that it does not have to be attached to the TLI, making it less intrusive than conventional tagging schemes. Key to Buddy Tag's capability is its motion sensing and analysis subsystem. Due to the nature of Buddy Tag's potential application, the motion sensing and analysis subsystem must be highly sensitive, extremely reliable, and capable of correctly distinguishing illegal movement of the Buddy Tag from inputs due to nearby cultural activity or low level seismic disturbances. This paper overviews the Buddy Tag concept and discusses its motion sensing and analysis subsystem.

Jordon, S.E. (Sandia National Labs., Albuquerque, NM (United States))

1991-01-01

185

Affinity chromatography using biocompatible and reusable biotinylated membranes.  

PubMed

A novel, reusable biotinylated affinity chromatography strategy for the bio-specific binding of bioactive avidin tagged enzymes or polypeptides is reported. Using an avidin coupled peroxidase fusion protein as a test system; non-specific protein shielding and matrix regeneration were also shown. The amphiphilic surfactant Pluronic F108 was used as an affinity linker, by non-covalent binding to membrane chromatographic matrices while the terminal hydroxyl groups of Pluronic were covalently coupled to the biological ligand biotin. Planar nonporous membranes of varying surface chemistry were synthesised to test the matrix dependent affinity binding of biotinylated Pluronic and their respective ability to resist non-specific protein adsorption. Membrane regeneration using sodium dodecyl sulphate (SDS) was capable of displacing both adsorbed proteins and Pluronic. SDS micelles (34 mM) were effective in desorbing membrane bound protein while 5mM SDS removed up to 85% of the bound ligand after 20 h incubation at 20 degrees C. In this study, polyvinylidene membranes had the highest ligand binding capacity of 0.22 mg cm(-2) and specific, competitive affinity binding of avidin-peroxidase was shown in the presence of up to 0.2 mg ml(-1) 'contaminant' proteins. The resultant biocompatible affinity chromatographic system was regenerated and reused with no significant change in performance for up to five cycles. PMID:17875407

Govender, S; Jacobs, E P; Bredenkamp, M W; Swart, P

2007-08-26

186

WhaleNet's Satellite Tagging Program  

NSDL National Science Digital Library

This extensive, easy to use site contains information on satellite tags and tagging, data from current and archived tagging projects on whales, porpoises, seals, and turtles, and questions and activities to help use the data in the classroom. Follow these marine mammals up and down the coast or use the archived data to study distribution and migration patterns of dolphins, seals, etc. Site also links to other tagging and monitoring programs.

187

Tag Gardening for Folksonomy Enrichment and Maintenance  

Microsoft Academic Search

As social tagging applications continuously gain in popularity, it becomes more and more accepted that models and tools for (re-)organizing tags are needed. Some first approaches are already practically implemented. Recently, activities to edit and organize tags have been described as \\

Isabella Peters; Katrin Weller

2008-01-01

188

Use of META Tags for Internet Documents  

Microsoft Academic Search

The number of web pages incorporating META tags into HTML coding was determined for web sites linked to the University of Nebraska Agricultural Network Information Center (AgNIC) Plant Science Page. META tags were examined by domain and year of last update using the View\\/Document Source option on Netscape. The “keywords” META tag was included in coding for 23% of the

Elaine A. Nowick

2002-01-01

189

Tagrank - Measuring tag importance for image annotation  

Microsoft Academic Search

Traditional image annotation approaches are only applicable for datasets with small and limited lexicon. Besides, annotation words are treated equally without considering the importance of each word in the real world. To address these problems, we propose TagRank, a method to model the relative importance of every candidate word. By exploiting tag clusters on Flickr, TagRank could be modeled as

Xiao Ling; Jimin Jia; Nenghai Yu; Mingjing Li

2008-01-01

190

Coupling passive sensors to UHF RFID tags  

Microsoft Academic Search

Coupling passive sensor data to existing UHF RFID tags explores the possibility for low volume applications without designing a new tag ASIC. The existing UHF RFID system can be used to convey additional data by overlaying a coupling loop on the tag antenna and modulating vector backscatter. The design of the RFID passive sensor prototype is presented and the feasibility

Huan-Yang Chen; Sangchul Bae; Atul Bhadkamkar; Yue Weng Mak; Daniel W. van der Weide

2012-01-01

191

QAM backscatter for passive UHF RFID tags  

Microsoft Academic Search

Traditional passive UHF RFID tags employ either ASK or PSK backscatter modulation to communicate data from memory or sensors on the tag to a remotely-located reader. These simple modulation schemes transfer data at a rate of one bit per symbol period, which for an integrated CMOS tag IC requires an on-chip oscillator with a frequency at least equal to the

Stewart Thomas; Matthew S. Reynolds

2010-01-01

192

ActiveTags: Making Tags More Useful Anywhere on the Web  

Microsoft Academic Search

Tags in social tagging systems store meaning for the taggers who have entered them, and other users often share this understanding. The result of this, a folksonomy, is typically used in several ways, including information retrieval and clustering, serendipitous information access, or visualization of folksonomic characteristics. For these uses tags work pretty well; however, the ambiguity of tags makes it

Stephan Hagemann; Gottfried Vossen

2009-01-01

193

In vivo investigation of protein-protein interactions for helicases using tandem affinity purification.  

PubMed

A key component in determining the functional role of any protein is the elucidation of its binding partners using protein-protein interaction (PPI) data. Here we examine the use of tandem affinity purification (TAP) tagging to study RNA/DNA helicase PPIs in Escherichia coli. The tag, which consists of a calmodulin-binding region, a TEV protease recognition sequence, and an IgG-binding domain, is introduced into E. coli using a lambdared recombination system. This method prevents the overproduction of the target protein, which could generate false interactions. The interacting proteins are then affinity purified using double affinity purification steps and are separated by SDS-PAGE followed by mass spectrometry identification. Each protein identified would represent a physical interaction in the cell. These interactions may potentially be mediated by an RNA/DNA template, for which the helicase would likely be needed to disrupt the secondary structures. PMID:20225144

Jessulat, Matthew; Buist, Terry; Alamgir, Md; Hooshyar, Mohsen; Xu, Jianhua; Aoki, Hiroyuki; Ganoza, M Clelia; Butland, Gareth; Golshani, Ashkan

2010-01-01

194

Mortality, Predation, and Tag Visibility of Fish Marked with Visible Implant Elastomer Tags  

Microsoft Academic Search

We evaluated tag-induced mortality (7 d), predation, and tag visibility (365 d) for age-0 largemouth bass (LMB) Micropterus salmoides (27–112 mm total length [TL]), age-0 channel catfish (CCF) Ictalurus punctatus (42–101 mm TL), and adult blacktail shiners (BTS) Cyprinella venusta (34–98 mm TL) tagged with three visible implant elastomer (VIE) tags. Tagging mortality after 7 d was 14.7% for age-0

Kerry S. Reeves; David L. Buckmeier

2009-01-01

195

A low-cost affinity purification system using ?-1,3-glucan recognition protein and curdlan beads  

PubMed Central

Silkworm ?-1,3-glucan recognition protein (?GRP) tightly and specifically associates with ?-1,3-glucan. We report here an affinity purification system named the ‘GRP system’, which uses the association between the ?-1,3-glucan recognition domain of ?GRP (GRP-tag), as an affinity tag, and curdlan beads. Curdlan is a water-insoluble ?-1,3-glucan reagent, the low cost of which (about 100 JPY/g) allows the economical preparation of beads. Curdlan beads can be readily prepared by solubilization in an alkaline solution, followed by neutralization, sonication and centrifugation. We applied the GRP system to preparation of several proteins and revealed that the expression levels of the GRP-tagged proteins in soluble fractions were two or three times higher than those of the glutathione S-transferase (GST)-tagged proteins. The purity of the GRP-tagged proteins on the curdlan beads was comparable to that of the GST-tagged proteins on glutathione beads. The chemical stability of the GRP system was more robust than conventional affinity systems under various conditions, including low pH (4–6). Biochemical and structural analyses revealed that proteins produced using the GRP system were structurally and functionally active. Thus, the GRP system is suitable for both the large- and small-scale preparation of recombinant proteins for functional and structural analyses.

Horiuchi, Masataka; Takahasi, Kiyohiro; Kobashigawa, Yoshihiro; Ochiai, Masanori; Inagaki, Fuyuhiko

2012-01-01

196

Myocardial Wall Tagging With Undersampled Projection Reconstruction  

PubMed Central

Azimuthally undersampled projection reconstruction (PR) acquisition is investigated for use in myocardial wall tagging with MR using grid tags to provide increased temporal and spatial resolution. PR can provide the high-resolution images required for tagging with very few projections, at the expense of artifact. Insight is provided into the PR undersampling artifact, in the context of measuring myocardial motion with tags. For Fourier transform imaging, at least 112 phase-encodings must be collected to image tagging grids spaced 7 pixels apart. PR requires about 80 projections, a 1.4-fold reduction in scan time.

Peters, Dana C.; Epstein, Frederick H.; McVeigh, Elliot R.

2007-01-01

197

Stop reconstruction with tagged tops  

NASA Astrophysics Data System (ADS)

At the LHC combinatorics make it unlikely that we will be able to observe stop pair production with a decay to a semi-leptonic top pair and missing energy for generic supersymmetric mass spectra. Using a Standard-Model top tagger on fully hadronic top decays we can not only extract the stop signal but also measure the top momentum. To illustrate the promise of tagging tops with moderate boost we include a detailed discussion of our HEPTopTagger algorithm.

Plehn, Tilman; Spannowsky, Michael; Takeuchi, Michihisa; Zerwas, Dirk

2010-10-01

198

Expressivity tag: a novel tool for increased expression in Escherichia coli.  

PubMed

Protein expression in Escherichia coli is rarely trivial as low expression and insolubility are common problems. In this work we define a fusion partner, which increases expression levels similarly to the distinct function of solubility and affinity tags. This type of fusion tag we term an expressivity tag. Our work is based on earlier observations where 3' deletions of the InfB gene displays strongly increased expression levels. We have constructed progressively shortened fragments of the InfB(1-471) gene and fused gene fragments to a gfp reporter gene. A 5-fold increase in GFP expression was seen for an optimal 21 nucleotide InfB(1-21) sequence compared to gfp independently. We defined the InfB(1-21) sequence as an expressivity tag. The tag was tested for improved expression of two biotechnological important proteins streptavidin and a single chain antibody (scFv). Expression of both streptavidin and scFv(L32) was improved as evaluated by SDS-PAGE. Calculation of folding energies in the translation initiation region gave higher free energies for gfp, L32 and streptavidin when linked to InfB(1-21) than independently. InfB(1-21) did however not improve the codon usage or codon adaptation index. The expressivity tag is an important addition to the box of tools available for optimizing heterologous protein expression. PMID:21801766

Hansted, Jon Gade; Pietikäinen, Laura; Hög, Friederike; Sperling-Petersen, Hans Uffe; Mortensen, Kim Kusk

2011-07-27

199

Approximation properties of haplotype tagging  

PubMed Central

Background Single nucleotide polymorphisms (SNPs) are locations at which the genomic sequences of population members differ. Since these differences are known to follow patterns, disease association studies are facilitated by identifying SNPs that allow the unique identification of such patterns. This process, known as haplotype tagging, is formulated as a combinatorial optimization problem and analyzed in terms of complexity and approximation properties. Results It is shown that the tagging problem is NP-hard but approximable within 1 + ln((n2 - n)/2) for n haplotypes but not approximable within (1 - ?) ln(n/2) for any ? > 0 unless NP ? DTIME(nlog log n). A simple, very easily implementable algorithm that exhibits the above upper bound on solution quality is presented. This algorithm has running time O((2m - p + 1)) ? O(m(n2 - n)/2) where p ? min(n, m) for n haplotypes of size m. As we show that the approximation bound is asymptotically tight, the algorithm presented is optimal with respect to this asymptotic bound. Conclusion The haplotype tagging problem is hard, but approachable with a fast, practical, and surprisingly simple algorithm that cannot be significantly improved upon on a single processor machine. Hence, significant improvement in computatational efforts expended can only be expected if the computational effort is distributed and done in parallel.

Vinterbo, Staal A; Dreiseitl, Stephan; Ohno-Machado, Lucila

2006-01-01

200

Reprint of: Immobilized-Metal Affinity Chromatography (IMAC): A Review.  

PubMed

This article reviews the development of immobilized-metal affinity chromatography (IMAC) and describes its most important applications. We provide an overview on the use of IMAC in protein fractionation and proteomics, in protein immobilization and detection, and on some special applications such as purification of immunoglobulins and the Chelex method. The most relevant application- purification of histidine-tagged recombinant proteins-will be reviewed in greater detail with focus of state-of-the-art materials, methods, and protocols, and the limitations of IMAC and recent advances to improve the technology and the methods will be described. PMID:21893200

Block, Helena; Maertens, Barbara; Spriestersbach, Anne; Brinker, Nicole; Kubicek, Jan; Fabis, Roland; Labahn, Jörg; Schäfer, Frank

2011-09-01

201

Co-Evolution of Multipartite Interactions Between an Extended tmRNA Tag and a Robust Lon Protease in Mycoplasma  

PubMed Central

Messenger RNAs that lack in-frame stop codons promote ribosome stalling and accumulation of aberrant and potentially harmful polypeptides. The SmpB-tmRNA quality control system has evolved to solve problems associated with nonstop mRNAs, by rescuing stalled ribosomes and directing the addition of a peptide tag to the C-termini of the associated proteins, marking them for proteolysis. In E. coli, the ClpXP system is the major contributor to disposal of tmRNA tagged proteins. We have shown that the AAA+ Lon protease can also degrade tmRNA tagged proteins, but with much lower efficiency. Here, we present a unique case of enhanced recognition and degradation of an extended Mycoplasma pneumoniae (MP) tmRNA tag by the MP-Lon protease. We demonstrate that MP-Lon can efficiently and selectively degrade MP-tmRNA tagged proteins. Most significantly, our studies reveal that the larger (27 amino acid long) MP-tmRNA tag contains multiple discrete signaling motifs for efficient recognition and rapid degradation by Lon. We propose that higher affinity multipartite interactions between MP-Lon and the extended MP-tmRNA tag have co-evolved from pre-existing weaker interactions, as exhibited by Lon in E. coli, to better fulfill the function of MP-Lon as the sole soluble cytoplasmic protease responsible for the degradation of tmRNA tagged proteins.

Ge, Zhiyun; Karzai, A. Wali

2009-01-01

202

Engineering a high-affinity scaffold for non-chromatographic protein purification via intein-mediated cleavage.  

PubMed

While protein purification has long been dominated by standard chromatography, the relatively high cost and complex scale-up have promoted the development of alternative non-chromatographic separation methods. Here we developed a new non-chromatographic affinity method for the purification of proteins expressed in Escherichia coli. The approach is to genetically fuse the target proteins with an affinity tag. Direct purification and recovery can be achieved using a thermo-responsive elastin-like protein (ELP) scaffold containing the capturing domain. Naturally occurring cohesin-dockerin pairs, which are high-affinity protein complex responsible for the formation of cellulosome in anaerobic bacteria, were used as the model. By exploiting the highly specific interaction between the dockerin and cohesin domain from Clostridium thermocellum and the reversible aggregation property of ELP, highly purified and active dockerin-tagged proteins, such as the endoglucanase CelA, chloramphenicol acetyl transferase (CAT), and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single thermal precipitation step with yields achieving over 90%. Incorporation of a self-cleaving intein domain enabled rapid removal of the affinity tag from the target proteins, which was subsequently removed by another cycle of thermal precipitation. This method offers great flexibility as a wide range of affinity tags and ligands can be used. PMID:22566125

Liu, Fang; Tsai, Shen-Long; Madan, Bhawna; Chen, Wilfred

2012-05-17

203

pAUL: a gateway-based vector system for adaptive expression and flexible tagging of proteins in Arabidopsis.  

PubMed

Determination of protein function requires tools that allow its detection and/or purification. As generation of specific antibodies often is laborious and insufficient, protein tagging using epitopes that are recognized by commercially available antibodies and matrices appears more promising. Also, proper spatial and temporal expression of tagged proteins is required to prevent falsification of results. We developed a new series of binary Gateway cloning vectors named pAUL1-20 for C- and N-terminal in-frame fusion of proteins to four different tags: a single (i) HA epitope and (ii) Strep-tagIII, (iii) both epitopes combined to a double tag, and (iv) a triple tag consisting of the double tag extended by a Protein A tag possessing a 3C protease cleavage site. Expression can be driven by either the 35 S CaMV promoter or, for C-terminal fusions, promoters from genes encoding the chloroplast biogenesis factors HCF107, HCF136, or HCF173. Fusions of the four promoters to the GUS gene showed that endogenous promoter sequences are functional and drive expression more moderately and consistently throughout different transgenic lines when compared to the 35 S CaMV promoter. By testing complementation of mutations affected in chloroplast biogenesis factors HCF107 and HCF208, we found that the effect of different promoters and tags on protein function strongly depends on the protein itself. Single-step and tandem affinity purification of HCF208 via different tags confirmed the integrity of the cloned tags. PMID:23326506

Lyska, Dagmar; Engelmann, Kerstin; Meierhoff, Karin; Westhoff, Peter

2013-01-10

204

Method for the affinity purification of covalently linked peptides following cyanogen bromide cleavage of proteins.  

PubMed

The low resolution structure of a protein can sometimes be inferred from information about existing disulfide bridges or experimentally introduced chemical crosslinks. Frequently, this task involves enzymatic digestion of a protein followed by mass spectrometry-based identification of covalently linked peptides. To facilitate this task, we developed a method for the enrichment of covalently linked peptides following the chemical cleavage of a protein. The method capitalizes on the availability of homoserine lactone moieties at the C-termini of cyanogen bromide cleavage products which support selective conjugation of affinity tags. The availability of two C-termini within covalently linked peptides allows for the conjugation of two distinct affinity tags and thereby enables subsequent removal of unmodified peptides by tandem affinity chromatography. Here, we demonstrate the stepwise implementation of this method using a polyhistidine tag and a biotin tag for the selective two-step purification of covalently linked cyanogen bromide fragments from increasingly complex protein samples. The method is independent of the nature of the covalent bond, is adaptable to fully denaturing conditions, and requires only low picomole quantities of starting material. PMID:19924875

Shi, Tujin; Weerasekera, Rasanjala; Yan, Chen; Reginold, William; Ball, Haydn; Kislinger, Thomas; Schmitt-Ulms, Gerold

2009-12-15

205

Improved native isolation of endogenous Protein A-tagged protein complexes.  

PubMed

Here we report a modified peptide reagent useful for the rapid, native elution of protein complexes containing a Protein-A-tagged component. We tested this reagent for the elution of tagged endogenous protein complexes from yeast (Nup53p/Nup170p dimer; Nup1p/Kap95p/Kap60p trimer; pentameric GINS complex) and bacteria (RNAPII holoenzyme). The majority of the affinity-isolated material is released within 15 minutes under mild conditions, and the elution reagent itself is readily depleted from the elution mixture by simple spin column gel filtration. This reagent is ideal for eluting protein complexes after Protein A / IgG affinity isolation when protease cleavage is not possible or not desirable and facile depletion of the elution reagent is needed. PMID:23581468

LaCava, John; Chandramouli, Nagarajan; Jiang, Hua; Rout, Michael P

2013-04-01

206

Improved Native Isolation of Endogenous Protein A-Tagged Protein Complexes  

PubMed Central

Here we report a modified peptide reagent useful for the rapid, native elution of protein complexes containing a Protein-A-tagged component. We tested this reagent for the elution of tagged endogenous protein complexes from yeast (Nup53p/Nup170p dimer; Nup1p/Kap95p/Kap60p trimer; pentameric GINS complex) and bacteria (RNAP holoenzyme). The majority of the affinity-isolated material is released within 15 minutes under mild conditions, and the elution reagent itself is readily depleted from the elution mixture by simple spin column gel filtration. This reagent is ideal for eluting protein complexes after Protein A/IgG affinity isolation when protease cleavage is not possible or not desirable and facile depletion of the elution reagent is needed.

LaCava, John; Chandramouli, Nagarajan; Jiang, Hua; Rout, Michael P.

2013-01-01

207

Endotoxin affinity for orthodontic brackets  

Microsoft Academic Search

Endotoxin, cell envelope lipopolysaccharide produced by gram-negative bacteria can activate an immune response through a variety of pathways. In addition, it can stimulate bone resorption and reduce the periodontal tissue’s healing capacity. Previous studies have documented the affinity of lipopolysaccharide for restorative materials. This study evaluated the affinity of lipopolysaccharide for commercially available orthodontic brackets. Stainless steel, ceramic, plastic, and

Kent L. Knoernschild; Holly M. Rogers; Carol A. Lefebvre; Weston M. Fortson; George S. Schuster

1999-01-01

208

Chemical tagging strategies for mass spectrometry-based phospho-proteomics.  

PubMed

The study of protein phosphorylation in combination with chemical methods may serve several purposes. The removal of the phosphate group from phosphoserine and -threonine residues by beta-elimination has been employed to improve sensitivity for mass spectrometric detection and to attach affinity tags for phosphopeptide enrichment. More recently, phosphoramidate chemistry has been shown to be another promising tool for enriching phosphorylated peptides, and other phosphate-directed reactions may also be applicable to the study of the phosphoproteome in the future. In recent years, the combination of large-scale phospho-proteomics studies with stable isotope labeling for quantification purposes has become of growing importance, frequently involving the introduction of chemical tags such as iTRAQ. In this chapter, we will highlight several key strategies that involve chemical tagging reactions. PMID:19241017

Leitner, Alexander; Lindner, Wolfgang

2009-01-01

209

In vivo expression and purification of aptamer-tagged small RNA regulators  

Microsoft Academic Search

Small non-coding RNAs (sRNAs) are an emerging class of post-transcriptional regulators of bacterial gene expression. To study sRNAs and their potential protein interaction partners, it is desirable to purify sRNAs from cells in their native form. Here, we used RNA-based affinity chromatography to purify sRNAs following their expression as aptamer-tagged variants in vivo. To this end, we developed a family

Nelly Said; Renate Rieder; Robert Hurwitz; Jochen Deckert; Henning Urlaub; Jorg Vogel

2009-01-01

210

Enhancement of drug delivery to bone: Characterization of human tissue-nonspecific alkaline phosphatase tagged with an acidic oligopeptide  

PubMed Central

Hypophosphatasia is caused by deficiency of activity of the tissue-nonspecific alkaline phosphatase (TNSALP), resulting in a defect of bone mineralization. Enzyme replacement therapy (ERT) with partially purified plasma enzyme was attempted but with little clinical improvement. Attaining clinical effectiveness with ERT for hypophosphatasia may require delivering functional TNSALP enzyme to bone. We tagged the C-terminal-anchorless TNSALP enzyme with an acidic oligopeptide (a six or eight residue stretch of L-Asp), and compared the biochemical properties of the purified tagged and untagged enzymes derived from Chinese hamster ovary cell lines. The specific activities of the purified enzymes tagged with the acidic oligopeptide were the same as the untagged enzyme. In vitro affinity experiments showed the tagged enzymes had 30-fold higher affinity for hydroxyapatite than the untagged enzyme. Lectin affinity chromatography for carbohydrate structure showed little difference among the three enzymes. Biodistribution pattern from single infusion of the fluorescence-labeled enzymes into mice showed delayed clearance from the plasma up to 18h post infusion and the amount of tagged enzyme retained in bone was 4-fold greater than that of the untagged enzyme. In vitro mineralization assays with the bone marrow from a hypophosphatasia patient using each of the three enzymes in the presence of high concentrations of pyrophosphate provided evidence of bone mineralization. These results show the anchorless enzymes tagged with an acidic oligopeptide are delivered efficiently to bone and function bioactively in bone mineralization, at least in vitro. They suggest potential advantages for use of these tagged enzymes in ERT for hypophosphatasia, which should be explored.

Nishioka, Tatsuo; Tomatsu, Shunji; Gutierrez, Monica A.; Miyamoto, Ken-ichi; Trandafirescu, Georgeta G.; Lopez, Patricia L.C.; Grubb, Jeffrey H.; Kanai, Rie; Kobayashi, Hironori; Yamaguchi, Seiji; Gottesman, Gary S.; Cahill, Richard; Noguchi, Akihiko; Sly, William S.

2008-01-01

211

Enhancement of drug delivery to bone: characterization of human tissue-nonspecific alkaline phosphatase tagged with an acidic oligopeptide.  

PubMed

Hypophosphatasia is caused by deficiency of activity of the tissue-nonspecific alkaline phosphatase (TNSALP), resulting in a defect of bone mineralization. Enzyme replacement therapy (ERT) with partially purified plasma enzyme was attempted but with little clinical improvement. Attaining clinical effectiveness with ERT for hypophosphatasia may require delivering functional TNSALP enzyme to bone. We tagged the C-terminal-anchorless TNSALP enzyme with an acidic oligopeptide (a six or eight residue stretch of L-Asp), and compared the biochemical properties of the purified tagged and untagged enzymes derived from Chinese hamster ovary cell lines. The specific activities of the purified enzymes tagged with the acidic oligopeptide were the same as the untagged enzyme. In vitro affinity experiments showed the tagged enzymes had 30-fold higher affinity for hydroxyapatite than the untagged enzyme. Lectin affinity chromatography for carbohydrate structure showed little difference among the three enzymes. Biodistribution pattern from single infusion of the fluorescence-labeled enzymes into mice showed delayed clearance from the plasma up to 18 h post infusion and the amount of tagged enzyme retained in bone was 4-fold greater than that of the untagged enzyme. In vitro mineralization assays with the bone marrow from a hypophosphatasia patient using each of the three enzymes in the presence of high concentrations of pyrophosphate provided evidence of bone mineralization. These results show the anchorless enzymes tagged with an acidic oligopeptide are delivered efficiently to bone and function bioactively in bone mineralization, at least in vitro. They suggest potential advantages for use of these tagged enzymes in ERT for hypophosphatasia, which should be explored. PMID:16616566

Nishioka, Tatsuo; Tomatsu, Shunji; Gutierrez, Monica A; Miyamoto, Ken-ichi; Trandafirescu, Georgeta G; Lopez, Patricia L C; Grubb, Jeffrey H; Kanai, Rie; Kobayashi, Hironori; Yamaguchi, Seiji; Gottesman, Gary S; Cahill, Richard; Noguchi, Akihiko; Sly, William S

2006-04-17

212

Scalable Faceted Ranking in Tagging Systems  

NASA Astrophysics Data System (ADS)

Nowadays, web collaborative tagging systems which allow users to upload, comment on and recommend contents, are growing. Such systems can be represented as graphs where nodes correspond to users and tagged-links to recommendations. In this paper we analyze the problem of computing a ranking of users with respect to a facet described as a set of tags. A straightforward solution is to compute a PageRank-like algorithm on a facet-related graph, but it is not feasible for online computation. We propose an alternative: (i) a ranking for each tag is computed offline on the basis of tag-related subgraphs; (ii) a faceted order is generated online by merging rankings corresponding to all the tags in the facet. Based on the graph analysis of YouTube and Flickr, we show that step (i) is scalable. We also present efficient algorithms for step (ii), which are evaluated by comparing their results with two gold standards.

Orlicki, José I.; Alvarez-Hamelin, J. Ignacio; Fierens, Pablo I.

213

Understanding why users tag: A survey of tagging motivation literature and results from an empirical study.  

PubMed

While recent progress has been achieved in understanding the structure and dynamics of social tagging systems, we know little about the underlying user motivations for tagging, and how they influence resulting folksonomies and tags. This paper addresses three issues related to this question. (1) What distinctions of user motivations are identified by previous research, and in what ways are the motivations of users amenable to quantitative analysis? (2) To what extent does tagging motivation vary across different social tagging systems? (3) How does variability in user motivation influence resulting tags and folksonomies? In this paper, we present measures to detect whether a tagger is primarily motivated by categorizing or describing resources, and apply these measures to datasets from seven different tagging systems. Our results show that (a) users' motivation for tagging varies not only across, but also within tagging systems, and that (b) tag agreement among users who are motivated by categorizing resources is significantly lower than among users who are motivated by describing resources. Our findings are relevant for (1) the development of tag-based user interfaces, (2) the analysis of tag semantics and (3) the design of search algorithms for social tagging systems. PMID:23471473

Strohmaier, Markus; Körner, Christian; Kern, Roman

2012-12-01

214

Understanding why users tag: A survey of tagging motivation literature and results from an empirical study  

PubMed Central

While recent progress has been achieved in understanding the structure and dynamics of social tagging systems, we know little about the underlying user motivations for tagging, and how they influence resulting folksonomies and tags. This paper addresses three issues related to this question. (1) What distinctions of user motivations are identified by previous research, and in what ways are the motivations of users amenable to quantitative analysis? (2) To what extent does tagging motivation vary across different social tagging systems? (3) How does variability in user motivation influence resulting tags and folksonomies? In this paper, we present measures to detect whether a tagger is primarily motivated by categorizing or describing resources, and apply these measures to datasets from seven different tagging systems. Our results show that (a) users’ motivation for tagging varies not only across, but also within tagging systems, and that (b) tag agreement among users who are motivated by categorizing resources is significantly lower than among users who are motivated by describing resources. Our findings are relevant for (1) the development of tag-based user interfaces, (2) the analysis of tag semantics and (3) the design of search algorithms for social tagging systems.

Strohmaier, Markus; Korner, Christian; Kern, Roman

2012-01-01

215

Two-dimensional phosphate-affinity gel electrophoresis for the analysis of phosphoprotein isotypes.  

PubMed

Herein, we describe three kinds of 2-DE using phosphate-affinity PAGE for the analysis of phosphoprotein isotypes. The first dimension is a urea-PAGE, IEF/NEPHGE, or SDS-PAGE, which are widely used. The second dimension is a phosphate-affinity SDS-PAGE using a phosphate-binding tag molecule, Phos-tag (Mn(2+)-Phos-tag SDS-PAGE). The first 2-D procedure coupling urea-PAGE and Mn(2+)-Phos-tag SDS-PAGE was applied to the separation of beta-casein phosphoisotypes. A typical protein sample containing multiple phosphoisotypes from beta-casein (with five phosphorylation sites) was prepared by partial dephosphorylation with alkaline phosphatase. The second procedure coupling IEF/NEPHGE and Mn(2+)-Phos-tag SDS-PAGE was applied to the separations of phosphoisotypes of caseins and in vitro kinase reaction products of Tau. The third procedure coupling normal SDS-PAGE and Mn(2+)-Phos-tag SDS-PAGE was applied to the separation of A431 cell lysates before and after stimulation with an epidermal growth factor. This procedure followed by immunoblotting with anti-mitogen-activated protein kinase(MAPK) and anti-Shc antibodies demonstrated the detection of phosphoisotypes in each protein isoform of MAPK1/2 (44 and 42 kDa) and Shc (66, 52, and 46 kDa) after the stimulation. By these novel 2-D procedures, the separations of phosphoprotein isotypes should be improved relative to those by current gel electrophoresis methods, including 1-D Mn(2+)-Phos-tag SDS-PAGE. PMID:19156764

Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Matsubara, Mamoru; Aoki, Yuri; Ohie, Shiori; Mouri, Yuka; Koike, Tohru

2009-02-01

216

Comparative Study of Three Proteomic Quantitative Methods, DIGE, cICAT, and iTRAQ, Using 2D Gel or LC?MALDI TOF\\/TOF  

Microsoft Academic Search

A comparative study on the three quantitative methods frequently used in proteomics, 2D DIGE (difference gel electrophoresis), cICAT (cleavable isotope-coded affinity tags) and iTRAQ (isobaric tags for relative and absolute quantification), was carried out. DIGE and cICAT are familiar techniques used in gel- and LC-based quantitative proteomics, respectively. iTRAQ is a new LC-based technique which is gradually gaining in popularity.

Wells W. Wu; Guanghui Wang; Seung Joon Baek; Rong-Fong Shen

2006-01-01

217

Backscattering improvement of UHF RFID tag efficiency  

Microsoft Academic Search

In this work, a tag-load selection methodology is proposed for optimized tag-to-reader backscatter communication. Derivation of the method is based on antenna\\/communication theory and applies to any tag-antenna, including minimum scattering antennas as a special case. In contrast to what is commonly believed, it is shown that amplitude maximization of complex reflection coefficient difference between the two states is not

Aggelos Bletsas; Antonis G. Dimitriou; John N. Sahalos

2010-01-01

218

Sensor-based material tagging system  

SciTech Connect

Electronic identification tags are being developed for tracking material and personnel. In applying electronic identification tags to radioactive materials safeguards, it is important to measure attributes of the material to ensure that the tag remains with the material. The addition of a microcontroller with an on-board analog-to-digital converter to an electronic identification tag application-specific integrated-circuit has been demonstrated as means to provide the tag with sensor data. Each tag is assembled into a housing, which serves as a scale for measuring the weight of a paint-can-sized container and its contents. Temperature rise of the can above ambient is also measured, and a piezoelectric detector detects disturbances and immediately puts the tag into its alarm and beacon mode. Radiation measurement was also considered, but the background from nearby containers was found to be excessive. The sensor-based tagging system allows tracking of the material in cans as it is stored in vaults or is moved through the manufacturing process. The paper presents details of the sensor-based material tagging system and describes a demonstration system.

Vercellotti, L.C.; Cox, R.W.; Ravas, R.J.; Schlotterer, J.C. [Westinghouse Electric Corp., Churchill, PA (United States). Science and Technology Center

1991-12-31

219

Sensor-based material tagging system  

SciTech Connect

Electronic identification tags are being developed for tracking material and personnel. In applying electronic identification tags to radioactive materials safeguards, it is important to measure attributes of the material to ensure that the tag remains with the material. The addition of a microcontroller with an on-board analog-to-digital converter to an electronic identification tag application-specific integrated-circuit has been demonstrated as means to provide the tag with sensor data. Each tag is assembled into a housing, which serves as a scale for measuring the weight of a paint-can-sized container and its contents. Temperature rise of the can above ambient is also measured, and a piezoelectric detector detects disturbances and immediately puts the tag into its alarm and beacon mode. Radiation measurement was also considered, but the background from nearby containers was found to be excessive. The sensor-based tagging system allows tracking of the material in cans as it is stored in vaults or is moved through the manufacturing process. The paper presents details of the sensor-based material tagging system and describes a demonstration system.

Vercellotti, L.C.; Cox, R.W.; Ravas, R.J.; Schlotterer, J.C. (Westinghouse Electric Corp., Churchill, PA (United States). Science and Technology Center)

1991-01-01

220

The biological activity of a recombinantly expressed (His) 6-tagged peanut allergen (rAra h 1) is unaffected by endotoxin removal  

Microsoft Academic Search

The application of recombinant (His)6-tagged proteins in cell culture assays is associated with problems due to lipopolysaccharide (LPS) contamination. LPS stimulates cells of the immune system, thereby masking antigen-specific activation of T cells. Due to the affinity of LPS for histidine it is associated with difficulties to remove LPS from recombinant (His)6-tagged proteins. Here we describe that the Triton X-114

Louise Bjerremann Jensen; Anna Maria Torp; Sven Bode Andersen; Per Stahl Skov; Lars K. Poulsen; Edward F. Knol; Els van Hoffen

2008-01-01

221

Heuristic Query Tree Protocol: Use of Known Tags for RFID Tag Anti-Collision  

NASA Astrophysics Data System (ADS)

Existing query tree protocols deal with RFID tags in a blind manner. They query tags in a fixed bit order based on the assumption that the tag ID numbers are uniformly distributed throughout the range of the entire ID space because readers have no prior knowledge of the tags. This paper attempts to distinguish RFID applications where readers are already aware of all tags used by the application. We propose a heuristic query tree (H-QT) protocol that uses heuristic to select effective bits from known tags for the best queries in a divide and conquer approach. The performance evaluation shows that the proposed protocol is superior to original query tree protocols because it significantly reduces the number of tag collisions and no tag response.

Sung, Jongwoo; Kim, Daeyoung; Kim, Taehong; Choi, Jinhyuk

222

Identification of Protein Interacting Partners Using Tandem Affinity Purification  

PubMed Central

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8. As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter. Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Thorne, Lucy; Goodfellow, Ian

2012-01-01

223

To tag or not to tag: A comparative evaluation of immunoaffinity-labeling and tandem mass spectrometry for the identification and localization of posttranslational protein carbonylation by 4-hydroxy-2-nonenal, an end-product of lipid peroxidation  

PubMed Central

Posttranslational carbonylation of proteins by the covalent attachment of the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) is a biomarker of oxidative stress. Tandem mass spectrometry (MS/MS) has become an essential tool for characterization of this modification. Chemical tagging methods have been used to facilitate the immunoaffinity-based enrichment or even quantification of HNE-modified peptides and proteins. With MS/MS spectra of the untagged modified peptides considered as references, a comparative evaluation is presented focusing on the impact of affinity-tagging with four carbonyl-specific reagents (2,4-dinitrophenyl hydrazine, biotin hydrazide, biotinamidohexanoic acid hydrazide and N’-aminooxymethylcarbonylhydrazino D-biotin) on collision-induced dissociation of the tagged HNE-carbonylated peptides. Our study has shown that chemical labeling may not be carried out successfully for all the peptides and with all the reagents. The attachment of a tag usually cannot circumvent the occurrence of strong neutral losses observed with untagged species and, in addition, fragmentation of the introduced tag may also be introduced. Chemical tagging of certain peptides may, nevertheless, afford more sequence ions upon MS/MS than the untagged carbonylated peptide, especially when Michael addition of the lipid peroxidation product occurs on cysteine residues. Therefore, tagging may increase the confidence of identifications of HNE-modified peptides by database searches.

Guo, Jia; Prokai, Laszlo

2011-01-01

224

Tag-based web photo retrieval improved by batch mode re-tagging  

Microsoft Academic Search

Web photos in social media sharing websites such as Flickr are generally accompanied by rich but noisy textual descriptions (tags, captions, categories, etc.). In this pa- per, we proposed a tag-based photo retrieval framework to improve the retrieval performance for Flickr photos by em- ploying a novel batch mode re-tagging method. The pro- posed batch mode re-tagging method can automatically

Dong Xu; Ivor Wai-Hung Tsang; Jiebo Luo

2010-01-01

225

Adsorptive detagging of poly-histidine tagged protein using hexa-histidine tagged exopeptidase  

Microsoft Academic Search

The ubiquitous use of poly-histidine fusion tags has made the purification of the recombinant target proteins much simpler, although the presence of residual fusion tags can generate immunogenic products or products with changed biological activities. This work presents a generic method of removing poly-histidine fusion tags from recombinant proteins through the use of a hexa-histidine tagged exopeptidase (DAPase) when both

Wen-Hui K. Kuo; Howard A. Chase

2010-01-01

226

CAMAR Tag Framework: Context-Aware Mobile Augmented Reality Tag Framework for Dual-reality Linkage  

Microsoft Academic Search

In this paper, we propose a novel tag framework for sharing information in dual-reality space, which is based on context-aware mobile augmented reality (CAMAR). When a user selects a target object to be tagged onto dual-reality, the proposed framework and procedures create CAMAR Tag with a userpsilas mobile device to be registered in virtual space. CAMAR Tag is able to

Hyejin Kim; Wonwoo Lee; Woontack Woo

2009-01-01

227

Survival of Fingerling Black Crappies Tagged with Microwire Tags Stocked in a Florida Lake  

Microsoft Academic Search

We evaluated 24-h poststocking survival of fingerling black crappies Pomoxis nigromaculatus (mean total length 117 mm) that were cheek-tagged with binary-coded microwire (CW) tags and estimated the percent contribution of stocked fish to a naturally occurring year-class in a Florida lake. Retention of CW tags at 15 d was high (96%), but posttagging survival of CW-tagged fish was low compared

Randall A. Myers; Micheal S. Allen; Douglas E. Colle

2000-01-01

228

Improved Phos-tag SDS-PAGE under neutral pH conditions for advanced protein phosphorylation profiling.  

PubMed

We describe an improved Phos-tag SDS-PAGE (Zn(2+)-Phos-tag SDS-PAGE) using a dizinc(II) complex of Phos-tag acrylamide in conjunction with a Bis-tris-buffered neutral-pH gel system to detect shifts in the mobility of phosphoproteins. An existing technique (Mn(2+)-Phos-tag SDS-PAGE) using a polyacrylamide-bound Mn(2+)-Phos-tag and a conventional Laemmli's buffer system under alkaline pH conditions has limitations for separating certain phosphoproteins. The major improvements were demonstrated by visualizing novel up-shifted bands of commercially available pepsin, recombinant Tau treated in vitro with tyrosine kinases, and endogeneous ?-catenin in whole-cell lysates. Additionally, the Zn(2+)-Phos-tag SDS-PAGE gels showed better long-term stability than the Mn(2+)-Phos-tag SDS-PAGE gels. We can therefore provide a simple, convenient, and more reliable homemade gel system for phosphate-affinity SDS-PAGE. PMID:21204258

Kinoshita, Eiji; Kinoshita-Kikuta, Emiko

2010-12-14

229

Feasibility of Tagging Walleye Pollock, Theragra chalcogramma, Captured with Hook and Line using External Tags.  

National Technical Information Service (NTIS)

Walleye pollock (Theragra chalcogramma) were captured with jigs near Auke Bay, southeast Alaska, to determine the feasibility of tagging them with external tags. Most (92%) were < 40 cm fork length (juvenile size). The fish were tagged with either lock-on...

2011-01-01

230

RNase One Gene Isolation, Expression, and Affinity Purification Models Research Experimental Progression and Culminates with Guided Inquiry-Based Experiments  

ERIC Educational Resources Information Center

|This new biochemistry laboratory course moves through a progression of experiments that generates a platform for guided inquiry-based experiments. RNase One gene is isolated from prokaryotic genomic DNA, expressed as a tagged protein, affinity purified, and tested for activity and substrate specificity. Student pairs present detailed explanations…

Bailey, Cheryl P.

2009-01-01

231

Efficient Framed Slotted Aloha Protocol for RFID Tag Anticollision  

Microsoft Academic Search

In this paper, we propose a novel efficient frame slotted aloha (EFSA) protocol for radio frequency identification (RFID) tag anticollision in this paper. After successfully iden- tifying each tag, the EFSA protocol will allocate the tag a slot number, which signifies when the tag could be identified during a read cycle. When no tags arrive and leave, idle slots and

Haifeng Wu; Yu Zeng

2011-01-01

232

Stable Isotope-Coded Proteomic Mass Spectrometry  

SciTech Connect

The ability to quantify the changes in protein abundance between cells subjected to a variety of extracellular stimuli or the onset of a diseased state remains an extremely active area of proteome research. Although advances in sample preparation, chromatographic separation, mass spectrometry instrumentation and bioinformatics contribute to producing a viable method for comparative proteome-wide analyses, the foundation of quantitation is based in part upon improved methods for chemical and metabolic stable isotope labeling of proteins and peptides. The ability to quantify differences in protein expression and post-translational modifications has been demonstrated, but insights into the biochemical mechanisms that will contribute to the development of new biotechnologies have yet to be realized.

Goshe, Michael B. (BATTELLE (PACIFIC NW LAB)); Smith, Richard D. (BATTELLE (PACIFIC NW LAB))

2003-02-01

233

In vitro affinity screening of protein and peptide binders by megavalent bead surface display  

PubMed Central

The advent of protein display systems has provided access to tailor-made protein binders by directed evolution. We introduce a new in vitro display system, bead surface display (BeSD), in which a gene is mounted on a bead via strong non-covalent (streptavidin/biotin) interactions and the corresponding protein is displayed via a covalent thioether bond on the DNA. In contrast to previous monovalent or low-copy bead display systems, multiple copies of the DNA and the protein or peptide of interest are displayed in defined quantities (up to 106 of each), so that flow cytometry can be used to obtain a measure of binding affinity. The utility of the BeSD in directed evolution is validated by library selections of randomized peptide sequences for binding to the anti-hemagglutinin (HA) antibody that proceed with enrichments in excess of 103 and lead to the isolation of high-affinity HA-tags within one round of flow cytometric screening. On-bead Kd measurements suggest that the selected tags have affinities in the low nanomolar range. In contrast to other display systems (such as ribosome, mRNA and phage display) that are limited to affinity panning selections, BeSD possesses the ability to screen and rank binders by their affinity in vitro, a feature that hitherto has been exclusive to in vivo multivalent cell display systems (such as yeast display).

Diamante, Letizia; Gatti-Lafranconi, Pietro; Schaerli, Yolanda; Hollfelder, Florian

2013-01-01

234

In vitro affinity screening of protein and peptide binders by megavalent bead surface display.  

PubMed

The advent of protein display systems has provided access to tailor-made protein binders by directed evolution. We introduce a new in vitro display system, bead surface display (BeSD), in which a gene is mounted on a bead via strong non-covalent (streptavidin/biotin) interactions and the corresponding protein is displayed via a covalent thioether bond on the DNA. In contrast to previous monovalent or low-copy bead display systems, multiple copies of the DNA and the protein or peptide of interest are displayed in defined quantities (up to 10(6) of each), so that flow cytometry can be used to obtain a measure of binding affinity. The utility of the BeSD in directed evolution is validated by library selections of randomized peptide sequences for binding to the anti-hemagglutinin (HA) antibody that proceed with enrichments in excess of 10(3) and lead to the isolation of high-affinity HA-tags within one round of flow cytometric screening. On-bead Kd measurements suggest that the selected tags have affinities in the low nanomolar range. In contrast to other display systems (such as ribosome, mRNA and phage display) that are limited to affinity panning selections, BeSD possesses the ability to screen and rank binders by their affinity in vitro, a feature that hitherto has been exclusive to in vivo multivalent cell display systems (such as yeast display). PMID:23980186

Diamante, Letizia; Gatti-Lafranconi, Pietro; Schaerli, Yolanda; Hollfelder, Florian

2013-08-26

235

Tagging text with a probabilistic model  

Microsoft Academic Search

Experiments on the use of a probabilistic model to tag English text, that is, to assign to each word the correct tag (part of speech) in the context of the sentence, are presented. A simple triclass Markov model is used, and the best way to estimate the parameters of this model, depending on the kind and amount of training data

B. Merialdo

1991-01-01

236

Tagging English Text with a Probabilistic Model  

Microsoft Academic Search

In this paper we present some experiments on the use of a probabilistic model to tag English text, i.e. to assign to each word the correct tag (part of speech) in the context of the sentence. The main novelty of these experiments is the use of untagged text in the training of the model. We have used a simple triclass

Bernard Merialdo

1994-01-01

237

Sense tagging: does it make sense?  

Microsoft Academic Search

Sense tagging is probably one of the challenges that corpus linguists have to face in the near future. So far, computerisation of this task has yielded very modest results despite numerous efforts, and sense tagging is turning out to be a touchy task. Difficulties stem from various sources, extracting disambiguating information from the context. However, one of the main problems

Jean Véronis

2001-01-01

238

Tag-Based Contextual Collaborative Filtering  

Microsoft Academic Search

In this paper, we introduce a new Collaborative Filtering (CF) model which takes into consideration users' context based upon tagging information such as available from recently popular social tagging systems. In numerous implementations, traditional CF systems have been proven to work well under certain circumstances. However, CF systems still suffer a weakness: They do not take context into consideration. Yet

Reyn NAKAMOTO; Shinsuke NAKAJIMA; Jun MIYAZAKI; Shunsuke UEMURA

2007-01-01

239

A Radio Tag for Big Whales  

ERIC Educational Resources Information Center

|Radio tags to track wildlife have been used for years. However, such tagging of whales has been more complicated and less successful. This article explores the latest technology that is designed to give information over a long period of time. (MA)|

Watkins, William A.

1978-01-01

240

Transfer tagging from image to video  

Microsoft Academic Search

Nowadays massive amount of web video datum has been emerging on the Internet. To achieve an effective and efficient video retrieval, it is critical to automatically assign semantic keywords to the videos via content analysis. However, most of the existing video tagging methods suffer from the problem of lacking sufficient tagged training videos due to high labor cost of manual

Yang Yang; Yi Yang; Zi Huang; Heng Tao Shen

2011-01-01

241

Exploiting lexical information for function tag labeling  

Microsoft Academic Search

This paper proposes an novel approach to annotate function tags for unparsed text. What distinguishes our work from other attempts in such task is that we assign function tags directly basing on lexical information other than on parsed trees. In order to demonstrate the effectiveness and versatility of our method, we investigate two statistical models for automatic annotation, one is

Caixia Yuan; Xiaojie Wang; Fuji Ren

2008-01-01

242

50 CFR 20.36 - Tagging requirement.  

Code of Federal Regulations, 2011 CFR

...WILDLIFE AND PLANTS (CONTINUED) MIGRATORY BIRD HUNTING Possession § 20.36 Tagging...person shall put or leave any migratory game birds at any place (other than at his personal...taxidermy services performed, unless such birds have a tag attached, signed by the...

2011-10-01

243

50 CFR 20.36 - Tagging requirement.  

Code of Federal Regulations, 2012 CFR

...WILDLIFE AND PLANTS (CONTINUED) MIGRATORY BIRD HUNTING Possession § 20.36 Tagging...person shall put or leave any migratory game birds at any place (other than at his personal...taxidermy services performed, unless such birds have a tag attached, signed by the...

2012-10-01

244

Fuel and control assembly tag gas  

SciTech Connect

This standard establishes the requirements for tag gas to be used for locating failed fuel pins and control rod absorber pins in the reactor by the failure monitoring system. Tag gas shall consist of varying isotopic mixtures of xenon, krypton, or other gases as specified in the Ordering Data.

Not Available

1986-01-01

245

Gateway vectors for the production of combinatorially-tagged His6-MBP fusion proteins in the cytoplasm and periplasm of Escherichia coli  

PubMed Central

Many proteins that accumulate in the form of insoluble aggregates when they are overproduced in Escherichia coli can be rendered soluble by fusing them to E. coli maltose binding protein (MBP), and this will often enable them to fold in to their biologically active conformations. Yet, although it is an excellent solubility enhancer, MBP is not a particularly good affinity tag for protein purification. To compensate for this shortcoming, we have engineered and successfully tested Gateway destination vectors for the production of dual His6MBP-tagged fusion proteins in the cytoplasm and periplasm of E. coli. The MBP moiety improves the yield and solubility of its fusion partners while the hexahistidine tag (His-tag) serves to facilitate their purification. The availability of a vector that targets His6MBP fusion proteins to the periplasm expands the utility of this dual tagging approach to include proteins that contain disulfide bonds or are toxic in the bacterial cytoplasm.

Nallamsetty, Sreedevi; Austin, Brian P.; Penrose, Kerri J.; Waugh, David S.

2005-01-01

246

Self-organization in social tagging systems  

NASA Astrophysics Data System (ADS)

Individuals often imitate each other to fall into the typical group, leading to a self-organized state of typical behaviors in a community. In this paper, we model self-organization in social tagging systems and illustrate the underlying interaction and dynamics. Specifically, we introduce a model in which individuals adjust their own tagging tendency to imitate the average tagging tendency. We found that when users are of low confidence, they tend to imitate others and lead to a self-organized state with active tagging. On the other hand, when users are of high confidence and are stubborn to change, tagging becomes inactive. We observe a phase transition at a critical level of user confidence when the system changes from one regime to the other. The distributions of post length obtained from the model are compared to real data, which show good agreement.

Liu, Chuang; Yeung, Chi Ho; Zhang, Zi-Ke

2011-06-01

247

Intrinsic-surface-tag image authentication  

SciTech Connect

The objective of this work is to further the development of a unique treaty limited item (TLI) intrinsic surface tag for arms control applications. This tag`s unique feature is the ability to capture the sub-micron scale topography of the TLI surface. The surface topography is captured by plastic castings of the surface as digitally imaged by an electron microscope. Tag authentication is accomplished by comparing digital castings images obtained in two different inspections. Surface replication experiments are described, as these experiments from the basis for the authentication algorithm. Both the experiments and the authentication algorithm are analyzed using the modulation transfer function. Recommendations for future improvements in tag authentication are also suggested by the modulation transfer function analysis. 4 refs.

Palm, R.G.; DeVolpi, A.

1991-12-01

248

Biotin-tag affinity purification of a centromeric nucleosome assembly complex.  

PubMed

Centromeres are chromosomal sites of microtubule binding that ensure correct mitotic segregation of chromosomes to daughter cells. This process is mediated by a special centromere-specific histone H3 variant (CenH3), which packages centromeric chromatin and epigenetically maintains the centromere at a distinct chromosomal location. However, CenH3 is present at low abundance relative to canonical histones, presenting a challenge for the isolation and characterization of the chaperone machinery that assembles CenH3 into nucleosomes at centromeres. To address this challenge, we used controlled overexpression of Drosophila CenH3 (CID) and an efficient biochemical purification strategy offered by in vivo biotinylation of CID to successfully purify and characterize the soluble CID nucleosome assembly complex. It consists of a single chaperone protein, RbAp48, complexed with CID and histone H4. RbAp48 is also found in protein complexes that assemble canonical histone H3 and replacement histone H3.3. Here, we highlight the benefits of our improved biotin-mediated purification method, and address the question of how the simple CID/H4-RbAp48 chaperone complex can mediate nucleosome assembly specifically at centromeres. PMID:16775420

Furuyama, Takehito; Henikoff, Steven

2006-06-15

249

In silico tandem affinity purification refines an Oct4 interaction list  

PubMed Central

Introduction Octamer-binding transcription factor 4 (Oct4) is a master regulator of early mammalian development. Its expression begins from the oocyte stage, becomes restricted to the inner cell mass of the blastocyst and eventually remains only in primordial germ cells. Unearthing the interactions of Oct4 would provide insight into how this transcription factor is central to cell fate and stem cell pluripotency. Methods In the present study, affinity-tagged endogenous Oct4 cell lines were established via homologous recombination gene targeting in embryonic stem (ES) cells to express tagged Oct4. This allows tagged Oct4 to be expressed without altering the total Oct4 levels from their physiological levels. Results Modified ES cells remained pluripotent. However, when modified ES cells were tested for their functionality, cells with a large tag failed to produce viable homozygous mice. Use of a smaller tag resulted in mice with normal development, viability and fertility. This indicated that the choice of tags can affect the performance of Oct4. Also, different tags produce a different repertoire of Oct4 interactors. Conclusions Using a total of four different tags, we found 33 potential Oct4 interactors, of which 30 are novel. In addition to transcriptional regulation, the molecular function associated with these Oct4-associated proteins includes various other catalytic activities, suggesting that, aside from chromosome remodeling and transcriptional regulation, Oct4 function extends more widely to other essential cellular mechanisms. Our findings show that multiple purification approaches are needed to uncover a comprehensive Oct4 protein interaction network.

2011-01-01

250

Adaptive tag switching reinforces the coevolution of contingent cooperation and tag diversity.  

PubMed

Most of the previous studies concerning the similarity-based interaction have assumed that the change of tags just happens in the imitation stage. Individuals actually can adjust their tags whenever the environments related to these tags grow nasty. We institute a spatial model to investigate the effect of the coevolution of tag and strategy on the evolution of cooperation in the context of the Prisoner's Dilemma game. Interactions just happen between tag-identical neighbors. Individuals exploited by defectors change their current tags at a certain cost. The time-scale ratio controls how fast interaction happens relatively to selection. Results show that whenever individuals have enough chance to adapt to the environment, cooperation is greatly improved even for quite large temptation to defect. Intensive exploration reveals that both little and large costs of tag switching can further favor the establishment of cooperation. Our work may add more into the literature concerning games on adaptive networks. PMID:23603056

Wu, Te; Fu, Feng; Zhang, Yanling; Wang, Long

2013-04-17

251

Preparation of dart tags for use in the field  

USGS Publications Warehouse

Tagging in the field requires an efficient method of preparing the tags for dispensation under a wide range of conditions. The method described here was very efficient in an extensive tagging program on Oahe Reservoir, South Dakota.

Higham, Joseph R.

1966-01-01

252

Direct Dynamic Protein-Affinity Selection Mass-Spectrometry  

PubMed Central

A new methodology is described enabling the affinity screening of potential ligands towards the human estrogen receptor alpha ligand binding domain (ER?-LBD). In-solution incubation is performed of the analyte and the His-tagged ER?-LBD. The bound complex is immobilized on a nickel-loaded protein-affinity selection column, where after the unbound fraction is removed. The immobilized protein–ligand complex is exposed to a decreased pH value and an increased organic modifier concentration releasing the ligand for MS detection, and precipitating the proteins on a filter positioned between the affinity column and the mass spectrometer. The trapping column can be regenerated for reuse at least 70 times. The advantages of the methodology over existing methodologies are the absence of a pre-concentration as well as a chromatographic separation step, resulting in a significantly shorter analysis time compared to previously described procedures, and in addition, allowing the determination of solutes with unfavorable chromatographic properties. The overall analysis time now can be reduced about 250% to approximately 6 min. Replacing the filters after every measurement results in an intra-day standard deviation of 14.8% and an inter-day standard deviation of 21.3%.

Jonker, Niels; Irth, Hubertus

2010-01-01

253

Identification of an Orthogonal Peptide Binding Motif for Biarsenical Multiuse Affinity Probes  

SciTech Connect

Biarsenical multiuse affinity probes (MAPs) complexed with ethanedithiol (EDT) permit the selective cellular labeling of proteins engineered with tetracysteine motifs, but are limited by the availability of a single binding motif (i.e., CCPGCC or PG tag) that prevents the differential labeling of co-expressed proteins. To overcome this problem, we have used a high-throughput peptide screen to identify an alternate binding motif (i.e., CCKACC or KA tag), which has a similar brightness to the classical sequence upon MAP binding, but displays altered rates and affinities of association that permit the differential labeling of these peptide sequences by the red probe 4,5-bis(1,3,2-dithiarsolan-2-yl)-resorufin (ReAsH-EDT2) or its green cognate 4’,5’-bis(1,3,2-dithoarsolan-2-yl)fluorescein-(1,2-ethanedithiol)2 (FLAsH-EDT2). The utility of this labeling strategy was demonstrated following the expression of PG- and KA-tagged subunits of RNA polymerase expressed in E. coli. Specific labeling of two subunits of RNA polymerase in cellular lysates was achieved, whereby ReAsH-EDT2 is shown to selectively label the PG-tag on RNA polymerase alpha subunit prior to the labeling of the KA-tag sequence of the beta subunit of RNA polymerase with FlAsH-EDT2. These results demonstrate the ability to selectively label multiple individual proteins with orthogonal sequence tags in complex cellular lystates with spectroscopically distinct MAPs, and indicate the absolute specificity of ReAsH to target expressed proteins with essentially no nonspecific binding interactions.

Chen, Baowei; Cao, Haishi; Yan, Ping; Mayer, M. Uljana; Squier, Thomas C.

2007-07-01

254

Mobility shift detection of phosphorylation on large proteins using a Phos-tag SDS-PAGE gel strengthened with agarose.  

PubMed

We describe a novel technique of phosphate-affinity SDS-PAGE using Phos-tag to analyze large phosphoproteins with molecular masses of more than 200 kDa. The protein phosphoisotypes were clearly separated as up-shifted migration bands in a 3% w/v polyacrylamide gel containing 20 microM Phos-tag and 0.5% w/v agarose. In subsequent immunoblotting, the procedure permitted the determination of the phosphoisotypes of high-molecular-mass proteins, such as mTOR (289 kDa), ATM kinase (350 kDa), and 53BP1 (213 kDa). PMID:19658103

Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Ujihara, Hiromi; Koike, Tohru

2009-08-01

255

DICOM involving XML path-tag  

NASA Astrophysics Data System (ADS)

Digital Imaging and Communications in Medicine (DICOM) is a standard for handling, storing, printing, and transmitting information in medical imaging. XML (Extensible Markup Language) is a set of rules for encoding documents in machine-readable form which has become more and more popular. The combination of these two is very necessary and promising. Using XML tags instead of numeric labels in DICOM files will effectively increase the readability and enhance the clear hierarchical structure of DICOM files. However, due to the fact that the XML tags rely heavily on the orders of the tags, the strong data dependency has a lot of influence on the flexibility of inserting and exchanging data. In order to improve the extensibility and sharing of DICOM files, this paper introduces XML Path-Tag to DICOM. When a DICOM file is converted to XML format, adding simple Path-Tag into the DICOM file in place of complex tags will keep the flexibility of a DICOM file while inserting data elements and give full play to the advantages of the structure and readability of an XML file. Our method can solve the weak readability problem of DICOM files and the tedious work of inserting data into an XML file. In addition, we set up a conversion engine that can transform among traditional DICOM files, XML-DCM and XML-DCM files involving XML Path-Tag efficiently.

Zeng, Qiang; Yao, Zhihong; Liu, Lei

2011-03-01

256

Tags and seals for arms control verification  

SciTech Connect

Tags and seals have long been recognized as important tools in arms control. The trend in control of armaments is to limit militarily significant equipment that is capable of being verified through direct and cooperative means, chiefly on-site inspection or monitoring. Although this paper will focus on the CFE treaty, the role of tags and seals for other treaties will also be addressed. Published technology and concepts will be reviewed, based on open sources. Arms control verification tags are defined as unique identifiers designed to be tamper-revealing; in that respect, seals are similar, being used as indicators of unauthorized access. Tamper-revealing tags might be considered as single-point markers, seals as two-point couplings, and nets as volume containment. The functions of an arms control tag can be considered to be two-fold: to provide field verification of the identity of a treaty-limited item (TLI), and to have a means of authentication of the tag and its tamper-revealing features. Authentication could take place in the field or be completed elsewhere. For CFE, the goal of tags and seals can be to reduce the overall cost of the entire verification system.

DeVolpi, A.

1990-09-18

257

A new generation of lock and tag  

SciTech Connect

The safety culture of an organization needs to change to achieve full implementation of Chapter 9, Lockout and Tagouts'' of DOE Order 5480.19, Conduct of Operations Requirements for DOE Facilities (DOE 1990). You can implement this change of culture through Conduct of Operations training in the classroom, in organized group discussions, and with on-the-job training. In many facilities, lock and tag is viewed as an administration function that is not directly tied to individual employee safety. Often, lock and tag is seen as an obstacle to getting the job done, a roadblock in the way of progress that has been placed there by unseen forces for unknown reasons. Because lock and tag is not always viewed as part of the personal safety standards of the employee, the necessary attention to detail is lacking. We are presenting you with three useful methods for introducing and reinforcing a new generation of safety culture and lock and tag safety. The method will help your fellow workers view lock and tag and as a safety tool. Lock and tag will become part of their safety foundation. However, you may need to do some foundation building regarding safety, personal standards, and worker attitude before the principles of lock and tag training can become an integral part of your safety culture.

Wells, P.A.; Bickford, J.C.

1992-04-01

258

A new generation of lock and tag  

SciTech Connect

The safety culture of an organization needs to change to achieve full implementation of Chapter 9, ``Lockout and Tagouts`` of DOE Order 5480.19, Conduct of Operations Requirements for DOE Facilities (DOE 1990). You can implement this change of culture through Conduct of Operations training in the classroom, in organized group discussions, and with on-the-job training. In many facilities, lock and tag is viewed as an administration function that is not directly tied to individual employee safety. Often, lock and tag is seen as an obstacle to getting the job done, a roadblock in the way of progress that has been placed there by unseen forces for unknown reasons. Because lock and tag is not always viewed as part of the personal safety standards of the employee, the necessary attention to detail is lacking. We are presenting you with three useful methods for introducing and reinforcing a new generation of safety culture and lock and tag safety. The method will help your fellow workers view lock and tag and as a safety tool. Lock and tag will become part of their safety foundation. However, you may need to do some foundation building regarding safety, personal standards, and worker attitude before the principles of lock and tag training can become an integral part of your safety culture.

Wells, P.A.; Bickford, J.C.

1992-04-01

259

Affine Invariant Medial Axis and Skew Symmetry  

Microsoft Academic Search

Affine invariant medial axes and symmetry sets of planar shapes are introduced and studied in this paper. Two different approaches are presented. The first one is based on affine invariant distances, and defines the symmetry set, a set containing the medial axis; as the closure of the locus of points on (at least) two affine normals an affine-equidistant from the

Peter J. Giblin; Guillermo Sapiro

1998-01-01

260

Engineering Escherichia coli BL21(DE3) derivative strains to minimize E. coli protein contamination after purification by immobilized metal affinity chromatography.  

PubMed

Recombinant His-tagged proteins expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) are commonly coeluted with native E. coli proteins, especially if the recombinant protein is expressed at a low level. The E. coli contaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineered E. coli BL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering, and characterization of two E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C terminus to a chitin binding domain (CBD) and the protein GlmS, with six surface histidines replaced by alanines. We show that each E. coli CBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flowthrough step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The "NiCo" strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein. PMID:21602383

Robichon, Carine; Luo, Jianying; Causey, Thomas B; Benner, Jack S; Samuelson, James C

2011-05-20

261

Tag gas capsule with magnetic piercing device  

DOEpatents

An apparatus for introducing a tag (i.e., identifying) gas into a tubular nuclear fuel element. A sealed capsule containing the tag gas is placed in the plenum in the fuel tube between the fuel and the end cap. A ferromagnetic punch having a penetrating point is slidably mounted in the plenum. By external electro-magnets, the punch may be caused to penetrate a thin rupturable end wall of the capsule and release the tag gas into the fuel element. Preferably the punch is slidably mounted within the capsule, which is in turn loaded as a sealed unit into the fuel element.

Nelson, Ira V. (Richland, WA)

1976-06-22

262

Use of a HEHEHE purification tag instead of a hexahistidine tag improves biodistribution of affibody molecules site-specifically labeled with (99m)Tc, (111)In, and (125)I.  

PubMed

Affibody molecules are a class of small (?7 kDa) robust scaffold proteins suitable for radionuclide molecular imaging in vivo. The attachment of a hexahistidine (His(6))-tag to the Affibody molecule allows facile purification by immobilized metal ion affinity chromatography (IMAC) but leads to high accumulation of radioactivity in the liver. Earlier, we have demonstrated that replacement of the His(6)-tag with the negatively charged histidine-glutamate-histidine-glutamate-histidine-glutamate (HEHEHE)-tag permits purification of Affibody molecules by IMAC, enables labeling with [(99m)Tc(CO)(3)](+), and provides low hepatic accumulation of radioactivity. In this study, we compared the biodistribution of cysteine-containing Affibody molecules site-specifically labeled with (111)In, (99m)Tc, and (125)I at the C-terminus, having a His(6)-tag at the N- or C-terminus or a HEHEHE-tag at the N-terminus. We show that the use of a HEHEHE-tag provides appreciable reduction of hepatic radioactivity, especially for radiometal labels. We hope that this information can also be useful for development of other scaffold protein-based imaging agents. PMID:21524142

Hofstrom, Camilla; Orlova, Anna; Altai, Mohamed; Wangsell, Fredrik; Graslund, Torbjorn; Tolmachev, Vladimir

2011-05-12

263

Comparative Performance of Acoustic-tagged and PIT-tagged Juvenile Salmonids  

SciTech Connect

Numerous research tools and technologies are currently being used to evaluate fish passage and survival to determine the impacts of the Federal Columbia River Power System (FCRPS) on endangered and threatened juvenile salmonids, including PIT tags, balloon tags, hydroacoustic evaluations, radio telemetry, and acoustic telemetry. Each has advantages and disadvantages, but options are restricted in some situations because of limited capabilities of a specific technology, lack of detection capability downstream, or availability of adequate numbers of fish. However, there remains concern about the comparative effects of the tag or the tagging procedure on fish performance. The recently developed Juvenile Salmonid Acoustic Telemetry System (JSATS) acoustic transmitter is the smallest active acoustic tag currently available. The goal of this study was to determine whether fish tagged with the JSATS acoustic-telemetry tag can provide unbiased estimates of passage behavior and survival within the performance life of the tag. We conducted both field and laboratory studies to assess tag effects. For the field evaluation we released a total of 996 acoustic-tagged fish in conjunction with 21,026 PIT-tagged fish into the tailrace of Lower Granite Dam on 6 and 13 May. Travel times between release and downstream dams were not significantly different for the majority of the reaches between acoustic-tagged and PIT-tagged fish. In addition to the field evaluation, a series of laboratory experiments were conducted to determine if growth and survival of juvenile Chinook salmon surgically implanted with acoustic transmitters is different than untagged or PIT tagged juvenile Chinook salmon. Only yearling fish with integrated and non-integrated transmitters experienced mortalities, and these were low (<4.5%). Mortality among sub-yearling control and PIT-tag treatments ranged up to 7.7% while integrated and non-integrated treatments had slightly higher rates (up to 8.3% and 7.9% respectively). No acoustic transmitters were shed by yearling fish during the course of the 90 day study. Up to 7.8% of subyearling fish expelled transmitters. Tags were expelled from 5 to 63 days post-surgery. The average time to expulsion was 27 days; few fish expelled transmitters within 14 days of implantation or less. Histological results suggest that inflammation associated with implantation of an acoustic transmitter can produce fibrous tissue which can invade and possibly damage internal organs soon after implantation. Reactions severe enough to damage organs however, were limited to only ~20% of subyearling Chinook salmon, all of which were under 101mm and 12g at tagging. The infiltration of the fibrous tissue into organs was observed most often in fish held for 21 days and appeared to decrease in subsequent holding times.

Hockersmith, Eric E.; Brown, Richard S.; Liedtke, Theresa L.

2008-02-01

264

Highly-Efficient Purification of Native Polyhistidine-tagged Proteins by Multivalent NTA-modified Magnetic Nanoparticles  

PubMed Central

A new bis-nitrilotriacetic acid (NTA) chelate with catechol anchor was synthesized and immobilized on superparamagnetic iron oxide nanoparticles. When loaded with Ni(II), these bis-NTA-immobilized nanoparticles were shown to bind polyhistidine (His×6-tagged) fusion proteins in their native, folded conformations that commercial microbeads failed to bind under identical conditions. Control experiments with a mono-NTA chelate immobilized on iron oxide nanoparticles indicate a similarly high affinity for His×6-tagged native proteins, suggesting that the high density of the mono-NTA chelate presented by the nanoparticles allows the binding of the His×6-tag to more than one Ni-NTA moiety on the surface. This study shows that the multivalency strategy can be utilized to enhance the binding of His×6-tagged proteins in their native, folded conformations. We further demonstrated the selective purification of His×6-tagged proteins from crude cell lysates by using the Ni(II)-loaded iron oxide nanoparticles. The present platform is capable of efficient purification of His×6-tagged proteins that are expressed at low levels in mammalian cells. This work thus presents a novel nanoparticle-based high-capacity protein purification system with shorter incubation times, proportionally large washes, and significantly smaller elution volumes compared to commercially available microbeads.

Kim, Jason S.; Valencia, C. Alexander; Liu, Rihe; Lin, Wenbin

2008-01-01

265

Mass-producible micro-holographic tags  

SciTech Connect

Microtags are microscopic computer-generated holograms with 130-nm features and are mass-producible with EUVL. This fabrication method renders microtags difficult to counterfeit. Applications includ tagging and tracking of microprocessors, memory chips, currencey, and credit cards.

Sweatt, W.C.; Ray-Chaudhuri, A.K.; Kravitz, S.H.; Warren, M.E.; Stulen, R.H.; Tichenor, D.A.; Krenz, K.D. [Sandia National Labs., Albuquerque, NM (United States)]|[Sandia National Labs., Livermore, CA (United States); Descour, M.R. [Arizona Univ., Tucson, AZ (United States). Optical Sciences Center; Underwood, J.H. [Lawrence Berkeley Lab., CA (United States)

1996-06-01

266

50 CFR 20.81 - Tagging requirement.  

Code of Federal Regulations, 2012 CFR

...EXPORTATION, AND IMPORTATION OF WILDLIFE AND PLANTS (CONTINUED) MIGRATORY BIRD HUNTING Migratory Bird Preservation Facilities § 20.81 Tagging requirement. No migratory bird preservation facility shall receive or have in custody any...

2012-10-01

267

50 CFR 20.81 - Tagging requirement.  

Code of Federal Regulations, 2011 CFR

...EXPORTATION, AND IMPORTATION OF WILDLIFE AND PLANTS (CONTINUED) MIGRATORY BIRD HUNTING Migratory Bird Preservation Facilities § 20.81 Tagging requirement. No migratory bird preservation facility shall receive or have in custody any...

2011-10-01

268

Intrinsic-surface-tag image authentication  

SciTech Connect

The objective of this work is to further the development of a unique treaty limited item (TLI) intrinsic surface tag for arms control applications. This tag's unique feature is the ability to capture the sub-micron scale topography of the TLI surface. The surface topography is captured by plastic castings of the surface as digitally imaged by an electron microscope. Tag authentication is accomplished by comparing digital castings images obtained in two different inspections. Surface replication experiments are described, as these experiments from the basis for the authentication algorithm. Both the experiments and the authentication algorithm are analyzed using the modulation transfer function. Recommendations for future improvements in tag authentication are also suggested by the modulation transfer function analysis. 4 refs.

Palm, R.G.; DeVolpi, A.

1991-12-01

269

RFID System Based on Fully Printable Chipless Tag for Paper\\/Plastic-ltem Tagging  

Microsoft Academic Search

An RFID system, utilizing a chipless RFID tag on a 90 µm thin Taconic TF290 laminate, is presented. The chipless tag consists of two cross-polarized ultra-wideband antennas and a multi-resonating circuit. The data encoding is performed in the multi-resonating circuit, which is composed of multiple stop-band spiral resonators. The chipless tag encodes data into the spectral signature in both the

Sfevan Preradovic; Sushim M. Roy; Nemai C. Karmakar

2011-01-01

270

Lock-Out / Tag-Out  

NSDL National Science Digital Library

Tags and locks can be the last line of defense against machinery accidents. This MATEC module leaves your learners solidly grounded in OSHA's six-step lockout/tagout procedures for preventing unexpected start-ups of equipment. They learn how to use energy-isolating devices and how to safely remove locks and tags. They can demonstrate their facility with the OHSA standards using a machine on the manufacturing floor.

2010-05-14

271

Lysosomal Degradation of Ubiquitin-Tagged Receptors  

NSDL National Science Digital Library

Access to the article is free, however registration and sign-in are required. Cytosolic proteins are tagged with the polypeptide ubiquitin for eventual destruction by the proteasome. A recent paper in Cell (L. Hicke and H. Riezman, vol. 84, p. 277) shows that in yeast ubiquitin also serves to tag membrane proteins for degradation by proteases in the vacuole, the yeast equivalent of the lysosome.

Stella M. Hurtley (AAAS;Science Magazine, Europe Office)

1996-02-02

272

MRI of myocardial infarction with tissue tagging  

Microsoft Academic Search

Myocardial tagging with MRI has been available for three decades as a method for direct noninvasive quantification of regional\\u000a myocardial motion for assessing the impact of ischemia, electrical asynchrony, and heart failure, among other conditions.\\u000a In recent years, new developments in imaging sequences, hybrid techniques, and automated postprocessing have brought tagging\\u000a closer to clinical application. Improvements in acquisition strategies have

Daniel A. Herzka; Elliot R. McVeigh

2009-01-01

273

A hypergraph model of social tagging networks  

NASA Astrophysics Data System (ADS)

The past few years have witnessed the great success of a new family of paradigms, so-called folksonomy, which allows users to freely associate tags with resources and efficiently manage them. In order to uncover the underlying structures and user behaviors in folksonomy, in this paper, we propose an evolutionary hypergraph model for explaining the emerging statistical properties. The present model introduces a novel mechanism that can not only assign tags to resources, but also retrieve resources via collaborative tags. We then compare the model with a real-world data set: Del.icio.us. Indeed, the present model shows considerable agreement with the empirical data in the following aspects: power-law hyperdegree distributions, negative correlation between clustering coefficients and hyperdegrees, and small average distances. Furthermore, the model indicates that most tagging behaviors are motivated by labeling tags on resources, and the tag plays a significant role in effectively retrieving interesting resources and making acquaintances with congenial friends. The proposed model may shed some light on the in-depth understanding of the structure and function of folksonomy.

Zhang, Zi-Ke; Liu, Chuang

2010-10-01

274

Microstructure and Crystal Structure in TAGS Compositions  

SciTech Connect

GeTe, a small bandgap semiconductor that has native p-type defects due to Ge vacancies, is an important constituent in the thermoelectric material known as TAGS. TAGS is an acronym for alloys of GeTe with AgSbTe{sub 2}, and compositions are normally designated as TAGS-x, where x is the fraction of GeTe. TAGS-85 is the most important with regard to applications, and there is also commercial interest in TAGS-80. The crystal structure of GeTe{sub 1+{delta}} has a composition-dependent phase transformation at a temperature ranging from 430 C ({delta} = 0) to {approx}400 C ({delta} = 0.02). The high-temperature form is cubic. The low-temperature form is rhombohedral for {delta} < 0.01, as is the case for good thermoelectric performance. Addition of AgSbTe{sub 2} shifts the phase transformation to lower temperatures, and one of the goals of this work is a systematic study of the dependence of transformation temperature on the parameter x. We present results on phase transformations and associated instabilities in TAGS compositions in the range of 70 at.% to 85 at.% GeTe.

Thompson, A. J. [Marlow Industries, Inc; Sharp, J [Marlow Industries, Inc; Rawn, Claudia J [ORNL

2009-01-01

275

Preparation of recombinant murine tumor necrosis factor-? in Escherichia coli: A rapid method to remove tags from fusion proteins by thrombin-cleavage and ion-exchange chromatography  

Microsoft Academic Search

A recombinant protein of murine tumor necrosis factor (TNF)-? was expressed in Escherichia coli (E. coli) by using a pET Trx Fusion System. The fusion protein was effectively solubilized and purified by Ni-affinity chromatography. A high concentration of thrombin quickly and specifically cleaved the introduced site between the tags and the target fragment. We found that thrombin tightly bound to

Hiroki Tsukamoto; Kenji Fukudome; Jun Kohara; Hiroshi Nakatake; Masao Kimoto

2007-01-01

276

The use of biotin tagging in Saccharomyces cerevisiae improves the sensitivity of chromatin immunoprecipitation  

PubMed Central

Affinity tagging has been used in many global studies towards protein function. We describe a highly efficient system for in vivo biotinylation of transcription factors in the yeast Saccharomyces cerevisiae, which is based on the bacterial BirA biotin ligase. The strength of the biotin–streptavidin interaction was exploited to improve detection of in vivo protein–DNA complexes in chromatin immunoprecipitation (ChIP) experiments. In a test system using the biotin-tagged LexA DNA-binding protein, we found that stringent washing conditions resulted in a strong improvement of the signal-to-noise ratios. Yeast strains with chromosomally integrated versions of tagged transcription factor genes were generated using N- or C-terminal biotin-tagging cassettes. ChIP experiments with biotinylated Rbp3p, a RNA polymerase II subunit, showed that Rbp3p-binding could even be detected at weakly expressed genes. Other methods failed to detect RNA polymerase II binding at such genes. Our results show that biotinylation of yeast transcription factors improves the detection of in vivo protein–DNA complexes.

van Werven, Folkert J.; Timmers, H. Th. Marc

2006-01-01

277

Construction, Verification and Experimental Use of Two Epitope-Tagged Collections of Budding Yeast Strains  

PubMed Central

A major challenge in the post-genomic era is the development of experimental approaches to monitor the properties of proteins on a proteome-wide level. It would be particularly useful to systematically assay protein subcellular localization, post-translational modifications and protein–protein interactions, both at steady state and in response to environmental stimuli. Development of new reagents and methods will enhance our ability to do so efficiently and systematically. Here we describe the construction of two collections of budding yeast strains that facilitate proteome-wide measurements of protein properties. These collections consist of strains with an epitope tag integrated at the C-terminus of essentially every open reading frame (ORF), one with the tandem affinity purification (TAP) tag, and one with the green fluorescent protein (GFP) tag. We show that in both of these collections we have accurately tagged a high proportion of all ORFs (approximately 75% of the proteome) by confirming expression of the fusion proteins. Furthermore, we demonstrate the use of the TAP collection in performing high-throughput immunoprecipitation experiments. Building on these collections and the methods described in this paper, we hope that the yeast community will expand both the quantity and type of proteome level data available.

Howson, Russell; Huh, Won-Ki; Ghaemmaghami, Sina; Falvo, James V.; Bower, Kiowa; Belle, Archana; Dephoure, Noah; Wykoff, Dennis D.; Weissman, Jonathan S.

2005-01-01

278

Affinity monolith preconcentrators for polymer microchip capillary electrophoresis  

PubMed Central

Developments in biology are increasing demands for rapid, inexpensive, and sensitive biomolecular analysis. In this study, polymer microdevices with monolithic columns and electrophoretic channels were used for biological separations. Glycidyl methacrylate-co-ethylene dimethacrylate monolithic columns were formed within poly(methyl methacrylate) microchannels by in situ photopolymerization. Flow experiments in these columns demonstrated retention and then elution of amino acids under conditions optimized for sample preconcentration. To enhance analyte selectivity, antibodies were immobilized on monoliths, and subsequent lysozyme treatment blocked nonspecific adsorption. The enrichment capability and selectivity of these affinity monoliths were evaluated by purifying fluorescently tagged amino acids from a mixture containing green fluorescent protein (GFP). Twenty-fold enrichment and 91% recovery were achieved for the labeled amino acids, with a <25,000-fold reduction in GFP concentration, as indicated by microchip electrophoresis analysis. These devices should provide a simple, inexpensive, and effective platform for trace analysis in complex biological samples.

Yang, Weichun; Sun, Xiuhua; Pan, Tao; Woolley, Adam T.

2008-01-01

279

Bio-orthogonal affinity purification of direct kinase substrates.  

PubMed

Protein phosphorylation is a major mechanism of post-translational protein modification used to control cellular signaling. A challenge in phosphoproteomics is to identify the direct substrates of each protein kinase. Herein, we describe a chemical strategy for delivery of a bio-orthogonal affinity tag to the substrates of an individual protein kinase. The kinase of interest is engineered to transfer a phosphorothioate moiety to phosphoacceptor hydroxyl groups on direct substrates. In a second nonenzymatic step, the introduced phosphorothioate is alkylated with p-nitrobenzylmesylate (PNBM). Antibodies directed against the alkylated phosphorothioate epitope recognize these labeled substrates, but not alkylation products of other cellular nucleophiles. This strategy is demonstrated with Cdk1/cyclinB substrates using ELISA, western blotting, and immunoprecipitation in the context of whole cell lysates. PMID:15826144

Allen, Jasmina J; Lazerwith, Scott E; Shokat, Kevan M

2005-04-20

280

Theory and measurement of backscattering from RFID tags  

Microsoft Academic Search

This paper presents a method for measuring signal backscattering from RFID tags, and for calculating a tag's radar cross section (RCS). We derive a theoretical formula for the RCS of an RFID tag with a minimum-scattering antenna. We describe an experimental measurement technique, which involves using a network analyzer connected to an anechoic chamber with and without the tag. The

Pavel V. Nikitin; K. V. S. Rao

2006-01-01

281

Shark Tagging: A Review Of Conventional Methods and Studies  

Microsoft Academic Search

The tagging of sharks using conventional tags has long been recognized as a valuable means for studying various aspects of their life history, migrations and movements, and population structure. Conventional tags are defined as those that can be identified visually without the use of special detection equipment. Tagging studies specifically targeting sharks began in the late 1920's, and today numerous

Nancy E. Kohler; Patricia A. Turner

2001-01-01

282

Shark tagging: a review of conventional methods and studies  

Microsoft Academic Search

Synopsis The tagging of sharks using conventional tags has long been recognized as a valuable means for studying various aspects of their life history, migrations and movements, and population structure. Conventional tags are defined as those that can be identified visually without the use of special detection equipment. Tagging studies specifically targeting sharks began in the late 1920's, and today

Nancy E. Kohler; Patricia A. Turner

2001-01-01

283

Tag-Based User Profiling for Social Media Recommendation  

Microsoft Academic Search

Making recommendations for social media presents special challenges. As tagging becomes common prac- tice at many social media sites, this research proposes a new approach to user profiling based on the tags as- sociated with one's personal collection of contents. To utilize the social interaction implied by tagging, a per- sonal profile can be further extended with the tags spec-

Chia-Chuan Hung; Yi-Ching Huang; Jane Yung-jen Hsu; David Kuan-Chun Wu

2008-01-01

284

Coded Wire Tag Placement Affects Homing Ability of Pink Salmon  

Microsoft Academic Search

Coded wire tags (CWTs) are routinely injected into the snouts of Pacific salmon Oncorhynchus spp. as fry to estimate contributions of tagged populations to spawning escapements and near-shore fisheries and to assess straying. Because not all fish are tagged, tag recoveries are extrapolated to include contributions of nontagged fish released at the same time and location. A key assumption in

Christopher Habicht; Samuel Sharr; David Evans; James E. Seeb

1998-01-01

285

Deckard: A System to Detect Change of RFID Tag Ownership  

Microsoft Academic Search

Summary Change of tag ownership compromises the security goals of Radio Frequency Identification (RFID). When an attacker clones or steals an authorized subject's tag, they are willingly granted access as RFID assumes the owner of a tag is always the authorized entity. We present Deckard, a new approach to preventing change of tag ownership. Deckard uses the principles of intrusion

Luke Mirowski; Jacky Hartnett

2007-01-01

286

Theoretical proton affinity and fluoride affinity of nerve agent VX.  

PubMed

Proton affinity and fluoride affinity of nerve agent VX at all of its possible sites were calculated at the RI-MP2/cc-pVTZ//B3LYP/6-31G* and RI-MP2/aug-cc-pVTZ//B3LYP/6-31+G* levels, respectively. The protonation leads to various unique structures, with H(+) attached to oxygen, nitrogen, and sulfur atoms; among which the nitrogen site possesses the highest proton affinity of -?E ? 251 kcal/mol, suggesting that this is likely to be the major product. In addition some H(2), CH(4) dissociation as well as destruction channels have been found, among which the CH(4) + [Et-O-P(?O)(Me)-S-(CH(2))(2)-N(+)(iPr)?CHMe] product and the destruction product forming Et-O-P(?O)(Me)-SMe + CH(2)?N(+)(iPr)(2) are only 9 kcal/mol less stable than the most stable N-protonated product. For fluoridization, the S-P destruction channel to give Et-O-P(?O)(Me)(F) + [S-(CH(2))(2)-N-(iPr)(2)](-) is energetically the most favorable, with a fluoride affinity of -?E ? 44 kcal. Various F(-) ion-molecule complexes are also found, with the one having F(-) interacting with two hydrogen atoms in different alkyl groups to be only 9 kcal/mol higher than the above destruction product. These results suggest VX behaves quite differently from surrogate systems. PMID:21117653

Bera, Narayan C; Maeda, Satoshi; Morokuma, Keiji; Viggiano, Al A

2010-11-30

287

Affinity chromatography and capillary electrophoresis for analysis of the yeast ribosomal proteins.  

PubMed

We present a top down separation platform for yeast ribosomal proteins using affinity chromatography and capillary electrophoresis which is designed to allow deposition of proteins onto a substrate. FLAG tagged ribosomes were affinity purified, and rRNA acid precipitation was performed on the ribosomes followed by capillary electrophoresis to separate the ribosomal proteins. Over 26 peaks were detected with excellent reproducibility (<0.5% RSD migration time). This is the first reported separation of eukaryotic ribosomal proteins using capillary electrophoresis. The two stages in this workflow, affinity chromatography and capillary electrophoresis, share the advantages that they are fast, flexible and have small sample requirements in comparison to more commonly used techniques. This method is a remarkably quick route from cell to separation that has the potential to be coupled to high throughput readout platforms for studies of the ribosomal proteome. PMID:22531133

Goyder, Miriam S; Willison, Keith R; Klug, David R; Demello, Andrew J; Ces, Oscar

2012-04-01

288

Neural net controlled tag gas sampling system for nuclear reactors  

DOEpatents

A method and system for providing a tag gas identifier to a nuclear fuel rod and analyze escaped tag gas to identify a particular failed nuclear fuel rod. The method and system include disposing a unique tag gas composition into a plenum of a nuclear fuel rod, monitoring gamma ray activity, analyzing gamma ray signals to assess whether a nuclear fuel rod has failed and is emitting tag gas, activating a tag gas sampling and analysis system upon sensing tag gas emission from a failed nuclear rod and evaluating the escaped tag gas to identify the particular failed nuclear fuel rod.

Gross, Kenneth C. (Bolingbrook, IL); Laug, Matthew T. (Idaho Fall, ID); Lambert, John D. B. (Wheaton, IL); Herzog, James P. (Downers Grove, IL)

1997-01-01

289

In Vivo Analysis of Proteomes and Interactomes Using Parallel Affinity Capture (iPAC) Coupled to Mass Spectrometry*  

PubMed Central

Affinity purification coupled to mass spectrometry provides a reliable method for identifying proteins and their binding partners. In this study we have used Drosophila melanogaster proteins triple tagged with Flag, Strep II, and Yellow fluorescent protein in vivo within affinity pull-down experiments and isolated these proteins in their native complexes from embryos. We describe a pipeline for determining interactomes by Parallel Affinity Capture (iPAC) and show its use by identifying partners of several protein baits with a range of sizes and subcellular locations. This purification protocol employs the different tags in parallel and involves detailed comparison of resulting mass spectrometry data sets, ensuring the interaction lists achieved are of high confidence. We show that this approach identifies known interactors of bait proteins as well as novel interaction partners by comparing data achieved with published interaction data sets. The high confidence in vivo protein data sets presented here add new data to the currently incomplete D. melanogaster interactome. Additionally we report contaminant proteins that are persistent with affinity purifications irrespective of the tagged bait.

Rees, Johanna S.; Lowe, Nick; Armean, Irina M.; Roote, John; Johnson, Glynnis; Drummond, Emma; Spriggs, Helen; Ryder, Edward; Russell, Steven; Johnston, Daniel St; Lilley, Kathryn S.

2011-01-01

290

A single-step method for purification of active His-tagged ribosomes from a genetically engineered Escherichia coli  

Microsoft Academic Search

With the rapid development of the ribosome field in recent years a quick, simple and high-throughput method for purification of the bacterial ribosome is in demand. We have designed a new strain of Escherichia coli (JE28) by an in-frame fusion of a nucleotide sequence encoding a hexa-histidine affinity tag at the 3'-end of the single copy rplL gene (encoding the

Josefine Ederth; Chandra Sekhar Mandava; Santanu Dasgupta; Suparna Sanyal

2008-01-01

291

Associated Particle Tagging (APT) in Magnetic Spectrometers  

SciTech Connect

Summary In Brief The Associated Particle Tagging (APT) project, a collaboration of Pacific Northwest National Laboratory (PNNL), Idaho National Laboratory (INL) and the Idaho State University (ISU)/Idaho Accelerator Center (IAC), has completed an exploratory study to assess the role of magnetic spectrometers as the linchpin technology in next-generation tagged-neutron and tagged-photon active interrogation (AI). The computational study considered two principle concepts: (1) the application of a solenoidal alpha-particle spectrometer to a next-generation, large-emittance neutron generator for use in the associated particle imaging technique, and (2) the application of tagged photon beams to the detection of fissile material via active interrogation. In both cases, a magnetic spectrometer momentum-analyzes charged particles (in the neutron case, alpha particles accompanying neutron generation in the D-T reaction; in the tagged photon case, post-bremsstrahlung electrons) to define kinematic properties of the relevant neutral interrogation probe particle (i.e. neutron or photon). The main conclusions of the study can be briefly summarized as follows: Neutron generator: • For the solenoidal spectrometer concept, magnetic field strengths of order 1 Tesla or greater are required to keep the transverse size of the spectrometer smaller than 1 meter. The notional magnetic spectrometer design evaluated in this feasibility study uses a 5-T magnetic field and a borehole radius of 18 cm. • The design shows a potential for 4.5 Sr tagged neutron solid angle, a factor of 4.5 larger than achievable with current API neutron-generator designs. • The potential angular resolution for such a tagged neutron beam can be less than 0.5o for modest Si-detector position resolution (3 mm). Further improvement in angular resolution can be made by using Si-detectors with better position resolution. • The report documents several features of a notional generator design incorporating the alpha-particle spectrometer concept, and outlines challenges involved in the magnetic field design. Tagged photon interrogation: • We investigated a method for discriminating fissile from benign cargo-material response to an energy-tagged photon beam. The method relies upon coincident detection of the tagged photon and a photoneutron or photofission neutron produced in the target material. The method exploits differences in the shape of the neutron production cross section as a function of incident photon energy in order to discriminate photofission yield from photoneutrons emitted by non-fissile materials. Computational tests of the interrogation method as applied to material composition assay of a simple, multi-layer target suggest that the tagged-photon information facilitates precise (order 1% thickness uncertainty) reconstruction of the constituent thicknesses of fissile (uranium) and high-Z (Pb) constituents of the test targets in a few minutes of photon-beam exposure. We assumed an 18-MeV endpoint tagged photon beam for these simulations. • The report addresses several candidate design and data analysis issues for beamline infrastructure required to produce a tagged photon beam in a notional AI-dedicated facility, including the accelerator and tagging spectrometer.

Jordan, David V.; Baciak, James E.; Stave, Sean C.; Chichester, David; Dale, Daniel; Kim, Yujong; Harmon, Frank

2012-10-16

292

A brief examination of optical tagging technologies.  

SciTech Connect

Presented within this report are the results of a brief examination of optical tagging technologies funded by the Laboratory Directed Research and Development (LDRD) program at Sandia National Laboratories. The work was performed during the summer months of 2002 with total funding of $65k. The intent of the project was to briefly examine a broad range of approaches to optical tagging concentrating on the wavelength range between ultraviolet (UV) and the short wavelength infrared (SWIR, {lambda} < 2{micro}m). Tagging approaches considered include such things as simple combinations of reflective and absorptive materials closely spaced in wavelength to give a high contrast over a short range of wavelengths, rare-earth oxides in transparent binders to produce a narrow absorption line hyperspectral tag, and fluorescing materials such as phosphors, dies and chemically precipitated particles. One technical approach examined in slightly greater detail was the use of fluorescing nano particles of metals and semiconductor materials. The idea was to embed such nano particles in an oily film or transparent paint binder. When pumped with a SWIR laser such as that produced by laser diodes at {lambda}=1.54{micro}m, the particles would fluoresce at slightly longer wavelengths, thereby giving a unique signal. While it is believed that optical tags are important for military, intelligence and even law enforcement applications, as a business area, tags do not appear to represent a high on return investment. Other government agencies frequently shop for existing or mature tag technologies but rarely are interested enough to pay for development of an untried technical approach. It was hoped that through a relatively small investment of laboratory R&D funds, enough technologies could be identified that a potential customers requirements could be met with a minimum of additional development work. Only time will tell if this proves to be correct.

Ackermann, Mark R.; Cahill, Paul A. (Aspecular Optics, Dayton, OH); Drummond, Timothy J.; Wilcoxon, Jess Patrick

2003-07-01

293

Gas-phase NO+ affinities  

PubMed Central

A scale of relative gas-phase NO+ binding energies (BEs) has been constructed by evaluation of NO+-transfer equilibria L1NO+ + L2 ? L2NO+ + L1 by Fourier-transform ion cyclotron resonance mass spectrometry and by application of the kinetic method, based on the metastable fragmentation of L1(NO+)L2 nitryl-ion bound dimers. The relative scale, anchored to the NO+ affinity of water, for 52 ligands, including alkyl halides, alkyl nitrates, alcohols, nitroalkanes, nitriles, aldehydes, ketones, and aromatic and heterocyclic compounds, led to an absolute NO+ affinity scale. The results are compared with those of an earlier study, and the apparent discrepancies are traced to a different choice of the absolute BE value used as the reference standard. The NO+ BEs fit a satisfactorily linear correlation when plotted versus the corresponding proton affinities (PAs). The NO+ BEs, while much lower than the PAs, are nevertheless higher than the corresponding BEs of the strictly related NO2+ cation, a result consistent with the experimental and theoretical results currently available on the structure and the stability of NO+ and NO2+ complexes. The NO+ BE vs. PA correlation allows one to estimate within 1–2 kcal·mol?1 the NO+ BE of the molecules included in the comprehensive PA compilations currently available. For example, the correlation gives the following NO+ affinities of the DNA bases, in kcal·mol?1 (1 kcal = 4.18 kJ): adenine, 40.3; cytosine, 40.4; guanine, 40.1; and thymine, 34.9. The experimental NO+ BE of thymine, the only one accessible to direct measurement, amounts to 35.6 ± 2 kcal·mol?1, which underlines the predictive value of the correlation. This study reports the second successful extension of the kinetic method to the evaluation of the absolute BEs of polyatomic cations, following our recent application to the strictly related NO2+ ion.

Cacace, F.; de Petris, G.; Pepi, F.

1997-01-01

294

Immobilized metal ion affinity chromatography  

Microsoft Academic Search

This article describes the technique of immobilized metal ion affinity chromatography (1MAC). The IMAC stationary phases are\\u000a designed to chelate certain metal ions that have selectivity for specific groups in peptides and on protein surfaces. The\\u000a number of stationary phases that can be synthesized for efficient chclation of metal ions is unlimited, but the critical consideration\\u000a is that there is

Tai-Tung Yip; T. William Hutchens

1994-01-01

295

Endotoxin affinity for orthodontic brackets.  

PubMed

Endotoxin, cell envelope lipopolysaccharide produced by gram-negative bacteria can activate an immune response through a variety of pathways. In addition, it can stimulate bone resorption and reduce the periodontal tissue's healing capacity. Previous studies have documented the affinity of lipopolysaccharide for restorative materials. This study evaluated the affinity of lipopolysaccharide for commercially available orthodontic brackets. Stainless steel, ceramic, plastic, and "gold" brackets were exposed to 10 EU/mm2radiolabeled Porphyromonas gingivalis or Escherichia coli lipolpoysaccharide in water and incubated for 24 hours at 37 degrees C. Brackets were then transferred to fresh lipopolysaccharide-free water and incubated for 24 hours at 37 degrees C to evaluate elution. This elution transfer was continued up to 96 hours total incubation. Lipopolysaccharide adherence and elution levels were calculated after treatment, and elution solutions were evaluated through liquid scintillation spectrometry. Mean initial lipopolysaccharide adherence ranged from 2.42 +/- 0.26 EU/mm2(E. coli, plastic) to 6.75 +/- 0.34 EU/mm2 (P. gingivalis, stainless steel). P. gingivalis lipopolysaccharide adherence was significantly greater than E. coli lipopolysaccharide adherence for all bracket types. Moreover, for each lipopolysaccharide type, stainless steel brackets exhibited significantly greater lipopolysaccharide adherence. Regarding elution, only the P. gingivalis lipopolysaccharide-exposed ceramic and plastic brackets at 24 hours and the stainless steel and ceramic brackets at 48 hours eluted measurable lipopolysaccharide. Results from this study demonstrate that P. gingivalis and E. coli LPS exhibit a high affinity for orthodontic brackets. In vivo, this affinity could affect the concentration of LPS in the gingival sulcus, thereby contributing to inflammation in tissues adjacent to the brackets. PMID:10358245

Knoernschild, K L; Rogers, H M; Lefebvre, C A; Fortson, W M; Schuster, G S

1999-06-01

296

The effect of PIT tagging on survival, tag retention, and weight gain in fingerling white bass  

Technology Transfer Automated Retrieval System (TEKTRAN)

We tagged fingerling white bass Morone chrysops with Passive Integrated Transponders (PIT) at two body locations (peritoneal cavity and dorsal musculature) and six weight classes (-6, 10, 14, 19, 25, and 30 g) to evaluate survival, tag retention, and weight gain during a 28-day experimental period. ...

297

Assistive tagging: A survey of multimedia tagging with human-computer joint exploration  

Microsoft Academic Search

Along with the explosive growth of multimedia data, automatic multimedia tagging has attracted great interest of various research communities, such as computer vision, multimedia, and information retrieval. However, despite the great progress achieved in the past two decades, automatic tagging technologies still can hardly achieve satisfactory performance on real-world multimedia data that vary widely in genre, quality, and content. Meanwhile,

Meng Wang; Bingbing Ni; Xian-Sheng Hua; Tat-Seng Chua

298

Electronic tagging and integrated product intelligence  

NASA Astrophysics Data System (ADS)

The advent of 'intelligent,' electronic data bearing tags is set to revolutionize the way industrial and retail products are identified and tracked throughout their life cycles. The dominant system for unique identification today is the bar code, which is based on printed symbology and regulated by the International Article Numbering Association. Bar codes provide users with significant operational advantages and generate considerable added value to packaging companies, product manufacturers, distributors and retailers, across supply chains in many different sectors, from retailing, to baggage handling and industrial components, e.g., for vehicles or aircraft. Electronic tags offer the potential to: (1) record and store more complex data about the product or any modifications which occur during its life cycle; (2) access (and up-date) stored data in real time in a way which does not involve contact with the product or article; (3) overcome the limitations imposed by systems which rely on line-of-sight access to stored data. Companies are now beginning to consider how electronic data tags can be used, not only to improve the efficiency of their supply chain processes, but also to revolutionize the way they do business. This paper reviews the applications and business opportunities for electronic tags and outlines CEST's strategy for achieving an 'open' standard which will ensure that tags from different vendors can co-exist on an international basis.

Swerdlow, Martin; Weeks, Brian

1996-03-01

299

Tags to Track Illicit Uranium and Plutonium  

SciTech Connect

With the expansion of nuclear power, it is essential to avoid nuclear materials from falling into the hands of rogue nations, terrorists, and other opportunists. This paper examines the idea of detection and attribution tags for nuclear materials. For a detection tag, it is proposed to add small amounts [about one part per billion (ppb)] of {sup 232}U to enriched uranium to brighten its radioactive signature. Enriched uranium would then be as detectable as plutonium and thus increase the likelihood of intercepting illicit enriched uranium. The use of rare earth oxide elements is proposed as a new type of 'attribution' tag for uranium and thorium from mills, uranium and plutonium fuels, and other nuclear materials. Rare earth oxides are chosen because they are chemically compatible with the fuel cycle, can survive high-temperature processing operations in fuel fabrication, and can be chosen to have minimal neutronic impact within the nuclear reactor core. The mixture of rare earths and/or rare earth isotopes provides a unique 'bar code' for each tag. If illicit nuclear materials are recovered, the attribution tag can identify the source and lot of nuclear material, and thus help police reduce the possible number of suspects in the diversion of nuclear materials based on who had access. (authors)

Haire, M. Jonathan; Forsberg, Charles W. [Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN, 37831-6166 (United States)

2007-07-01

300

Social Tagging for Personalized Web Search  

NASA Astrophysics Data System (ADS)

Social networks and collaborative tagging systems are rapidly gaining popularity as primary means for sorting and sharing data: users tag their bookmarks in order to simplify information dissemination and later lookup. Social Bookmarking services are useful in two important respects: first, they can allow an individual to remember the visited URLs, and second, tags can be made by the community to guide users towards valuable content. In this paper we focus on the latter use: we present a novel approach for personalized web search using query expansion. We further extend the family of well-known co-occurence matrix technique models by using a new way of exploring social tagging services. Our approach shows its strength particularly in the case of disambiguation of word contexts. We show how to design and implement such a system in practice and conduct several experiments. To the best of our knowledge this is the first study centered on using social bookmarking and tagging techniques for personalization of web search and its evaluation in a real-world scenario.

Biancalana, Claudio

301

Purification of His-tagged proteins using Ni 2+–poly(2-acetamidoacrylic acid) hydrogel  

Microsoft Academic Search

In this study, a new matrix for immobilized metal affinity chromatography (IMAC) using poly(2-acetamidoacrylic acid) (PAAA) hydrogels complexed with Ni2+ was developed for the purification of the recombinant histidine-tagged green fluorescence protein (His6–GFP). The Ni2+-complexed PAAA hydrogel was prepared by polymerizing 2-acetamidoacrylic acid (AAA) and 2,2?-[(1,4-dioxo-1,4-butanediyl)diamino] bis(2-propenoic acid) (DBDBPA) with potassium persulfate in DMSO, followed by Ni2+ complexation. Confocal laser

Eun-Ju Ha; Yu-Jin Kim; Seong Soo A. An; Young-Rok Kim; Jang-Oo Lee; Sun-Gu Lee; Hyun-jong Paik

2008-01-01

302

Purification of lysozyme by multistage affinity filtration  

Microsoft Academic Search

A multistage affinity filtration process was developed for the purification of proteins. An affinity adsorbent was prepared by immobilizing Cibacron Blue 3GA to TSK gel HW-65F. Adsorption equilibrium experiments showed that the blue TSK gel had a high affinity for lysozyme, while its binding to bovine serum albumin (BSA) was weaker. Using a three-stage affinity filtration system, lysozyme was purified

L. He; Y. Sun

2002-01-01

303

Affine collineations in space-time  

SciTech Connect

The existence of affine collineations in space-time is discussed and the types of space-time admitting proper affine collineations is displayed. The close connection between such space-times and their holonomy structure and local decomposability is established. Affine collineations with fixed points are also considered as is the problem of extending local affine collineations to the whole of space-time.

Hall, G.S.; da Costa, J.

1988-11-01

304

Androgen receptor binding affinity of pesticide \\  

Microsoft Academic Search

The COREPA approach for identifying the COmmon REactivity PAttern of biologically similar chemicals was employed to upgrade the recently derived affinity pattern for high androgen receptor (AR) binding affinity. The training set consisted of 28 steroidal and nonsteroidal ligands whose AR binding affinity was determined in competitive binding assays (in terms of p K i ). The interatomic distances between

R. Serafimova; J. Walker; O. Mekenyan

2002-01-01

305

Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II.  

PubMed

Affinity purification is a useful approach for purification of recombinant proteins. Eukaryotic expression systems have become more frequently used at the expense of prokaryotic systems since they afford recombinant eukaryotic proteins with post-translational modifications similar or identical to the native ones. Here, we present a one-step affinity purification set-up suitable for the purification of secreted proteins. The set-up is based on the interaction between biotin and mutated streptavidin. Drosophila Schneider 2 cells are chosen as the expression host, and a biotin acceptor peptide is used as an affinity tag. This tag is biotinylated by Escherichia coli biotin-protein ligase in vivo. We determined that localization of the ligase within the ER led to the most effective in vivo biotinylation of the secreted proteins. We optimized a protocol for large-scale expression and purification of AviTEV-tagged recombinant human glutamate carboxypeptidase II (Avi-GCPII) with milligram yields per liter of culture. We also determined the 3D structure of Avi-GCPII by X-ray crystallography and compared the enzymatic characteristics of the protein to those of its non-tagged variant. These experiments confirmed that AviTEV tag does not affect the biophysical properties of its fused partner. Purification approach, developed here, provides not only a sufficient amount of highly homogenous protein but also specifically and effectively biotinylates a target protein and thus enables its subsequent visualization or immobilization. PMID:22178733

Tykvart, J; Sácha, P; Ba?inka, C; Knedlík, T; Starková, J; Lubkowski, J; Konvalinka, J

2011-12-08

306

Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II  

SciTech Connect

Affinity purification is a useful approach for purification of recombinant proteins. Eukaryotic expression systems have become more frequently used at the expense of prokaryotic systems since they afford recombinant eukaryotic proteins with post-translational modifications similar or identical to the native ones. Here, we present a one-step affinity purification set-up suitable for the purification of secreted proteins. The set-up is based on the interaction between biotin and mutated streptavidin. Drosophila Schneider 2 cells are chosen as the expression host, and a biotin acceptor peptide is used as an affinity tag. This tag is biotinylated by Escherichia coli biotin-protein ligase in vivo. We determined that localization of the ligase within the ER led to the most effective in vivo biotinylation of the secreted proteins. We optimized a protocol for large-scale expression and purification of AviTEV-tagged recombinant human glutamate carboxypeptidase II (Avi-GCPII) with milligram yields per liter of culture. We also determined the 3D structure of Avi-GCPII by X-ray crystallography and compared the enzymatic characteristics of the protein to those of its non-tagged variant. These experiments confirmed that AviTEV tag does not affect the biophysical properties of its fused partner. Purification approach, developed here, provides not only a sufficient amount of highly homogenous protein but also specifically and effectively biotinylates a target protein and thus enables its subsequent visualization or immobilization.

Tykvart, J.; Sacha, P.; Barinka, C.; Knedlik, T.; Starkova, J.; Lubkowski, J.; Konvalinka, J. (Gilead); (NCI); (Czech Academy)

2012-02-07

307

Affinity Purification of Protein Complexes in C. elegans  

PubMed Central

C. elegans is a powerful metazoan model system to address fundamental questions in cell and developmental biology. Research in C. elegans has traditionally focused on genetic, physiological, and cell biological approaches. However, C. elegans is also a facile system for biochemistry: worms are easy to grow in large quantities, the functionality of tagged fusion proteins can be assessed using mutants or RNAi, and the relevance of putative interaction partners can be rapidly tested in vivo. Combining biochemistry with function-based genetic and RNA interference screens can rapidly accelerate the delineation of protein networks and pathways in diverse contexts. In this chapter, we focus on two strategies to identify protein–protein interactions: single-step immunoprecipitation and tandem affinity purification. We describe methods for growth of worms in large-scale liquid culture, preparation of worm and embryo extracts, immunoprecipitation, and tandem affinity purification. In addition, we describe methods to test specificity of antibodies, strategies for optimizing starting material, and approaches to distinguish specific from non-specific interactions.

Zanin, Esther; Dumont, Julien; Gassmann, Reto; Cheeseman, Iain; Maddox, Paul; Bahmanyar, Shirin; Carvalho, Ana; Niessen, Sherry; Yates, John R.; Oegema, Karen; Desai, Arshad

2012-01-01

308

Molecular tagging velocimetry in turbulence using biacetyl.  

PubMed

We evaluate various molecular tagging velocimetry (MTV) techniques for application in turbulent flows of gases where the smallest length scales must be resolved. We argue that tracer diffusion dictates the use of large complex molecules and discuss a few candidate molecules. The accuracy of MTV is determined by the profile of written lines which widen due to molecular dynamics, including both diffusion and chemical reaction. We evaluate these profiles for tagging with phosphorescing biacetyl molecules, which is a commonly used probe in MTV. For relatively large laser power, these profiles are determined not by molecular diffusion, but by the triplet-triplet annihilation reaction of excited biacetyl molecules. We identify a new reaction pathway, and present a model for the observed line shapes. The rapid widening of tagged lines of biacetyl molecules due to chemical reaction restricts this MTV technique to large-scale turbulent motion in gases of comparable molecular weight. PMID:23214688

Mirzaei, M; Dam, N J; van de Water, W

2012-10-19

309

Molecular tagging velocimetry in turbulence using biacetyl  

NASA Astrophysics Data System (ADS)

We evaluate various molecular tagging velocimetry (MTV) techniques for application in turbulent flows of gases where the smallest length scales must be resolved. We argue that tracer diffusion dictates the use of large complex molecules and discuss a few candidate molecules. The accuracy of MTV is determined by the profile of written lines which widen due to molecular dynamics, including both diffusion and chemical reaction. We evaluate these profiles for tagging with phosphorescing biacetyl molecules, which is a commonly used probe in MTV. For relatively large laser power, these profiles are determined not by molecular diffusion, but by the triplet-triplet annihilation reaction of excited biacetyl molecules. We identify a new reaction pathway, and present a model for the observed line shapes. The rapid widening of tagged lines of biacetyl molecules due to chemical reaction restricts this MTV technique to large-scale turbulent motion in gases of comparable molecular weight.

Mirzaei, M.; Dam, N. J.; van de Water, W.

2012-10-01

310

Phosphate-affinity electrophoresis on a microchip for determination of protein kinase activity.  

PubMed

We describe microchip-based phosphate-affinity electrophoresis (microPAE) for separation of peptides aimed at determination of kinase activity. The microPAE exploits two recently published technologies: autonomous sample injection for PDMS microchips and a phosphate-specific affinity ligand, Phos-tag. We prepared a fluorescently labeled substrate peptide, specific to human c-Src, and its phosphorylated form. We synthesized a Phos-tag-poly(dimethylacrylamide) conjugate. The conjugate and the sample solutions were autonomously injected into a PDMS-glass hybrid microchip. The two solutions were contacted together in the microchannel. When the peptides were electrophoresed into the Phos-tag-poly(dimethylacrylamide) region, the phosphorylated peptide was specifically trapped, and separated from the nonphosphorylated peptide in 10 s. The results were quantified by the areas of the fluorescence peaks. The calibration plot obtained with standard samples showed an excellent linearity and a LOD of 0.9% phosphorylated peptide among the total peptides. For c-Src-reacted samples, the results from the microPAE were in good agreement with those from matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The microPAE was also successful in the presence of inhibitors for c-Src. The measured 50% inhibitory concentration values for staurosporine, PP2, and SU6656 were in good agreement with the literature values. PMID:19784951

Han, Aishan; Hosokawa, Kazuo; Maeda, Mizuo

2009-10-01

311

Comparison of magnetic carboxymethyl chitosan nanoparticles and cation exchange resin for the efficient purification of lysine-tagged small ubiquitin-like modifier protease.  

PubMed

A fusion tag that can be purified by the cheap ion-exchanger based on the ionic binding force may provide a cost-effective scheme over other affinity fusion tags. Small ubiquitin-like modifier (SUMO) protease derived from Saccharomyces cerevisiae was fused with a poly lysine tag containing 10 lysine residues at its C-terminus and then expressed in Escherichia coli. The ionic binding force provided by the ploy lysine tag allowed the selective recovery of the small ubiquitin-like modifier protease from recombinant E. coli cell extracts. A preliminary comparative study of the adsorption and elution of poly lysine tagged SUMO protease on Amberlite Cobalamion and magnetite carboxymethyl chitosan nanoparticles was performed. Amberlite Cobalamion and magnetite nanoparticles had the similar elution profile due to the common functional groups - carboxyl groups. The maximum dynamic adsorption capacity of Amberlite Cobalamion and magnetite nanoparticles reached 36.8 and 211.4 mg/g, respectively. The lysine-tagged protease can be simply purified by magnetite nanoparticles from cell extracts with higher purity than that by Amberlite Cobalamion. The superparamagnetic nanoparticles possess the advantages of highly specific, fast and excellent binding of a larger amount of lysine tagged SUMO modifier protease, and it is also easier to separate from the crude biological process liquors compared with the conventional separation techniques of polycationic amino acids fusion proteins. PMID:22995375

Li, Junhua; Zhang, Yang; Shen, Fei; Yang, Yanjun

2012-08-30

312

Imaging proteins inside cells with fluorescent tags  

PubMed Central

Watching biological molecules provides clues to their function and regulation. Some of the most powerful methods of labeling proteins for imaging use genetically encoded fluorescent fusion tags. There are four standard genetic methods of covalently tagging a protein with a fluorescent probe for cellular imaging. These use I) auto-fluorescent proteins, II) self-labeling enzymes, III) enzymes that catalyze the attachment of a probe to a target sequence, and IV) biarsenical dyes that target tetracysteine motifs. Each of these techniques has advantages and disadvantages. In this review, we cover new developments in these methods and discuss practical considerations for their use in imaging proteins inside living cells.

Crivat, Georgeta; Taraska, Justin W.

2011-01-01

313

B mixing and flavor tagging at CDF  

SciTech Connect

The CDF Collaboration has made a preliminary measurement of B{sub d} mixing as a first step toward measuring mixing in the B{sub s} system. Flavor tagging using opposite-side jets and muons as well as same-side tagging schemes have been applied. Results agree well with precise results from the B-factories. They use these results to estimate CDF's B{sub s} mixing range using the present data set ({approx} 250 pb{sup -1}) and extrapolate to the potential from larger data sets in future running.

Russ, James S.; /Carnegie Mellon U.

2004-12-01

314

Proteomic Studies of Syk-Interacting Proteins Using a Novel Amine-Specific Isotope Tag and GFP Nanotrap  

NASA Astrophysics Data System (ADS)

Green fluorescent protein (GFP) and variants have become powerful tools to study protein localization, interactions, and dynamics. We present here a mass spectrometry-based proteomics strategy to examine protein-protein interactions using anti-GFP single-chain antibody VHH in a combination with a novel stable isotopic labeling reagent, isotope tag on amino groups (iTAG). We demonstrate that the single-chain VHH (GFP nanotrap) allows us to identify interacting partners of the Syk protein-tyrosine kinase bearing a GFP epitope tag with high efficiency and high specificity. Interacting proteins identified include CrkL, BLNK, ?- and ?-tubulin, Csk, RanBP5 and DJ-1. The iTAG reagents were prepared with simple procedures and characterized with high accuracy in the determination of peptides in model peptide mixtures and as well as in complex mixture. Applications of the iTAG method and GFP nanotrap to an analysis of the nucleocytoplasmic trafficking of Syk led to the identification of location-specific associations between Syk and multiple proteins. While the results reveal that the new quantitative proteomic strategy is generally applicable to integrate protein interaction data with subcellular localization, extra caution should be taken in evaluating the results obtained by such affinity purification strategies as many interactions appear to occur following cell lysis.

Galan, Jacob A.; Paris, Leela L.; Zhang, Hua-jie; Adler, Jacob; Geahlen, Robert L.; Tao, W. Andy

2011-02-01

315

Proteomic Studies of Syk-Interacting Proteins Using a Novel Amine-Specific Isotope Tag and GFP Nanotrap  

PubMed Central

Green fluorescent protein (GFP) and variants have become powerful tools to study protein localization, interactions, and dynamics. We present here a mass spectrometry-based proteomics strategy to examine protein–protein interactions using anti-GFP single-chain antibody VHH in a combination with a novel stable isotopic labeling reagent, isotope tag on amino groups (iTAG). We demonstrate that the single-chain VHH (GFP nanotrap) allows us to identify interacting partners of the Syk protein-tyrosine kinase bearing a GFP epitope tag with high efficiency and high specificity. Interacting proteins identified include CrkL, BLNK, ?- and ?-tubulin, Csk, RanBP5 and DJ-1. The iTAG reagents were prepared with simple procedures and characterized with high accuracy in the determination of peptides in model peptide mixtures and as well as in complex mixture. Applications of the iTAG method and GFP nanotrap to an analysis of the nucleocytoplasmic trafficking of Syk led to the identification of location-specific associations between Syk and multiple proteins. While the results reveal that the new quantitative proteomic strategy is generally applicable to integrate protein interaction data with subcellular localization, extra caution should be taken in evaluating the results obtained by such affinity purification strategies as many interactions appear to occur following cell lysis.

Galan, Jacob A.; Paris, Leela L.; Zhang, Hua-jie; Adler, Jacob; Geahlen, Robert L.; Tao, W. Andy

2011-01-01

316

Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice  

NASA Astrophysics Data System (ADS)

Proteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin/streptavidin for biotinylated templates. Here, we describe an approach for the single-step purification of transcription factor complex(es) based on specific in vivo biotinylation. We expressed the bacterial BirA biotin ligase in mammalian cells and demonstrated very efficient biotinylation of a hematopoietic transcription factor bearing a small (23-aa) artificial peptide tag. Biotinylation of the tagged transcription factor altered neither the factor's protein interactions or DNA binding properties in vivo nor its subnuclear distribution. Using this approach, we isolated the biotin-tagged transcription factor and at least one other known interacting protein from crude nuclear extracts by direct binding to streptavidin beads. Finally, this method works efficiently in transgenic mice, thus raising the prospect of using biotinylation tagging in protein complex purification directly from animal tissues. Therefore, BirA-mediated biotinylation of tagged proteins provides the basis for the single-step purification of proteins from mammalian cells.

de Boer, Ernie; Rodriguez, Patrick; Bonte, Edgar; Krijgsveld, Jeroen; Katsantoni, Eleni; Heck, Albert; Grosveld, Frank; Strouboulis, John

2003-06-01

317

Seamless Gene Tagging by Endonuclease-Driven Homologous Recombination  

PubMed Central

Gene tagging facilitates systematic genomic and proteomic analyses but chromosomal tagging typically disrupts gene regulatory sequences. Here we describe a seamless gene tagging approach that preserves endogenous gene regulation and is potentially applicable in any species with efficient DNA double-strand break repair by homologous recombination. We implement seamless tagging in Saccharomyces cerevisiae and demonstrate its application for protein tagging while preserving simultaneously upstream and downstream gene regulatory elements. Seamless tagging is compatible with high-throughput strain construction using synthetic genetic arrays (SGA), enables functional analysis of transcription antisense to open reading frames and should facilitate systematic and minimally-invasive analysis of gene functions.

Duishoev, Nurlanbek; Delhomme, Nicolas; Knop, Michael

2011-01-01

318

Glutamate Dehydrogenase Isoforms with N-Terminal (His)6- or FLAG-Tag Retain Their Kinetic Properties and Cellular Localization.  

PubMed

Glutamate dehydrogenase (GDH) is a crucial enzyme on the crossroads of amino acid and energy metabolism and it is operating in all domains of life. According to current knowledge GDH is present only in one functional isoform in most animals, including mice. In addition to this housekeeping enzyme (hGDH1 in humans), humans and apes have acquired a second isoform (hGDH2) with a distinct tissue expression profile. In the current study we have cloned both mouse and human GDH constructs containing FLAG and (His)6 small genetically-encoded tags, respectively. The hGDH1 and hGDH2 constructs containing N-terminal (His)6 tags were successfully expressed in Sf9 cells and the recombinant proteins were isolated to ?95 % purity in a two-step procedure involving ammonium sulfate precipitation and Ni(2+)-based immobilized metal ion affinity chromatography. To explore whether the presence of the FLAG and (His)6 tags affects the cellular localization and functionality of the GDH isoforms, we studied the subcellular distribution of the expressed enzymes as well as their regulation by adenosine diphosphate monopotassium salt (ADP) and guanosine-5'-triphosphate sodium salt (GTP). Through immunoblot analysis of the mitochondrial and cytosolic fraction of the HEK cells expressing the recombinant proteins we found that neither FLAG nor (His)6 tag disturbs the mitochondrial localization of GDH. The addition of the small tags to the N-terminus of the mature mitochondrial mouse GDH1 or human hGDH1 and hGDH2 did not change the ADP activation or GTP inhibition pattern of the proteins as compared to their untagged counterparts. However, the addition of FLAG tag to the C-terminus of the mouse GDH left the recombinant protein fivefold less sensitive to ADP activation. This finding highlights the necessity of the functional characterization of recombinant proteins containing even the smallest available tags. PMID:23619558

Paj?cka, Kamilla; Nielsen, Camilla Wendel; Hauge, Anne; Zaganas, Ioannis; Bak, Lasse K; Schousboe, Arne; Plaitakis, Andreas; Waagepetersen, Helle S

2013-04-26

319

Tag-to-Tag Mesh Network Using Dual-Radio RFID System for Port Logistics  

Microsoft Academic Search

\\u000a This paper consists of two parts: the protocol for tag-to-tag mesh network (T2T-MN) and the implementation of dual-radio RFID\\u000a system. Recently, RFID has been adopted in ports or warehouse, being attached to containers and palettes for loading\\/unloading\\u000a automation. However, the RFID system has encountered one problem—some tags cannot receive any command from reader intermittently\\u000a due to signal interference by containers

Jinhwan Kim; Hyocheol Jeong; Myungjae Kim; Haosong Gou; Munseok Choi; Younghwan Yoo

320

Review and Alignment of Tag Ontologies for Semantically-Linked Data in Collaborative Tagging Spaces  

Microsoft Academic Search

Abstract—As the number,of Web,2.0 sites offering tagging facilities for the users’ voluntary content annotation increases, so do the efforts to analyze,social phenomena,resulting from generated,tagging and folksonomies. Most of these efforts provide different views for the understanding,of various web,activities. Results from,various,experimental,research,should,be utilized to improve,existing approaches,underlying,tagging,data,and contribute further to weaving the Web. However, in practice, there are not enough,solutions taking advantage,of these

Hak Lae Kim; Alexandre Passant; John G. Breslin; Simon Scerri; Stefan Decker

2008-01-01

321

Finding Errors Automatically in Semantically Tagged Dialogues.  

National Technical Information Service (NTIS)

We describe a novel method for detecting errors in task-based human- computer (HC) dialogues by automatically deriving them from semantic tags. We examined 27 HC dialogues from the DARPA Communicator air travel domain, comparing user inputs to system resp...

J. Aberdeen C. Doran L. Damianos S. Bayer L. Hirschman

2006-01-01

322

Retromodulator for Optical Tagging for LEO Consumables.  

National Technical Information Service (NTIS)

In this paper, we report the results of a recent demonstration in which a Multiple Quantum Well retromodulator array was used as a low power, lightweight means to provide optical tagging of a remotely located object. A laser diode integrated on a tracker/...

G. C. Gilbreath M. J. Vilcheck R. Mahon T. J. Meehan W. S. Rabinovich

2007-01-01

323

Runtime Tags Aren't Necessary  

Microsoft Academic Search

Many modern programming environments use tag bits at runtime to distinguish objects of different types. This is particularly common in systems with garbage collection, since the garbage collector must be able to distinguish pointers from non-pointers, and to learn the length of records pointed to.

Andrew W. Appel; William Streets

1989-01-01

324

A review of shark satellite tagging studies  

Microsoft Academic Search

Recent advances in satellite tagging technologies have provided scientists growing opportunities to resolve previously unknown spatial ecology of marine predators, including sharks. Such an understanding is particularly important at this time given recent declines in shark populations worldwide. Here we reviewed 48 studies published in the primary literature between 1984 and 2010, addressing the most basic questions regarding the use

N. Hammerschlag; A. J. Gallagher; D. M. Lazarre

2011-01-01

325

Item Recommendation in Collaborative Tagging Systems  

Microsoft Academic Search

Along with the new opportunities introduced by Web 2.0 and collaborative tagging systems, several challenges have to be addressed too, notably, the problem of information overload. Recommender systems are among the most successful approaches for increasing the level of relevant content over the \\

Alexandros Nanopoulos

2011-01-01

326

Tag Based Models of English Text  

Microsoft Academic Search

The problem of compressing English text is important both because of the ubiquity of English as a target for compression and because of the light that compression can shed on the structure of English. English text is examined in conjunction with additional information about the parts of speech of each word in the text (these are referred to as “tags”).

W. J. Teahan; John G. Cleary

1998-01-01

327

Novel and efficient tag SNPs selection algorithms.  

PubMed

SNPs are the most abundant forms of genetic variations amongst species; the association studies between complex diseases and SNPs or haplotypes have received great attention. However, these studies are restricted by the cost of genotyping all SNPs; thus, it is necessary to find smaller subsets, or tag SNPs, representing the rest of the SNPs. In fact, the existing tag SNP selection algorithms are notoriously time-consuming. An efficient algorithm for tag SNP selection was presented, which was applied to analyze the HapMap YRI data. The experimental results show that the proposed algorithm can achieve better performance than the existing tag SNP selection algorithms; in most cases, this proposed algorithm is at least ten times faster than the existing methods. In many cases, when the redundant ratio of the block is high, the proposed algorithm can even be thousands times faster than the previously known methods. Tools and web services for haplotype block analysis integrated by hadoop MapReduce framework are also developed using the proposed algorithm as computation kernels. PMID:24092115

Chen, Wen-Pei; Hung, Che-Lun; Tsai, Suh-Jen Jane; Lin, Yaw-Ling

2013-01-01

328

Cardiac motion simulator for tagged MRI  

Microsoft Academic Search

Describes a computational simulator for use in cardiac imaging using tagged magnetic resonance imaging. The simulator incorporates a 13-parameter model of left-ventricular motion due to Arts et al. (1992) and applies it to a confocal prolate spherical shell, resembling the shape of the left ventricle. Using parameters determined in other work, our model can be made to assume a configuration

Edo Waks; Jerry L. Prince; Andrew S. Douglas

1996-01-01

329

Reverse Indexing for Reading Graffiti Tags  

Microsoft Academic Search

In this paper, we consider the problem of automatically reading graffiti tags. As a preparatory step, we create a large set of synthetic graffiti-like characters, generated from publicly available true type fonts. For each character in the database, we extract a number of scale independent local binary descriptors. Then, using binary non negative matrix factorization, a sufficient number of basis

Christian Thurau; Christian Bauckhage

2010-01-01

330

Prospects for Barium Tagging in Gaseous Xenon  

Microsoft Academic Search

Tagging events with the coincident detection of a barium ion would greatly reduce the background for a neutrino-less double beta decay search in xenon. This paper describes progress towards realizing this goal. It outlines a source that can produce large quantities of Ba++ in gas, shows that this can be extracted to vacuum, and demonstrates a mechanism by which the

D. Sinclair; E. Rollin; J. Smith; A. Mommers; N. Ackerman; B. Aharmim; M. Auger; P. S. Barbeau; C. Benitez-Medina; M. Breidenbach; A. Burenkov; S. Cook; A. Coppens; T. Daniels; R. DeVoe; A. Dobi; M. J. Dolinski; K. Donato; Fairbank W. Jr; J. Farine; G. Giroux

2012-01-01

331

Metropolitan Edison Company switching and tagging procedure  

SciTech Connect

Metropolitan Edison Company provides service to over 350,000 customers in an area diagonally across Southeastern Pennsylvania from North of Stroudsburg near the New York State line to the Maryland border South of Gettysburg. This area encompasses 3300 square miles, 7% of Pennsylvania, and includes all or part of 14 counties. Dispatching organization, safety factors, tagging list and switching are discussed.

Slater, H.J.

1980-05-01

332

Augmenting Wikipedia with Named Entity Tags  

Microsoft Academic Search

Wikipedia is the largest organized knowledge repository on the Web, increasingly employed by natural language processing and search tools. In this paper, we investigate the task of labeling Wikipedia pages with standard named entity tags, which can be used further by a range of information extraction and language processing tools. To train the classifiers, we manually annotated a small set

Wisam Dakka; Silviu Cucerzan

2008-01-01

333

Beliefs and Uses of Tagging among Undergraduates  

ERIC Educational Resources Information Center

|Context: This dissertation examines beliefs and uses regarding tagging among current undergraduate students, and examines the ecology of communications practice and implications for formation and maintenance of identity within the population. Currently enrolled undergraduate students at UNC-Chapel Hill formed the population for examination. …

Kramer-Duffield, Jacob

2010-01-01

334

Nitric oxide flow tagging in unseeded air  

Microsoft Academic Search

A scheme for molecular tagging velocimetry is presented that can be used in air f lows without any kind of seeding. The method is based on the local and instantaneous creation of nitric oxide (NO) molecules from N2 and O2 in the waist region of a focused ArF excimer laser beam. This NO distribution is advected by the f low

Nico Dam; R. J. H. Klein-Douwel; Nanna M. Sijtsema; J. J. ter Meulen

2001-01-01

335

Measurement Protocols for Optimized Fuel Assembly Tags  

SciTech Connect

This report describes the measurement protocols for optimized tags that can be applied to standard fuel assemblies used in light water reactors. This report describes work performed by the authors at Pacific Northwest National Laboratory for NA-22 as part of research to identify specific signatures that can be developed to support counter-proliferation technologies.

Gerlach, David C.; Mitchell, Mark R.; Reid, Bruce D.; Gesh, Christopher J.; Hurley, David E.

2008-11-01

336

TAGGING, TRACKING AND LOCATING WITHOUT GPS  

Microsoft Academic Search

The Savannah River National Laboratory (SRNL) was requested to lead a Law Enforcement Working Group that was formed to collaborate on common operational needs. All agencies represented on the working group ranked their need to tag, track, and locate a witting or unwitting target as their highest priority. Specifically, they were looking for technologies more robust than Global Positioning Satellite

J. Cordaro; T. Coleman; D. Shull

2012-01-01

337

Tagging, Tracking and Locating without GPS.  

National Technical Information Service (NTIS)

The Savannah River National Laboratory (SRNL) was requested to lead a Law Enforcement Working Group that was formed to collaborate on common operational needs. All agencies represented on the working group ranked their need to tag, track, and locate a wit...

J. Shuler J. V. Cordaro

2012-01-01

338

Linear reduction methods for tag SNP selection.  

PubMed

It is widely hoped that constructing a complete human haplotype map will help to associate complex diseases with certain SNP's. Unfortunately, the number of SNP's is huge and it is very costly to sequence many individuals. Therefore, it is desirable to reduce the number of SNP's that should be sequenced to considerably small number of informative representatives, so called tag SNP's. In this paper, we propose a new linear algebra based method for selecting and using tag SNP's. Our method is purely combinatorial and can be combined with linkage disequilibrium (LD) and block based methods. We measure the quality of our tag SNP selection algorithm by comparing actual SNP's with SNP's linearly predicted from linearly chosen tag SNP's. We obtain an extremely good compression and prediction rates. For example, for long haplotypes (>25000 SNP's), knowing only 0.4% of all SNP's we predict the entire unknown haplotype with 2% accuracy while the prediction method is based on a 10% sample of the population. PMID:17270869

He, Jingwu; Zelikovsky, Alex

2004-01-01

339

Convergent evidence identifying MAP\\/microtubule affinity-regulating kinase 1 (MARK1) as a susceptibility gene for autism  

Microsoft Academic Search

Autism spectrum disorders (ASDs) are common, heritable, but genetically heterogeneous neurodevelopmen- tal conditions. We recently defined a susceptibility locus for ASDs on chromosome 1q41-q42. High- resolution single-nucleotide polymorphisms (126 SNPs) genotyping across the chromosome 1q41-q42 region, followed by a MARK1 (microtubule affinity-regulating kinase 1)-tagged-SNP association study in 276 families with autism from the Autism Genetic Research Exchange, showed that several

Gilles Maussion; J erome Carayol; Aude-Marie Lepagnol-Bestel; Frederic Tores; Yann Loe-Mie; Ulla Milbreta; Francis Rousseau; Karine Fontaine; Julie Renaud; Jean-Marie Moalic; Anne Philippi; Alain Chedotal; Philip Gorwood; Nicolas Ramoz; Jorg Hager; Michel Simonneau

2008-01-01

340

A mobility shift detection method for DNA methylation analysis using phosphate affinity polyacrylamide gel electrophoresis.  

PubMed

We describe a procedure for DNA methylation analysis using the bisulfite-mediated cytosine-to-uracil conversion of a target DNA followed by methylation-specific polymerase chain reaction (MSP) and phosphate affinity polyacrylamide gel electrophoresis (PAGE). The MSP was performed using a 1:1 mixture of 5'-phosphorylated methylation-specific and 5'-OH non-methylation-specific primers. The PAGE using an immobilized phosphate-binding tag molecule (i.e., a polyacrylamide-bound dizinc(II) complex, Zn(2+)-Phos-tag), which selectively captures the 5'-phosphorylated DNA fragment, enabled the mobility shift detection of the methylation-specific product as a slower migration band. Using this novel procedure, we demonstrated the detection of a methylated cytosine base in a pUC19 plasmid. PMID:18394999

Kinoshita-Kikuta, Emiko; Kinoshita, Eiji; Koike, Tohru

2008-03-16

341

Virginia Game Fish Tagging Program: 2007 Annual Report.  

National Technical Information Service (NTIS)

Partial Contents: Introduction; Operations; Targeted Species 2002-2007 ; Special Virginia Institute of Marine Science (VIMS) Tagging Projects ; Utilization of Results and Key Data ; How the Program Works - Training Experienced Anglers to Tag Select Target...

J. A. Lucy L. Gillingham

2008-01-01

342

The 1971 Lake Washington Sockeye Salmon Tagging Study.  

National Technical Information Service (NTIS)

During June and July of 1971, 3709 sockeye salmon (Oncorhynchus nerka) were tagged in the area from Discovery Bay to Seattle, Washington. Sport, commercial , and Indian fisheries returned 1120 tags, while spawning migration observations at a weir and coun...

D. C. Pratt E. D. Jewell

1973-01-01

343

Recoil-fission tagging of the transfermium nucleus 252No  

NASA Astrophysics Data System (ADS)

An in-beam study of the transfermium nucleus 252No has been performed using the JUROSPHERE II array of germanium detectors coupled to the gas-filled recoil separator RITU. A new technique of recoil-fission tagging was used to extract tagged ?-ray data. Having significant spontaneous fission and ?-decay branches, 252No is an ideal candidate for a comparative study. In a similar manner to ?-decay tagging the fission events can be used to obtain ?-ray data. The recoil-fission tagged ?-ray spectrum showed a similar structure to the ?-decay tagged ?-ray spectrum. By comparing the ?-tagged and fission-tagged spectra and decay curves, it was shown that the spontaneous fission originates from the same initial state as the ? decay. This extension of the tagging method allows in-beam spectroscopic data to be obtained from heavy nuclei with significant spontaneous-fission branches.

Leppänen, A.-P.; Uusitalo, J.; Greenlees, P. T.; Herzberg, R.-D.; Amzal, N.; Becker, F.; Butler, P. A.; Chewter, A. J. C.; Cocks, J. F. C.; Dorvaux, O.; Eeckhaudt, S.; Eskola, K.; Gerl, J.; Grahn, T.; Hammond, N. J.; Hauschild, K.; Helariutta, K.; Heßberger, F. P.; Houry, M.; Jones, G. D.; Jones, P. M.; Julin, R.; Juutinen, S.; Kankaanpää, H.; Kettunen, H.; Khoo, T. L.; Korten, W.; Kuusiniemi, P.; Le Coz, Y.; Leino, M.; Lister, C. J.; Lucas, R.; Muikku, M.; Nieminen, P.; Nyman, M.; Page, R. D.; Pakarinen, J.; Rahkila, P.; Reiter, P.; Sarén, J.; Schlegel, Ch.; Scholey, C.; Stezowski, O.; Theisen, Ch.; Trzaska, W. H.; Wollersheim, H. J.

2006-06-01

344

Tagging of functional ribosomes in living cells by HaloTag® technology  

Microsoft Academic Search

Ribosomal proteins and ribosomal associated proteins are complicated subjects to target and study because of their high conservation\\u000a through evolution which led to highly structured and regulated proteins. Tagging of ribosomal proteins may allow following\\u000a of protein synthesis in vivo and isolating translated mRNAs. HaloTag® is a new technology which allows detection in living\\u000a cells, biochemical purification, and localization studies.

Simone Gallo; Anne Beugnet; Stefano Biffo

2011-01-01

345

Public Applications of SpaceTag and Their Impacts  

Microsoft Academic Search

SpaceTag is an object that can be accessed only from limited locations and time period. SpaceTags are served and distributed\\u000a from a central server which should be managed by a service provider. Users of the SpaceTag system can access SpaceTags with\\u000a portable terminals equipped with location sensors and wireless communication device such as mobile phones. Users walk around\\u000a in a

Hiroyuki Tarumi; Ken Morishita; Yahiko Kambayashi

2000-01-01

346

Implementation of RFID Tag for Metal Surface Mount  

Microsoft Academic Search

This paper described a metal mount RFID tag that works reliably on metallic surface. The proposed method is to use commercial RFID tags, Styrofoam103.7 material is attached on back side of RFID tag. Styrofoam103.7 material which has 2.5 mm thickness and 1.03 of relative permittivity was attached on back side of RFID tag. In order to verify the performance of

Chong Ryol Park; Sang Won Yoon; Kyung Kwon Jung; Ki Hwan Eom

2010-01-01

347

Implementation of RFID Tag for Metal Surface Mount  

Microsoft Academic Search

\\u000a This paper described a metal mount RFID tag that works reliably on metallic surface. The proposed method is to use commercial\\u000a RFID tags, Styrofoam103.7 material is attached on back side of RFID tag. Styrofoam103.7 material which has 2.5 mm thickness\\u000a and 1.03 of relative permittivity was attached on back side of RFID tag. In order to verify the performance of

Chong Ryol Park; Sang Won Yoon; Kyung Kwon Jung; Ki Hwan Eom

348

Design and analysis of UHF tag antenna structure  

Microsoft Academic Search

For passive RFID tags, one of the most important parameter is the read range. The power transferred to the tag IC and the tag read range are maximized when the reactance of the tag and IC chip are conjugate-matched. Structures effecting on antenna performance are studied in this paper that is based on the dipole antenna: Meander-line and tip-loading may

Tiling Hu; Caifeng Liu; Zhongyu Wang

2011-01-01

349

Platform-tolerant PIFA-type UHF RFID tag antenna  

Microsoft Academic Search

Platform-tolerant tag antennas are desired for ubiquitous RFID systems. Metal-mountable or wideband tag antennas can not guarantee platform-tolerance. This paper presents the design approaches of platform-tolerant tag antennas. A compact PIFA-type UHF tag antenna is proposed accordingly. Simulation and measurement results are provided to demonstrate the platform-tolerance feature of the proposed antenna and to validate the design approaches presented.

Jingtian Xi; Hailong Zhu; Terry T. YE

2010-01-01

350

Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE.  

PubMed

We provide a standard phosphate-affinity SDS-PAGE (Mn(2+)-Phos-tag SDS-PAGE) protocol, in which Phos-tag is used to analyze large phosphoproteins with molecular masses of more than 200 kDa. A previous protocol required a long electrophoresis time of 12 h for separation of phosphoisotypes of large proteins ( approximately 150 kDa). This protocol, which uses a 3% (wt/vol) polyacrylamide gel strengthened with 0.5% (wt/vol) agarose, permits the separation of protein phosphoisotypes larger than 200 kDa within 2 h. In subsequent immunoblotting, phosphoisotypes of high-molecular-mass proteins, such as mammalian target of rapamycin (289 kDa), ataxia telangiectasia-mutated kinase (350 kDa) and p53-binding protein 1 (213 kDa), can be clearly detected as up-shifted migration bands on the improved Mn(2+)-Phos-tag SDS-PAGE gel. The procedure from the beginning of gel preparation to the end of electrophoresis requires about 4 h in this protocol. PMID:19798084

Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

2009-09-24

351

Characterization and purification of recombinant bovine viral diarrhea virus particles with epitope-tagged envelope proteins.  

PubMed

Bovine viral diarrhea virus (BVDV) belongs to the genus Pestivirus within the family Flaviviridae. The lipid membrane of the virions is supposed to contain the three glycosylated envelope proteins E(rns), E1 and E2, but detailed studies of virus assembly are complicated because no efficient purification method for pestiviruses has been described so far. In this study, we generated infectious BVDV with N-terminally FLAG-tagged E(rns) or E2 proteins, respectively. The expression of the epitope-tagged E(rns) and E2 proteins could be shown by immunofluorescence and Western blot experiments. Furthermore, an affinity tag purification protocol for the isolation and concentration of infectious BVDV was established. In the preparation with a titre of 10(8.75) TCID(50) ml(-1), spherical particles with a diameter of 43-58 nm (mean diameter: 48 nm) could be detected by negative staining electron microscopy, and immunogold labelling located both E(rns) and E2 proteins at the virus membrane. PMID:21346033

Wegelt, Anne; Reimann, Ilona; Granzow, Harald; Beer, Martin

2011-02-23

352

Stable transformation of an episomal protein-tagging shuttle vector in the piscine diplomonad Spironucleus vortens  

PubMed Central

Background Diplomonads are common free-living inhabitants of anoxic aquatic environments and are also found as intestinal commensals or parasites of a wide variety of animals. Spironucleus vortens is a putatively commensal diplomonad of angelfish that grows to high cell densities in axenic culture. Genomic sequencing of S. vortens is in progress, yet little information is available regarding molecular and cellular aspects of S. vortens biology beyond descriptive ultrastructural studies. To facilitate the development of S. vortens as an additional diplomonad experimental model, we have constructed and stably transformed an episomal plasmid containing an enhanced green fluorescent protein (GFP) tag, an AU1 epitope tag, and a tandem affinity purification (TAP) tag. This construct also contains selectable antibiotic resistance markers for both S. vortens and E. coli. Results Stable transformants of S. vortens grew relatively rapidly (within 7 days) after electroporation and were maintained under puromycin selection for over 6 months. We expressed the enhanced GFP variant, eGFP, under transcriptional control of the S. vortens histone H3 promoter, and visually confirmed diffuse GFP expression in over 50% of transformants. Next, we generated a histone H3::GFP fusion using the S. vortens conventional histone H3 gene and its native promoter. This construct was also highly expressed in the majority of S. vortens transformants, in which the H3::GFP fusion localized to the chromatin in both nuclei. Finally, we used fluorescence in situ hybridization (FISH) of the episomal plasmid to show that the transformed plasmid localized to only one nucleus/cell and was present at roughly 10–20 copies per nucleus. Because S. vortens grows to high densities in laboratory culture, it is a feasible diplomonad from which to purify native protein complexes. Thus, we also included a TAP tag in the plasmid constructs to permit future tagging and subsequent purification of protein complexes by affinity chromatography via a two-step purification procedure. Conclusion Currently, progress in protistan functional and comparative genomics is hampered by the lack of free-living or commensal protists in axenic culture, as well as a lack of molecular genetic tools with which to study protein function in these organisms. This stable transformation protocol combined with the forthcoming genome sequence allows Spironucleus vortens to serve as a new experimental model for cell biological studies and for comparatively assessing protein functions in related diplomonads such as the human intestinal parasite, Giardia intestinalis.

Dawson, Scott C; Pham, Jonathan K; House, Susan A; Slawson, Elizabeth E; Cronembold, Daniela; Cande, W Zacheus

2008-01-01

353

Electrochemical immobilization of Fluorescent labelled probe molecules on a FTO surface for affinity detection based on photo-excited current  

NASA Astrophysics Data System (ADS)

Photo-excited current can be generated at a molecular interface between a photo-excited molecules and a semi-conductive material in appropriate condition. The system has been recognized for promoting photo-energy devices such as an organic dye sensitized solar-cell. The photo-current generated reactions are totally dependent on the interfacial energy reactions, which are in a highly fluctuated interfacial environment. The authors investigated the photo-excited current reaction to develop a smart affinity detection method. However, in order to perform both an affinity reaction and a photo-excited current reaction at a molecular interface, ordered fabrications of the functional (affinity, photo-excitation, etc.) molecules layer on a semi-conductive surface is required. In the present research, we would like to present the fabrication and functional performance of photo-excited current-based affinity assay device and its application for detection of endocrine disrupting chemicals. On the FTO surface, fluorescent pigment labelled affinity peptide was immobilized through the EC tag (electrochemical-tag) method. The modified FTO produced a current when it was irradiated with diode laser light. However, the photo current decreased drastically when estrogen (ES) coexisted in the reaction solution. In this case, immobilized affinity probe molecules formed a complex with ES and estrogen receptor (ER). The result strongly suggests that the photo-excited current transduction between probe molecule-labelled cyanine pigment and the FTO surface was partly inhibited by a complex that formed at the affinity oligo-peptide region in a probe molecule on the FTO electrode. The bound bulky complex may act as an impediment to perform smooth transduction of photo-excited current in the molecular interface. The present system is new type of photo-reaction-based analysis. This system can be used to perform simple high-sensitive homogeneous assays.

Haruyama, Tetsuya; Wakabayashi, Ryo; Cho, Takeshi; Matsuyama, Sho-taro

2011-10-01

354

Synaptic Tagging, Evaluation of Memories, and the Distal Reward Problem  

ERIC Educational Resources Information Center

Long-term synaptic plasticity exhibits distinct phases. The synaptic tagging hypothesis suggests an early phase in which synapses are prepared, or "tagged," for protein capture, and a late phase in which those proteins are integrated into the synapses to achieve memory consolidation. The synapse specificity of the tags is consistent with…

Papper, Marc; Kempter, Richard; Leibold, Christian

2011-01-01

355

TagiCoFi: tag informed collaborative filtering  

Microsoft Academic Search

Besides the rating information, an increasing number of mod- ern recommender systems also allow the users to add per- sonalized tags to the items. Such tagging information may provide very useful information for item recommendation, because the users' interests in items can be implicitly re- flected by the tags that they often use. Although some content-based recommender systems have made

Yi Zhen; Wu-jun Li; Dit-yan Yeung

2009-01-01

356

Developing a Better Method of Tag Attachment for Cetaceans.  

National Technical Information Service (NTIS)

The goal is to develop, test, and use some novel methods of tag attachments to cetaceans (1) to increase attachment duration, (2) minimize the negative effects to the individual, and (3) to increase types of tags thus broadening the options for tag deploy...

J. T. Harvey

2009-01-01

357

9 CFR 2.51 - Form of official tag.  

Code of Federal Regulations, 2013 CFR

...Identification of Animals § 2.51 Form of official tag. (a) The official tag shall be made of a durable alloy such as brass, bronze, or steel, or of a durable plastic. Aluminum of a sufficient thickness to assure the tag is durable and...

2013-01-01

358

Synaptic Tagging, Evaluation of Memories, and the Distal Reward Problem  

ERIC Educational Resources Information Center

|Long-term synaptic plasticity exhibits distinct phases. The synaptic tagging hypothesis suggests an early phase in which synapses are prepared, or "tagged," for protein capture, and a late phase in which those proteins are integrated into the synapses to achieve memory consolidation. The synapse specificity of the tags is consistent with…

Papper, Marc; Kempter, Richard; Leibold, Christian

2011-01-01

359

POS Tags and Decision Trees for Language Modeling  

Microsoft Academic Search

Language models for speech recognition con- centrate solely on recognizing the words that were spoken. In this paper, we advocate re- defining the speech recognition problem so that its goal is to find both the best sequence of words and their POS tags, and thus incorpo- rate POS tagging. To use POS tags effectively, we use clustering and decision tree

Peter A. Heeman

1999-01-01

360

RCS and read range of a UHF RFID tag  

Microsoft Academic Search

The development of a novel RFID tag operating at 869 MHz and its performance evaluation using simulated Radar Cross Section (RCS) are presented. The RCS variations of the tag antenna are evaluated over azimuth and elevation angular ranges for three cases of load: short, matched load and open. The tag exhibits appreciably good differential RCS between its switching states over

Thomaskutty Mathew; M. A. Ziai; John Batchelor

2010-01-01

361

Large-scale music tag recommendation with explicit multiple attributes  

Microsoft Academic Search

Social tagging can provide rich semantic information for large-scale retrieval in music discovery. Such collaborative intelligence, however, also generates a high degree of tags unhelpful to discovery, some of which obfuscate critical information. Towards addressing these shortcomings, tag recommendation for more robust music discovery is an emerging topic of significance for researchers. However, current methods do not consider diversity of

Zhendong Zhao; Xinxi Wang; Qiaoliang Xiang; Andy M. Sarroff; Zhonghua Li; Ye Wang

2010-01-01

362

Approaches to analyse corporate tags for business intelligence purposes  

Microsoft Academic Search

The information overload in business organizations hampers the information analysis process. Business intelligence tools can be used to analyse large amounts of information, however in most cases they only focus on structured information. More and more companies annotate tags to unstructured information to improve the information retrieval. We propose to exploit tags and tagging data to generate business intelligence. We

Céline Van Damme

2008-01-01

363

A model-based approach to selection of tag SNPs  

Microsoft Academic Search

BACKGROUND: Single Nucleotide Polymorphisms (SNPs) are the most common type of polymorphisms found in the human genome. Effective genetic association studies require the identification of sets of tag SNPs that capture as much haplotype information as possible. Tag SNP selection is analogous to the problem of data compression in information theory. According to Shannon's framework, the optimal tag set maximizes

Pierre Nicolas; Fengzhu Sun; Lei M. Li

2006-01-01

364

Tag Normalization and Prediction for Effective Social Media Retrieval  

Microsoft Academic Search

In this paper, we propose a tag normalization algorithm to unify the userspsila annotations. Meanwhile, we explore some general phenomena in a social annotation system and propose a supervised tag prediction model to predict the stabilized tag set of a resource, with feedback of a small amount of user annotation records. The experiments show that a large potion of the

Ming-hung Hsu; Hsin-hsi Chen

2008-01-01

365

Active contour models for tracking magnetic resonance tags  

Microsoft Academic Search

Cardiac magnetic resonance imaging with orthogonal stripes of magnetic tags (SPAMM) has enabled quantitative noninvasive analysis of regional myocardial motion and deformation ( 11. Manual detection and tracking of cardiac tags by visual inspection remains a time- consuming process. We have developed an interactively guided semi-automated method of detecting and tracking cardiac tags. A template matching approach combined with a

Dara L. Kraitchman; Alistair A. Young; Leon Axel

1993-01-01

366

Physical-layer identification of UHF RFID tags  

Microsoft Academic Search

In this work, we study physical-layer identification of passive UHF RFID tags. We collect signals from a population of 70 tags using a purpose-built reader and we analyze time domain and spectral features of the collected signals. We show that, based on timing features of the signals, UHF RFID tags can be classified, independently of the location and distance to

Davide Zanetti; Boris Danev; Srdjan Capkun

2010-01-01

367

Epitope tagging of endogenous genes in diverse human cell lines  

Microsoft Academic Search

Epitope tagging is a powerful and commonly used approach for studying the physical properties of proteins and their functions and localization in eukaryotic cells. In the case of Saccharomyces cerevisiae, it has been possible to exploit the high efficiency of homologous recombination to tag proteins by modifying their endogenous genes, making it possible to tag virtually every endogenous gene and

Jung-Sik Kim; Challice Bonifant; Fred Bunz; Todd Waldman

2008-01-01

368

High gain Yagi-Uda UHF RFID tag antennas  

Microsoft Academic Search

In this paper, 5-elements Yagi-Uda type UHF tag antennas, resonated at 915MHz, are designed to increase the gain and FBR (front-back-ratio) of tag antennas and extend the reading range. The designs have better FBR than a conventional Yagi-Uda tag antenna designs.

Kyounghwan Lee; You Chung Chung

2007-01-01

369

Electronic tagging and population structure of Atlantic bluefin tuna  

Microsoft Academic Search

Electronic tags that archive or transmit stored data to satellites have advanced the mapping of habitats used by highly migratory fish in pelagic ecosystems. Here we report on the electronic tagging of 772 Atlantic bluefin tuna in the western Atlantic Ocean in an effort to identify population structure. Reporting electronic tags provided accurate location data that show the extensive migrations

Barbara A. Block; Steven L. H. Teo; Andreas Walli; Andre Boustany; Michael J. W. Stokesbury; Charles J. Farwell; Kevin C. Weng; Heidi Dewar; Thomas D. Williams

2005-01-01

370

The Searching Effectiveness of Social Tagging in Museum Websites  

ERIC Educational Resources Information Center

This paper explores the search effectiveness of social tagging which allows the public to freely tag resources, denoted as keywords, with any words as well as to share personal opinions on those resources. Social tagging potentially helps users to organize, manage, and retrieve resources. Efficient retrieval can help users put more of their focus…

Cho, Chung-Wen; Yeh, Ting-Kuang; Cheng, Shu-Wen; Chang, Chun-Yen

2012-01-01

371

Searching and Tagging: Two Sides of the Same Coin?  

Microsoft Academic Search

This paper presents the duality hypothesis of search and tagging, two important behaviors of web users. The hypothesis states that if a user views a document D in the search results for query Q, the user would tend to assign document $D$ a tag identical to or similar to Q; similarly, if a user tags a document D with a

Qiaozhu MEI; Jing JIANG; Hang SU; ChengXiang ZHAI

2007-01-01

372

Searching and Tagging: Two Sides of the Same Coin?  

Microsoft Academic Search

This paper presents the duality hypothesis of search and tagging, two important behaviors of web users. The hy- pothesis states that if a user views a document D in the search results for query Q, the user would tend to assign document D a tag identical to or similar to Q; similarly, if a user tags a document D with

Qiaozhu Mei; Jing Jiang; Hang Su; ChengXiang Zhai

373

Conformal field theory on affine Lie groups  

SciTech Connect

Working directly on affine Lie groups, we construct several new formulations of the WZW model, the gauged WZW model, and the generic affine-Virasoro action. In one formulation each of these conformal field theories (CFTs) is expressed as a one-dimensional mechanical system whose variables are coordinates on the affine Lie group. When written in terms of the affine group element, this formulation exhibits a two-dimensional WZW term. In another formulation each CFT is written as a two-dimensional field theory, with a three- dimensional WZW term, whose fields are coordinates on the affine group. On the basis of these equivalent formulations, we develop a translation dictionary in which the new formulations on the affine Lie group are understood as mode formulations of the conventional formulations on the Lie group. Using this dictionary, we also express each CFT as a three-dimensional field theory on the Lie group with a four-dimensional WZW term. 36 refs.

Clubok, K.S.

1996-04-01

374

Affine models for motion and shape recovery  

NASA Astrophysics Data System (ADS)

This paper presents an affine model for 3-D motion and shape recovery using two perspective views and their relative 2-D displacement field. The 2-D displacement vectors are estimated as parameters of a 2-D affine model that generalizes standard block matching by allowing affine shape deformations of image blocks and affine intensity transformations. The matching block size is effectively found via morphological size histograms. The parameters of the 3-D affine model are estimated using a least-squares algorithm that requires solving a system of linear equations with rank three. Some stabilization of the recovered motion parameters under noise is achieved through a simple form of MAP estimation. A multi-scale searching in the parameter space is also used to improve accuracy without high computational cost. Experiments on applying these affine models to various real world image sequences demonstrate that they can estimate dense displacement fields and recover motion parameters and object shape with relatively small errors.

Fuh, Chiou-Shann; Maragos, Petros

1992-11-01

375

Stream Cipher Using Optical Affine Transformation  

NASA Astrophysics Data System (ADS)

In this chapter, a method for generating a sequence of pseudorandom patterns using two-dimensional affine transformation is presented. The method is called the pseudorandom pattern generation with affine transformation (PPGA). A parallel affine-transform feedback system is introduced as the platform of the PPGA. As an example of the application, a stream cipher is described. The procedure to implement the PPGA is explained with an evaluation of the results. Experimental result of optical implementation show the capabilities of the method.

Tanida, Jun; Sasaki, Toru

376

Structural determinants of sigma receptor affinity  

SciTech Connect

The structural determinants of sigma receptor affinity have been evaluated by examining a wide range of compounds related to opioids, neuroleptics, and phenylpiperidine dopaminergic structures for affinity at sigma receptor-binding sites labeled with (+)-(/sup 3/H)3-PPP. Among opioid compounds, requirements for sigma receptor affinity differ strikingly from the determinants of affinity for conventional opiate receptors. Sigma sites display reverse stereoselectivity to classical opiate receptors. Multi-ringed opiate-related compounds such as morphine and naloxone have negligible affinity for sigma sites, with the highest sigma receptor affinity apparent for benzomorphans which lack the C ring of opioids. Highest affinity among opioids and other compounds occurs with more lipophilic N-substituents. This feature is particularly striking among the 3-PPP derivatives as well as the opioids. The butyrophenone haloperidol is the most potent drug at sigma receptors we have detected. Among the series of butyrophenones, receptor affinity is primarily associated with the 4-phenylpiperidine moiety. Conformational calculations for various compounds indicate a fairly wide range of tolerance for distances between the aromatic ring and the amine nitrogen, which may account for the potency at sigma receptors of structures of considerable diversity. Among the wide range of structures that bind to sigma receptor-binding sites, the common pharmacophore associated with high receptor affinity is a phenylpiperidine with a lipophilic N-substituent.

Largent, B.L.; Wikstroem, H.G.; Gundlach, A.L.; Snyder, S.H.

1987-12-01

377

Non-affine deformations in polymer hydrogels  

PubMed Central

Most theories of soft matter elasticity assume that the local strain in a sample after deformation is identical everywhere and equal to the macroscopic strain, or equivalently that the deformation is affine. We discuss the elasticity of hydrogels of crosslinked polymers with special attention to affine and non-affine theories of elasticity. Experimental procedures to measure non-affine deformations are also described. Entropic theories, which account for gel elasticity based on stretching out individual polymer chains, predict affine deformations. In contrast, simulations of network deformation that result in bending of the stiff constituent filaments generally predict non-affine behavior. Results from experiments show significant non-affine deformation in hydrogels even when they are formed by flexible polymers for which bending would appear to be negligible compared to stretching. However, this finding is not necessarily an experimental proof of the non-affine model for elasticity. We emphasize the insights gained from experiments using confocal rheoscope and show that, in addition to filament bending, sample micro-inhomogeneity can be a significant alternative source of non-affine deformation.

Wen, Qi; Basu, Anindita; Janmey, Paul A.; Yodh, A. G.

2012-01-01

378

Tag loss and short-term mortality associated with passive integrated transponder tagging of juvenile Lost River suckers  

USGS Publications Warehouse

Passive integrated transponder (PIT) tags are commonly used to mark small catostomids, but tag loss and the effect of tagging on mortality have not been assessed for juveniles of the endangered Lost River sucker Deltistes luxatus. I evaluated tag loss and short-term (34-d) mortality associated with the PIT tagging of juvenile Lost River suckers in the laboratory by using a completely randomized design and three treatment groups (PIT tagged, positive control, and control). An empty needle was inserted into each positive control fish, whereas control fish were handled but not tagged. Only one fish expelled its PIT tag. Mortality rate averaged 9.8 ± 3.4% (mean ± SD) for tagged fish; mortality was 0% for control and positive control fish. All tagging mortalities occurred in fish with standard lengths of 71 mm or less, and most of the mortalities occurred within 48 h of tagging. My results indicate that 12.45- × 2.02-mm PIT tags provide a viable method of marking juvenile Lost River suckers that are 72 mm or larger.

Burdick, Summer M.

2011-01-01

379

Sampling and detection of tagged dredged material  

SciTech Connect

Systems for sampling and detecting tagged dredged sand in the Upper Mississippi River were developed by Argonne National Laboratory for the U.S. Army Corps of Engineers, Rock Island District. The Corps plans to demonstrate main-channel disposal of dredged material, and it requires systems to detect the movement of the dredged material after the material has been placed in a deep reach of river. The dredged material in the demonstration will be tagged with sand particles coated with fluorescent dye. Argonne designed systems for: sampling of the bottom surficial sediments at discrete points along transects from a boat that employs a precision navigation system; on-board visual inspection of the samples for dyed sand in an ultraviolet light box; and photography of ultraviolet-illuminated samples. This report describes the systems and their use as well as the results of tests to determine proper settings for photographic equipment.

Van Loon, L.S.; McCown, D.L.; Ditmars, J.D.

1982-01-01

380

Selected Isotopes for Optimized Fuel Assembly Tags  

SciTech Connect

In support of our ongoing signatures project we present information on 3 isotopes selected for possible application in optimized tags that could be applied to fuel assemblies to provide an objective measure of burnup. 1. Important factors for an optimized tag are compatibility with the reactor environment (corrosion resistance), low radioactive activation, at least 2 stable isotopes, moderate neutron absorption cross-section, which gives significant changes in isotope ratios over typical fuel assembly irradiation levels, and ease of measurement in the SIMS machine 2. From the candidate isotopes presented in the 3rd FY 08 Quarterly Report, the most promising appear to be Titanium, Hafnium, and Platinum. The other candidate isotopes (Iron, Tungsten, exhibited inadequate corrosion resistance and/or had neutron capture cross-sections either too high or too low for the burnup range of interest.

Gerlach, David C.; Mitchell, Mark R.; Reid, Bruce D.; Gesh, Christopher J.; Hurley, David E.

2008-10-01

381

Molecular Tagging Velocimetry in a Microchannel  

NASA Astrophysics Data System (ADS)

The majority of velocity measurements in microchannel geometries have been typically obtained using particle-based techniques. We report preliminary data from Molecular Tagging Velocimetry (MTV) in high aspect ratio rectangular channels with a gap width of order 100 micron. The aqueous flow is driven either electroosmotically or by a pressure differential. Results will be presented for the variation of flow speed versus applied potential, for the electroosmotic flow, and friction factor versus Reynolds number for the pressure driven flow.

Lum, C.; Koochesfahani, M.

2003-11-01

382

A fractal circular polarized RFID tag antenna  

NASA Astrophysics Data System (ADS)

In this paper, we present a novel fractal antenna for radiofrequency identification (RFID) tags. The proposed antenna has a resonant frequency equal to 2.45GHz and circular polarization. The fractal technique was very useful to obtain a miniaturization of antenna size by more than 30%. The gain and directivity of the antenna are acceptable for the desired RFID application. All the results are obtained using CST Microwave simulation tool.

Chaouki, Guesmi; Ferchichi, Abdelhak; Gharsallah, Ali

2013-09-01

383

In the Mood: Tagging Music with Affects  

Microsoft Academic Search

Music and mood carry a strong relationship, which is employed very effectively by classical music, Hollywood’s soundtracks,\\u000a and pop bands. Affective computing can provide support in selecting music that fits to a given mood. We describe a system\\u000a that addresses a full range of functionality. It allows the user to semi-automatically tag music with mood descriptions, determines\\u000a mood from sensors

Jörn Loviscach; David Oswald

2008-01-01

384

Tagged volume rendering of the heart.  

PubMed

We present a novel system for 3-D visualisation of the heart and coronary arteries. Binary tags (generated offline) are combined with value-gradient transfer functions (specified online) allowing for interactive visualisation, while relaxing the offline segmentation criteria. The arteries are roughly segmented using a Hessian-based line filter and the pericardial cavity using a Fast Marching active contour. A comparison of different contour initialisations reveals that simple geometric shapes (such as spheres or extruded polygons) produce suitable results. PMID:18051059

Mueller, Daniel; Maeder, Anthony; O'Shea, Peter

2007-01-01

385

Prospects for Barium Tagging in Gaseous Xenon  

NASA Astrophysics Data System (ADS)

Tagging events with the coincident detection of a barium ion would greatly reduce the background for a neutrino-less double beta decay search in xenon. This paper describes progress towards realizing this goal. It outlines a source that can produce large quantities of Ba++ in gas, shows that this can be extracted to vacuum, and demonstrates a mechanism by which the Ba++ can be efficiently converted to Ba+ as required for laser identification.

Sinclair, D.; Rollin, E.; Smith, J.; Mommers, A.; Ackeran, N.; Aharmin, B.; Auger, M.; Barbeau, P. S.; Benitez-Medina, C.; Breidenbach, M.; Burenkov, A.; Cook, S.; Coppens, A.; Daniels, T.; DeVoe, R.; Dobi, A.; Dolinski, M. J.; Donato, K.; Fairbank, W., Jr.; Farine, J.; Giroux, G.; Gornea, G.; Graham, K.; Gratta, G.; Green, M.; Hagemann, C.; Hall, C.; Hall, K.; Hallman, D.; Hargrove, C.; Herrin, S.; Kaufman, L. K.; Leonard, D. S.; LePort, F.; Mackay, D.; MacLennan, R.; Mong, B.; Montero Díez, M.; Müller, A. R.; Neilson, R.; Niner, E.; Odian, A.; O'Sullivan, K.; Ouellet, C.; Piepke, A.; Pocar, A.; Prescott, C. Y.; Pushkin, K.; Rowson, P. C.; Slutsky, S.; Stekhanov, V.; Twelker, K.; Voskanian, N.; Vuilleumier, J.-L.; Wichoski, U.; Wodin, J.; Yang, L.; Yen, Y.-R.

2011-08-01

386

Physics with tagged forward protons at RHIC  

SciTech Connect

The physics reach of the STAR detector at RHIC has been extended to include elastic and inelastic diffraction measurements with tagged forward protons. This program has started at RHIC in p+p collisions with a special optics run of {beta}* {approx} 21 m at STAR, at the center-of-mass energy {radical}s = 200 GeV during the last week of the RHIC 2009 run.

Yip,K.

2009-08-30

387

The tagged RIBs facility of LNS  

SciTech Connect

Radioactive Ion Beams (RIBs) are produced In-Flight at the Laboratori Nazionali del Sud (LNS) by projectile fragmentation on light targets at intermediate energies. RIBs rates up to 10{sup 5} ions/sec have been measured and about 95% of secondary beam has been transported up to one of the experimental caves. The {delta}E-ToF identification method was successfully applied to tag, event-by-event, the RIBs before the interaction with a secondary reaction target.

De Napoli, M.; Raciti, G.; Rapisarda, E.; Cardella, G. [Dipartimento di Fisica, Universita degli studi di Catania and Sezione INFN, 64, Via S. Sofia, I-95123 Catania (Italy); Amorini, F.; Calabretta, L. [Laboratori Nazionali del Sud-INFN, 62, Via S. Sofia, I-95123 Catania (Italy); Sfienti, C. [GSI, Darmstadt D-64291 (Germany)

2007-11-30

388

Hypergraph model of social tagging networks  

Microsoft Academic Search

The past few years have witnessed the great success of a new family of\\u000aparadigms, so-called folksonomy, which allows users to freely associate tags to\\u000aresources and efficiently manage them. In order to uncover the underlying\\u000astructures and user behaviors in folksonomy, in this paper, we propose an\\u000aevolutionary hypergrah model to explain the emerging statistical properties.\\u000aThe present model

Zi-Ke Zhang; Chuang Liu

2010-01-01

389

Scanning Cargo Containers with Tagged Neutrons  

NASA Astrophysics Data System (ADS)

A new Tagged Neutron Inspection System (TNIS) able to detect illicit materials such as explosives and narcotics in cargo containers has been developed within the EURopean Illicit TRAfficing Countermeasures Kit (EURITRACK) project. After the R&D phase, the inspection portal has been installed and commissioned at the Rijeka seaport in Croatia, where it has been operated in connection with the existing X-ray scanner for a first two-month demonstration campaign. Results obtained are presented and discussed in this paper.

Viesti, G.; Botosso, C.; Fabris, D.; Lunardon, M.; Moretto, S.; Nebbia, G.; Pesente, S.; Zenoni, A.; Donzella, A.; Perot, B.; Carasco, C.; Bernard, S.; Mariani, A.; Szabo, J.-L.; Sannie, G.; Valkovic, V.; Sudac, D.; Nad, K.; Peerani, P.; Sequeira, V.; Salvato, M.; Moszynski, M.; Gierlik, M.; Klamra, W.; Le Tourneur, P.; Lhuissier, M.; Colonna, A.; Tintori, C.

2007-10-01

390

Geographic location tags on digital images  

Microsoft Academic Search

We describe an end-to-end system that capitalizes on geographic location tags for digital photographs. The World Wide Media eXchange (WWMX) database indexes large collections of image media by several pieces of metadata including timestamp, owner, and critically, location stamp. The location where a photo was shot is important because it says much about its semantic content, while being relatively easy

Kentaro Toyama; Ron Logan; Asta Roseway

2003-01-01

391

Scanning Cargo Containers with Tagged Neutrons  

SciTech Connect

A new Tagged Neutron Inspection System (TNIS) able to detect illicit materials such as explosives and narcotics in cargo containers has been developed within the EURopean Illicit TRAfficing Countermeasures Kit (EURITRACK) project. After the R and D phase, the inspection portal has been installed and commissioned at the Rijeka seaport in Croatia, where it has been operated in connection with the existing X-ray scanner for a first two-month demonstration campaign. Results obtained are presented and discussed in this paper.

Viesti, G.; Botosso, C.; Fabris, D.; Lunardon, M.; Moretto, S.; Nebbia, G.; Pesente, S. [INFN and Universita di Padova, Via Marzolo 8, I-35131 Padova (Italy); Zenoni, A.; Donzella, A. [INFN and Universita di Brescia, 38 Via Branze 25123 Brescia (Italy); Perot, B.; Carasco, C.; Bernard, S.; Mariani, A. [Commissariat a l'Energie Atomique, 13108 St Paul-lez-Durance (France); Szabo, J.-L.; Sannie, G. [Commissariat a l'Energie Atomique, 91191 Gif-Sur-Yvette (France); Valkovic, V.; Sudac, D.; Nad, K. [Institute Ruder Boskovic, 54 Bijenicka c. 10000 Zagreb (Croatia); Peerani, P.; Sequeira, V. [European Commission, Joint Research Centre, I-21020 Ispra (Italy)] (and others)

2007-10-26

392

Monodisperse, "highly" positively charged protein polymer drag-tags generated in an intein-mediated purification system used in free-solution electrophoretic separations of DNA  

PubMed Central

Free-solution conjugate electrophoresis (FSCE) is a method of DNA sequencing that eliminates the need for viscous polymer solutions by tethering a carefully designed, mobility modifying “drag-tag” to each DNA molecule to achieve size-based separations of DNA. The most successful drag-tags to date are genetically engineered, highly repetitive polypeptides (“protein polymers”) that are designed to be large, water-soluble, and completely monodisperse. Positively charged arginines were deliberately introduced at regular intervals into the amino acid sequence to increase the hydrodynamic drag without increasing drag-tag length. Additionally, a one-step purification method that combines affinity chromatography and on-column tag cleavage was devised to achieve the required drag-tag monodispersity. Sequencing with a read length of approximately 180 bases was successfully achieved with a known sequence in free-solution electrophoresis using one of these positively charged drag-tags. This preliminary result is expected to lead to further progress in FSCE sequencing with ~400 bases read length possible when more “highly” positively charged protein polymers of larger size are generated with the intein system.

Wang, Xiaoxiao; Albrecht, Jennifer Coyne; Lin, Jennifer S.; Barron, Annelise E.

2012-01-01

393

A Functional Histidine-Tagged Replication Initiator Protein: Implications for the Study of Single-Stranded DNA Virus Replication In Planta†  

PubMed Central

Replication initiation of nanoviruses, plant viruses with a multipartite circular single-stranded DNA genome, is triggered by the master Rep (M-Rep) protein. To enable the study of interactions between M-Rep and viral or host factors involved in replication, we designed oligohistidine-tagged variants of the nanovirus Faba bean necrotic yellows virus (FBNYV) M-Rep protein that allow affinity purification of enzymatically active M-Rep from plant tissue. The tagged M-Rep protein was able to initiate replication of its cognate and other FBNYV DNAs in Nicotiana benthamiana leaf disks and plants. The replicon encoding the tagged M-Rep protein multiplied and moved systemically in FBNYV-infected Vicia faba plants and was transmitted by the aphid vector of the virus. Using the tagged M-Rep protein, we demonstrated the in planta interaction between wild-type M-Rep and its tagged counterpart. Such a tagged and fully functional replication initiator protein will have bearings on the isolation of protein complexes from plants.

Vega-Arreguin, Julio C.; Timchenko, Tatiana; Gronenborn, Bruno; Ramirez, Bertha Cecilia

2005-01-01

394

The accelerating growth of online tagging systems  

NASA Astrophysics Data System (ADS)

Research on the growth of online tagging systems not only is interesting in its own right, but also yields insights for website management and semantic web analysis. Traditional models that describing the growth of online systems can be divided between linear and nonlinear versions. Linear models, including the BA model [A.L. Barabasi, R. Albert, Science 286, 509 (1999)], assume that the average activity of users is a constant independent of population. Hence the total activity is a linear function of population. On the contrary, nonlinear models suggest that the average activity is affected by the size of the population and the total activity is a nonlinear function of population. In the current study, supporting evidences for the nonlinear growth assumption are obtained from data on Internet users' tagging behavior. A power law relationship between the number of new tags (F) and the population (P), which can be expressed as F~P? (? > 1), is found. I call this pattern accelerating growth and find it relates the to time-invariant heterogeneity in individual activities. I also show how a greater heterogeneity leads to a faster growth.

Wu, L. F.

2011-09-01

395

An approach to sequence DNA without tagging  

NASA Astrophysics Data System (ADS)

Microarray technology is playing an increasingly important role in biology and medicine and its application to genomics for gene expression analysis has already reached the market with a variety of commercially available instruments. In these combinatorial analysis methods, known probe single-strand DNA (ssDNA) 'primers' are attached in clusters of typically 100 µm × 100 µm pixels. Each pixel of the array has a slightly different sequence. On exposure to 'unknown' target ssDNA, the pixels with the right complementary probe ssDNA sequence convert to double-stranded DNA (dsDNA) by a hybridization reaction. To transduct the conversion of the pixel to dsDNA, the target ssDNA is labelled with a photoluminescent tag during the polymerase chain reaction (PCR) amplification process. Due to the statistical distribution of the tags in the target ssDNA, it becomes significantly difficult to implement these methods as a diagnostic tool in a pathology laboratory. A method to sequence DNA without tagging the molecule is developed. The fabrication process is compatible with current microelectronics and (emerging) soft-material fabrication technologies, allowing the method to be integrable with micro-electromechanical systems (MEMS) and lab-on-a-chip devices. An estimated sensitivity of 10-12 g on a 1 cm2 device area is obtained.

Niu, Sanjun; Saraf, Ravi F.

2002-10-01

396

Stable expression and characterization of N-terminal tagged recombinant human bone morphogenetic protein 15.  

PubMed

Oocyte-derived growth factors are critically involved in multiple ovarian processes via paracrine actions. Although recombinant proteins have been applied to dissect the physiological functions of these factors, variation of activities among different protein preparations remains an issue. To further elucidate the roles of one of these growth factors, bone morphogenetic protein 15 (BMP15), in mediating oocyte-regulated molecular and cellular events and to explore its potential clinical application, we engineered the human BMP15 sequence to efficiently produce bioactive recombinant human BMP15 (rhBMP15). The proteolytic cleavage site of the hBMP15 precursor was optimized to facilitate the production of the mature protein, and a FLAG-tag was placed at the N-terminus of the mature region to ease purification and avoid potential interference of the tag with the cystine knot structure. The rhBMP15 protein was purified using anti-FLAG M2 affinity gel. Our results demonstrated that the N-terminal tagged rhBMP15 was efficiently processed in HEK-293 cells. Furthermore, the purified rhBMP15 could activate SMAD1/5/8 and induce the transcription of genes encoding cumulus expansion-related transcripts (Ptx3, Has2, Tnfaip6 and Ptgs2), inhibitory SMADs (Smad6 and Smad7), BMP antagonists (Grem1 and Fst), activin/inhibin betaA (Inhba) and betaB (Inhbb) subunits, etc. Thus, our rhBMP15 containing a genetically modified cleavage sequence and an N-terminal FLAG-tag can be efficiently produced, processed and secreted in a mammalian expression system. The purified rhBMP15 is also biologically active and very stable, and can induce the expression of a variety of mouse granulosa cell genes. PMID:19651638

Li, Qinglei; Rajanahally, Saneal; Edson, Mark A; Matzuk, Martin M

2009-08-03

397

Specific inhibitors of HCV polymerase identified using an NS5B with lower affinity for template/primer substrate  

PubMed Central

The interaction of the hepatitis C virus (HCV) RNA-dependent RNA polymerase with RNA substrate is incompletely defined. We have characterized the activities of the HCV NS5B polymerase, modified by different deletions and affinity tags, with a routinely used homopolymeric substrate, and established apparent affinities of the various NS5B constructs both for the NTP and the template/primer substrates. We identified a uniquely tagged HCV NS5B RNA polymerase construct with a lower affinity (higher Km) than mature HCV NS5B for template/ primer substrate and highlighted the use of such a polymerase for the identification of inhibitors of NS5B activity, particularly inhibitors of productive RNA binding. The characterization of specific benzimidazole-5-carboxamide-based inhibitors, identified in a screening campaign, revealed that this class of compounds was non-competitive with regard to NTP incorporation and had no effect on processive elongation, but inhibited an initiation phase of the HCV polymerase activity. The potency of these compounds versus a panel of different NS5B polymerase constructs was inversely proportional to the enzymes’ affinities for template/primer substrate. The benzimidazole-5-carboxamide compounds also inhibited the full-length, untagged NS5B de novo initiation reaction using HCV 3?-UTR substrate RNA and expand the diversifying pool of potential HCV replication inhibitors.

McKercher, Ginette; Beaulieu, Pierre L.; Lamarre, Daniel; LaPlante, Steven; Lefebvre, Sylvain; Pellerin, Charles; Thauvette, Louise; Kukolj, George

2004-01-01

398

The use of tags in monitoring limits on mobile missiles  

SciTech Connect

Three tagging systems were considered in this paper: as a supplement to on-site inspection (OSI), as a supplement to national technical means (NTM), and as a supplement to site surveillance systems. Each system would require a different type of tag, perhaps ranging from microchip tags with infrared transponders to navigation receivers. Use of tags as a supplement to OSIs may be the simplest system to implement because it places the least demands on technology. Tags may make OSI more acceptable by replacing humans with remote sensors, thereby decreasing the perceived potential for espionage. Using tags as a supplement to NTM decreases the necessity for human OSI even further, but places higher demands on technology and may affect the normal operation of deployment areas. Site surveillance systems using tags have the potential for excellent missile verification, but they may be excessively intrusive and expensive, and could have a large effect on the normal operation of declared facilities.

Fetter, S.

1987-03-01

399

Strong Authentication Protocol for Secure RFID Tag Search without Help of Central Database  

Microsoft Academic Search

RFID tag search is to find a particular tag in a group of tags by using a portable reader. Since a portable reader has own identifier and secret values associated with each tag, the portable reader can wirelessly communicate with the tag without help of central database. The RFID tag search can be useful to find a lost book among

Tae Youn Won; Ji Young Chun; Dong Hoon Lee

2008-01-01

400

Braid group action and quantum affine algebras  

Microsoft Academic Search

We lift the lattice of translations in the extended affine Weyl group to a braid group action on the quantum affine algebra. This action fixes the Heisenberg subalgebra pointwise. Loop-like generators of the algebra are obtained which satisfy the relations of Drinfel'd's new realization. Coproduct formulas are given and a PBW type basis is constructed.

Jonathan Beck

1994-01-01

401

An affine invariant interest point detector  

Microsoft Academic Search

This paper presents a novel approach for detecting affine invariant interest points. Our method can deal with significant affine transformations including large scale changes. Such transformations in- troduce significant changes in the point location as well as in the scale and the shape of the neighbourhood of an interest point. Our approach allows to solve for these problems simultaneously. It

Krystian Mikolajczyk; Cordelia Schmid

2002-01-01

402

A Comparison of Affine Region Detectors  

Microsoft Academic Search

The paper gives a snapshot of the state of the art in affine covariant region detectors, and compares their performance on a set of test images under varying imaging conditions. Six types of detectors are included: detectors based on affine normalization around Harris (Mikolajczyk and Schmid, 2002; Schaffalitzky and Zisserman, 2002) and Hessian points (Mikolajczyk and Schmid, 2002), a detector

Krystian Mikolajczyk; Tinne Tuytelaars; Cordelia Schmid; Andrew Zisserman; Jiri Matas; Frederik Schaffalitzky; Timor Kadir; Luc J. Van Gool; J. Matas

2005-01-01

403

Lipopolysaccharide affinity for titanium implant biomaterials  

Microsoft Academic Search

Statement of problem. Lipopolysaccharide (LPS) affinity for titanium implant biomaterials could affect crevicular LPS concentrations and thereby influence periimplant inflammation.Purpose of study. The purpose of this study was to evaluate Porphyromonas gingivalis and Escherichia coli LPS affinity for titanium biomaterials groups that differed in surface oxide composition and surface roughness.Material and method. Polished and abraded grade 1 commercially pure titanium

Steven K. Nelson; Kent L. Knoernschild; Fonda G. Robinson; George S. Schuster

1997-01-01

404

Androgen receptor binding affinity: a QSAR evaluation  

Microsoft Academic Search

The multiparameter formulation of the COmmon REactivity PAttern (COREPA) approach has been used to describe the structural requirements for eliciting rat androgen receptor (AR) binding affinity, accounting for molecular flexibility. Chemical affinity for AR binding was related to the distances between nucleophilic sites and structural features describing electronic and hydrophobic interactions between the receptor and ligands. Categorical models were derived

M. Todorov; E. Mombelli; S. Aït-Aïssa; O. Mekenyan

2011-01-01

405

Affine root systems and dual numbers  

NASA Astrophysics Data System (ADS)

The root systems in Carroll spaces with degenerate metric are defined. It is shown that their Cartan matrices and reflection groups are affine. Due to the geometric consideration the root system structure of affine algebras is determined by a sufficiently simple algorithm.

Kostyakov, I. V.; Gromov, N. A.; Kuratov, V. V.

406

Affine\\/ Photometric Invariants for Planar Intensity Patterns  

Microsoft Academic Search

The paper contributes to the viewpoint invariant recognition of planar patterns, especially labels and signs under affine deformations. By their nature, the information of such eye-catchers is not contained in the outline or frame — they often are affinely equivalent like parallelograms and ellipses — but in the intensity content within. Moment invariants are well suited for their recognition. They

Luc J. Van Gool; Theo Moons; Dorin Ungureanu

1996-01-01

407

Sample simplification through affinity selection in proteomics  

Microsoft Academic Search

In “bottom up” approach in proteomics, it is always challenging to analyze the large number of peptides generated by protein digestion. Affinity selection of specific peptides provides a quick way to reduce sample complexity to match the analytical capacity of the system. In this thesis, agarose based immobilized metal affinity chromatography (IMAC) columns loaded with copper (II) were evaluated for

Diya Ren

2004-01-01

408

Loop realizations of quantum affine algebras  

SciTech Connect

We give a simplified description of quantum affine algebras in their loop presentation. This description is related to Drinfeld's new realization via halves of vertex operators. We also define an idempotent version of the quantum affine algebra which is suitable for categorification.

Cautis, Sabin [Department of Mathematics, University of Southern California, Los Angeles, California, 90089 (United States); Licata, Anthony [Department of Mathematics, Australian National University, Canberra (Australia)

2012-12-15

409

The affinity?seeking function of communication  

Microsoft Academic Search

A model of the affinity?seeking function of communication is introduced and explicated. The affinity?seeking construct describes ways people get others to like and feel positive about them. The research is grounded in the presumption that people attempt to generate liking by using various communication strategies. Four questions were addressed in six studies: (1) How do people attempt to generate liking?

Robert A. Bell; John A. Daly

1984-01-01

410

Improving image segmentation by learning region affinities  

SciTech Connect

We utilize the context information of other regions in hierarchical image segmentation to learn new regions affinities. It is well known that a single choice of quantization of an image space is highly unlikely to be a common optimal quantization level for all categories. Each level of quantization has its own benefits. Therefore, we utilize the hierarchical information among different quantizations as well as spatial proximity of their regions. The proposed affinity learning takes into account higher order relations among image regions, both local and long range relations, making it robust to instabilities and errors of the original, pairwise region affinities. Once the learnt affinities are obtained, we use a standard image segmentation algorithm to get the final segmentation. Moreover, the learnt affinities can be naturally unutilized in interactive segmentation. Experimental results on Berkeley Segmentation Dataset and MSRC Object Recognition Dataset are comparable and in some aspects better than the state-of-art methods.

Prasad, Lakshman [Los Alamos National Laboratory; Yang, Xingwei [TEMPLE UNIV.; Latecki, Longin J [TEMPLE UNIV.

2010-11-03

411

Integrated management and visualization of electronic tag data with Tagbase.  

PubMed

Electronic tags have been used widely for more than a decade in studies of diverse marine species. However, despite significant investment in tagging programs and hardware, data management aspects have received insufficient attention, leaving researchers without a comprehensive toolset to manage their data easily. The growing volume of these data holdings, the large diversity of tag types and data formats, and the general lack of data management resources are not only complicating integration and synthesis of electronic tagging data in support of resource management applications but potentially threatening the integrity and longer-term access to these valuable datasets. To address this critical gap, Tagbase has been developed as a well-rounded, yet accessible data management solution for electronic tagging applications. It is based on a unified relational model that accommodates a suite of manufacturer tag data formats in addition to deployment metadata and reprocessed geopositions. Tagbase includes an integrated set of tools for importing tag datasets into the system effortlessly, and provides reporting utilities to interactively view standard outputs in graphical and tabular form. Data from the system can also be easily exported or dynamically coupled to GIS and other analysis packages. Tagbase is scalable and has been ported to a range of database management systems to support the needs of the tagging community, from individual investigators to large scale tagging programs. Tagbase represents a mature initiative with users at several institutions involved in marine electronic tagging research. PMID:21750734

Lam, Chi Hin; Tsontos, Vardis M

2011-07-05

412

Development of techniques for tagging precursor and essential chemicals  

SciTech Connect

The ability to identify the manufacturers and distributors of chemicals seized in raids of illicit drug labs would be of great value in controlling the diversion of these chemicals. We developed a tagging scheme based on the addition of sub-ppM concentrations of various combinations of rare-earth elements to the target chemicals and evaluated a number of techniques for detecting the tags. We developed soluble tags for tagging liquids and selected Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) as the preferred detection technique. We developed insoluble tags for tagging solids and developed methods to analyze them and mix them into solid precursors. We have successfully demonstrated the tagging of several solvents and two of the precursor chemicals used in one of the most popular clandestine methamphetamine syntheses (ephedrine reacting with hydriodic acid/red phosphorus). The tagging scheme is capable of yielding tens of thousands of signatures (using holmium as an internal standard and up to 9 rare-earths at up to 3 concentrations yields 3{sup 9} {minus} 1 = 19,682 signatures) and is applicable to most of the chemicals on the precursor and essential chemicals list. In the concentrations employed, the tags are safe enough to be added to pharmaceuticals and cheap enough to tag tanker loads of chemicals.

Swansiger, W.A.; Shepodd, T.J. [Sandia National Labs., Livermore, CA (United States); Phillips, M.L.F. [Sandia National Labs., Albuquerque, NM (United States)

1994-01-01

413

Identification of new tag sequences with differential and selective recognition properties for the anti-FLAG monoclonal antibodies M1, M2 and M5  

Microsoft Academic Search

Summary The FLAG® peptides DYKDDDDK and MDYKDDDDK are widely used affinity tags. Here we describe new variants of the FLAG peptides which, in direct ELISA, showed selective and differential binding to the commercially available anti-FLAG monoclonal antibodies M1, M2 and M5. Variants of the FLAG peptides were synthesized on polymer-grafted plastic pins, and in an ELISA incubated with mAbs M1,

J. W. Slootstra; D. Kuperus; A. Plückthun; R. H. Meloen

1997-01-01

414

Expression in Pichia pastoris X33 of His-tagged lipase from a novel strain of Rhizopus oryzae and its mutant Asn 134 His: purification and characterization  

Microsoft Academic Search

The sequence corresponding to the mature lipase of Rhizopus oryzae WPG (ROLw) was subcloned in the pPIC9K expression vector, with a strong AOX1 promoter, to construct a recombinant lipase protein containing six histidine residues at the N-terminal. The His-tagged lipase\\u000a was expressed in Pichia Pastoris X33 and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity

Riadh Ben Salah; Ali Gargouri; Robert Verger; Youssef Gargouri; Hafedh Mejdoub

2009-01-01

415

Microcalorimetric Study of the Effect of Hexa-histidine Tag and Denaturant on the Interaction Mechanism between Protein and Metal-Chelating Gel  

Microsoft Academic Search

A recombinant protein, Schistosoma japonicum glutathione-S-transferase (SjGST), was fused with a C-terminal hexa-histidine tag to obtain SjGST\\/His. Both proteins were used to probe the interaction mechanisms with the metal ions immobilized on chromatography gels. Isothermal titration calorimetry was used to directly measure the adsorption enthalpies (?Hads) of both proteins with Ni-NTA and TALON (Co2+) commercial affinity resins, under the conditions

Fu-Yung Lin; Wen-Yih Chen; Hsiu-Mei Chen

2001-01-01

416

Synthesis of Ni–Zn ferrite nanoparticles in radiofrequency thermal plasma reactor and their use for purification of histidine-tagged proteins  

Microsoft Academic Search

Superparamagnetic Ni–Zn ferrite nanoparticles were synthesized in radiofrequency thermal plasma reactor from aqueous solutions\\u000a of Ni- and Zn-nitrates. The nanoparticles were studied for protein purification performance in both quantitative and qualitative\\u000a terms. For comparison, experiments were also performed by Ni-charged affinity chromatography. It was proved that the Ni–Zn\\u000a ferrite nanoparticles effectively purified histidine-tagged proteins with a maximum protein binding capacity

Tivadar Feczkó; Adél Muskotál; Loránd Gál; János Szépvölgyi; Anett Sebestyén; Ferenc Vonderviszt

2008-01-01

417

Affinity Purification Strategies for Proteomic Analysis of Transcription Factor Complexes  

PubMed Central

Affinity purification (AP) coupled to mass spectrometry (MS) has been successful in elucidating protein molecular networks of mammalian cells. These approaches have dramatically increased the knowledge of the interconnectivity present among proteins and highlighted biological functions within different protein complexes. Despite significant technical improvements reached in the past years, it is still challenging to identify the interaction networks and the subsequent associated functions of nuclear proteins such as transcription factors (TFs). A straightforward and robust methodology is therefore required to obtain unbiased and reproducible interaction data. Here we present a new approach for TF AP-MS, exemplified with the CCAAT/enhancer binding protein alpha (C/EBPalpha). Utilizing the advantages of a double tag and three different MS strategies, we conducted a total of six independent AP-MS strategies to analyze the protein–protein interactions of C/EBPalpha. The resultant data were combined to produce a cohesive C/EBPalpha interactome. Our study describes a new methodology that robustly identifies specific molecular complexes associated with transcription factors. Moreover, it emphasizes the existence of TFs as protein complexes essential for cellular biological functions and not as single, static entities.

2013-01-01

418

High-Throughput Analysis of Protein-DNA Binding Affinity.  

PubMed

Sequence-specific protein-DNA interactions mediate most regulatory processes underlying gene expression, such as transcriptional regulation by transcription factors (TFs) or chromatin organization. Current knowledge about DNA-binding specificities of TFs is based mostly on low- to medium-throughput methodologies that are time-consuming and often fail to identify DNA motifs recognized by a TF with lower affinity but retaining biological relevance. The use of protein-binding microarrays (PBMs) offers a high-throughput alternative for the identification of protein-DNA specificities. PBM consists in an array of pseudorandomized DNA sequences that are optimized to include all the possible 10- or 11-mer DNA sequences, allowing the determination of binding specificities of most eukaryotic TFs. PBMs that can be synthesized by several manufacturing companies as single-stranded DNA are converted into double-stranded in a simple primer extension reaction. The protein of interest fused to an epitope tag is then incubated onto the PBM, and specific DNA-protein complexes are revealed in a series of immunological reactions coupled to a fluorophore. After scanning and quantifying PBMs, specific DNA motifs recognized by the protein are identified with ready-to-use scripts, generating comprehensive but accessible information about the DNA-binding specificity of the protein. This chapter describes detailed procedures for preparation of double-stranded PBMs, incubation with recombinant protein, and detection of protein-DNA complexes. Finally, we outline some cues for evaluating the biological role of DNA motifs obtained in vitro. PMID:24057393

Franco-Zorrilla, José M; Solano, Roberto

2014-01-01

419

Development of translating ribosome affinity purification for zebrafish.  

PubMed

The regulation of transcription and translation by specific cell types is essential to generate the cellular diversity that typifies complex multicellular organisms. Tagging and purification of ribosomal proteins has been shown to be an innovative and effective means of characterizing the ribosome bound transcriptome of highly specific cell populations in vivo. To test the feasibility of using translating ribosome affinity purification (TRAP) in zebrafish, we have generated both a ubiquitous TRAP line and a melanocyte-specific TRAP line using the native zebrafish rpl10a ribosomal protein. We have demonstrated the capacity to capture mRNA transcripts bound to ribosomes, and confirmed the expected enrichment of melanocyte specific genes and depletion of non-melanocyte genes when expressing the TRAP construct with a cell specific promoter. We have also generated a generic EGFP-rpl10a Tol2 plasmid construct (Tol2-zTRAP) that can be readily modified to target any additional cell populations with characterized promoters in zebrafish. PMID:23281262

Tryon, Robert C; Pisat, Nilambari; Johnson, Stephen L; Dougherty, Joseph D

2013-02-26

420

N2O molecular tagging velocimetry  

NASA Astrophysics Data System (ADS)

A new seeded velocity measurement technique, N2O molecular tagging velocimetry (MTV), is developed to measure velocity in wind tunnels by photochemically creating an NO tag line. Nitrous oxide "laughing gas" is seeded into the air flow. A 193 nm ArF excimer laser dissociates the N2O to O(1D) that subsequently reacts with N2O to form NO. O2 fluorescence induced by the ArF laser "writes" the original position of the NO line. After a time delay, the shifted NO line is "read" by a 226-nm laser sheet and the velocity is determined by time-of-flight. At standard atmospheric conditions with 4% N2O in air, ˜1000 ppm of NO is photochemically created in an air jet based on experiment and simulation. Chemical kinetic simulations predict 800-1200 ppm of NO for 190-750 K at 1 atm and 850-1000 ppm of NO for 0.25-1 atm at 190 K. Decreasing the gas pressure (or increasing the temperature) increases the NO ppm level. The presence of humid air has no significant effect on NO formation. The very short NO formation time (<10 ns) makes the N2O MTV method amenable to low- and high-speed air flow measurements. The N2O MTV technique is demonstrated in air jet to measure its velocity profile. The N2O MTV method should work in other gas flows as well (e.g., helium) since the NO tag line is created by chemical reaction of N2O with O(1D) from N2O photodissociation and thus does not depend on the bulk gas composition.

ElBaz, A. M.; Pitz, R. W.

2012-03-01

421

Turbulent structure measurements by RELIEF flow tagging  

NASA Astrophysics Data System (ADS)

Molecular tagging by Raman excitation + laser-induced electronic fluorescence (RELIEF) is applied to an isotropically turbulent air flow to demonstrate the utility of the technique for measurements of fundamental turbulence parameters. Lines which are 120 ?m in diameter are written into the flow and their displacement is recorded after several microseconds. From these images, the average flow velocity profile, the average turbulence intensity profile, and the lateral velocity correlation function are computed. The lines themselves can be examined for coherent structure, eddy diffusion, line stretching, curvature, etc.

Miles, R.; Lempert, W.; Zhang, B.

1991-10-01

422

Personalized tag prediction via social influence in social networks  

NASA Astrophysics Data System (ADS)

Currently, social tagging systems have been adopted by many social websites. As tags help users to browse social content effectively, personalized tag prediction problem becomes important in social networks. In this paper, we present a new generative probabilistic model to solve personalized tag prediction problem. Differently with previous methods, we consider social influence between users and friends into this model. We bring two major contributions: 1) We propose a new probabilistic model which considers in social influence to describe users' actual tagging activities; 2) Based on this model, we propose a new approach to perform personalized tag prediction task. Experimental results on a real-world dataset crawled from Last.fm show that our method outperforms other methods.

Yan, Zhenlei; Zhou, Jie

2011-11-01

423

A Surface Trawl to Detect Migrating Juvenile Salmonids Tagged with Passive Integrated Transponder Tags  

Microsoft Academic Search

We developed a surface pair-trawl system to detect juvenile Pacific salmon Oncorhynchus spp. marked with passive integrated transponder (PIT) tags as they migrate through the upper Columbia River estuary. The trawl was fitted with a detection antenna in its cod end and was deployed by two vessels. Fish entering the trawl body exit after passing by the detection antenna. Detection

Richard D. Ledgerwood; Brad A. Ryan; Earl M. Dawley; Edward P. Nunnallee; John W. Ferguson

2004-01-01

424

Meaning Of A Tag: A Collaborative Approach to Bridge the Gap Between Tagging and Linked Data  

Microsoft Academic Search

This paper introduces MOAT, a lightweight Semantic Web framework that provides a collaborative way to let Web 2.0 content producers give meanings to their tags in a machine- readable way. To achieve this goal, this approach relies on Linked Data principles, using URIs from existing resources to define these meanings. That way, users can create inter- linked RDF data and

Alexandre Passant; Philippe Laublet

2008-01-01

425

Tags Help Make Libraries Del.icio.us: Social Bookmarking and Tagging Boost Participation  

ERIC Educational Resources Information Center

Traditional library web products, whether online public access catalogs, library databases, or even library web sites, have long been rigidly controlled and difficult to use. Patrons regularly prefer Google's simple interface. Now social bookmarking and tagging tools help librarians bridge the gap between the library's need to offer authoritative,…

Rethlefsen, Melissa L.

2007-01-01

426

A New Simulation Platform for UHF RFID Tag Development  

Microsoft Academic Search

This paper presents a new simulation platform for UHF RFID tag development, which efficiently reduces the development time and cost to achieve rapid, flexible and efficient simulation and design for UHF RFID tag. The simulation platform includes the RF analog front end and the tag control logic, which is implemented in the Altera FPGA. Besides coinciding with ISO\\/IEC 18000-6B\\/C series

Liying Chen; Shilin Zhang; Zheng Wang; Lei Li

2011-01-01

427

Susceptibility of UHF RFID Tags to Electromagnetic Analysis  

Microsoft Academic Search

The number of applications that use radio-frequency identification (RFID) technology has grown continually in the last few\\u000a years. Current RFID tags are mainly used for identification purposes and do not include crypto functionality. Therefore, classical\\u000a RFID tags are not designed as secure devices and do not contain countermeasures against side-channel analysis (SCA). The lack\\u000a of such countermeasures makes RFID tags

Thomas Plos

2008-01-01

428

Compounds with High Monoamine Transporter Affinity.  

National Technical Information Service (NTIS)

Featured compounds have high monoamine transport affinity and are characterized by one of the following two general formulas set out above. The compounds bind selectively or non-selectively to monoamine transporters. The compounds are useful to treat vari...

B. K. Madras P. Blundell P. Wang P. C. Meltzer

2005-01-01

429

PRINCIPLES OF AFFINITY-BASED BIOSENSORS  

EPA Science Inventory

Despite the amount of resources that have been invested by national and international academic, government, and commercial sectors to develop affinity-based biosensor products, little obvious success has been realized through commercialization of these devices for specific applic...

430

Electron Affinities of Atoms, Molecules, and Radicals.  

National Technical Information Service (NTIS)

We review briefly but comprehensively the theoretical, semiempirical and experimental methods employed to determine electron affinities (EAs) of atoms, molecules and radicals, and summarize the EA data obtained by these methods. The detailed processes und...

A. A. Christodoulides D. L. McCorkle L. G. Christophorou

1982-01-01

431

On the Nonsymmetric Purely Affine Gravity  

NASA Astrophysics Data System (ADS)

We review the vacuum purely affine gravity with the nonsymmetric connection and metric. We also examine dynamical effects of the second Ricci tensor and covariant second-rank tensors constructed from the torsion tensor in the gravitational Lagrangian.

Pop?awski, Nikodem J.

432

Affinity Bioadhesives Engineered for Cell Culture Applications.  

National Technical Information Service (NTIS)

The objective of the program is to develop, through genetic engineering technology, affinity bioadhesives to be used in applications requiring specific eucaryotic cell attachment. A series of recombinant adhesive proteins, based on the polyphenolic protei...

R. P. Link

1990-01-01

433

Current test results for the Athena radar responsive tag  

NASA Astrophysics Data System (ADS)

Sandia National Laboratories has teamed with General Atomics and Sierra Monolithics to develop the Athena tag for the Army's Radar Tag Engagement (RaTE) program. The radar-responsive Athena tag can be used for Blue Force tracking and Combat Identification (CID) as well as data collection, identification, and geolocation applications. The Athena tag is small (~4.5" x 2.4" x 4.2"), battery-powered, and has an integral antenna. Once remotely activated by a Synthetic Aperture Radar (SAR) or Moving Target Indicator (MTI) radar, the tag transponds modulated pulses to the radar at a low transmit power. The Athena tag can operate Ku-band and X-band airborne SAR and MTI radars. This paper presents results from current tag development testing activities. Topics covered include recent field tests results from the AN/APY-8 Lynx, F16/APG-66, and F15E/APG-63 V(1) radars and other Fire Control radars. Results show that the Athena tag successfully works with multiple radar platforms, in multiple radar modes, and for multiple applications. Radar-responsive tags such as Athena have numerous applications in military and government arenas. Military applications include battlefield situational awareness, combat identification, targeting, personnel recovery, and unattended ground sensors. Government applications exist in nonproliferation, counter-drug, search-and-rescue, and land-mapping activities.

Ormesher, Richard C.; Martinez, Ana; Plummer, Kenneth W.; Erlandson, David; Delaware, Sheri; Clark, David R.

2006-06-01

434

Stereospecific analysis of TAG from sunflower seed oil  

Microsoft Academic Search

Stereospecific analysis of TAG from a sunflower seed oil of Tunisian origin was performed. The TAG were first fractionated\\u000a according to chain length and degree of unsaturation by RP-HPLC. The four major diacid- and triacid-TAG fractions were palmitoyldilinoleoyl-glycerol,\\u000a dioleoyllinoleoylglycerol, oleoyldilinoleoylglycerol, and palmitoyloleoyl-linoleoyl-glycerol, amounting to 7.2, 16.6, 29.5,\\u000a and 12 mol%, respectively. The TAG of the four fractions were individually submitted

Sadok Boukhchina; Joseph Gresti; Habib Kallel; Jean Bézard

2003-01-01

435

Remote object authentication using distortion-invariant ID tags  

NASA Astrophysics Data System (ADS)

A number of applications in security or inventory control may benefit from an authentication system able to identify a remote object viewed from different perspectives or distances. Object identification can be accomplished by using optical ID tags, which include relevant information of the target and are located on a visible part of the object under surveillance. Encryption of the information codified in the ID tag allows increasing security and deters from unauthorized usage of optical tags. The identification process encompasses several steps such as detection, information decoding and verification which are all detailed in this work. Design of distortion-invariant ID tags has to be taken into account to achieve a correct object authentication even if the ID tag is detected and captured at different distances (i.e. different scales) or from different views (i.e. rotated versions of the original ID tag). Description of diverse distortion-invariant ID tags and authentication results using the proposed ID tags are provided. We show that distortion-tolerance is achieved by the described identification system. Information encrypted on the tested ID tags is correctly decoded and verified even if variations in scale and rotations are considered. The effects of environmental degradation are taken into account in the recognition process.

Perez-Cabre, Elisabet; Millan, Maria S.; Javidi, Bahram

2005-09-01

436

SNPPicker: High quality tag SNP selection across multiple populations  

PubMed Central

Background Linkage Disequilibrium (LD) bin-tagging algorithms identify a reduced set of tag SNPs that can capture the genetic variation in a population without genotyping every single SNP. However, existing tag SNP selection algorithms for designing custom genotyping panels do not take into account all platform dependent factors affecting the likelihood of a tag SNP to be successfully genotyped and many of the constraints that can be imposed by the user. Results SNPPicker optimizes the selection of tag SNPs from common bin-tagging programs to design custom genotyping panels. The application uses a multi-step search strategy in combination with a statistical model to maximize the genotyping success of the selected tag SNPs. User preference toward functional SNPs can also be taken into account as secondary criteria. SNPPicker can also optimize tag SNP selection for a panel tagging multiple populations. SNPPicker can optimize custom genotyping panels including all the assay-specific constraints of Illumina's GoldenGate and Infinium assays. Conclusions A new application has been developed to maximize the success of custom multi-population genotyping panels. SNPPicker also takes into account user constraints including options for controlling runtime. Perl Scripts, Java source code and executables are available under an open source license for download at http://mayoresearch.mayo.edu/mayo/research/biostat/software.cfm

2011-01-01

437

TORSORS OVER THE PUNCTURED AFFINE LINE  

Microsoft Academic Search

We classify G-torsors over the punctured affine line Spec( k(t±)) where G is a reductive algebraic group defined over a field k of good char- acteristic. Our classification is in terms of the Galois cohomology of the complete field k((t)) with values in G. Keywords: Linear algebraic group, group scheme, torsor, punctured affine line, non-abelian cohomology. MSC 2000 11E72, 14L30,

V. CHERNOUSOV; P. GILLE; A. PIANZOLA

438

Electron affinity of the HC2 radical  

Microsoft Academic Search

Large scale abinitio self-consistent field and configuration–interaction calculations were carried out to evaluate the electron affinity of the HC2 radical. The adiabatic electron affinity is calculated to be 2.14, the vertical 2.09, and the vertical detachment energy 2.17 eV. Corrections for the zero-point vibrational energy amount to less than 0.01 eV. From these values, the heat of formation of HC2,

K. Vasudevan; F. Grein

1978-01-01

439

Hairy Root-activation Tagging: a High-throughput System for Activation Tagging in Transformed Hairy Roots  

Microsoft Academic Search

Activation tagging is a powerful technique for generating gain-of-function mutants in plants. We developed a new vector system for activation tagging of genes in “transformed hairy roots”. The binary vector pHR-AT (Hairy Root-Activation Tagging) and its derivative pHR-AT-GFP contain a cluster of rol (rooting locus) genes together with the right border facing four tandem repeats of the cauliflower mosaic virus

Hikaru Seki; Tomoko Nishizawa; Nobukazu Tanaka; Yasuo Niwa; Shigeo Yoshida; Toshiya Muranaka

2005-01-01

440

Affinity Purification and Characterization of a G-Protein Coupled Receptor, Saccharomyces cerevisiae Ste2p  

SciTech Connect

We present a rare example of a biologically active G protein coupled receptor (GPCR) whose purity and identity were verified by mass spectrometry after being purified to near homogeneity from its native system. An overexpression vector was constructed to encode the Saccharomyces cerevisiae GPCR -factor receptor (Ste2p, the STE2 gene product) containing a 9-amino acid sequence of rhodopsin that served as an epitope/affinity tag. In the construct, two glycosylation sites and two cysteine residues were removed to aid future structural and functional studies. The receptor was expressed in yeast cells and was detected as a single band in a western blot indicating the absence of glycosylation. Tests of the epitope-tagged, mutated receptor showed it maintained its full biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5 % n-dodecyl maltoside (DM). Approximately 120 g of purified -factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (Kd) of the purified -factor receptor in DM micelles was 28 nM as compared to Kd = 12.7 nM for Ste2p in cell membranes, and approximately 40 % of the purified receptor was correctly folded as judged by ligand saturation binding. About 50 % of the receptor sequence was retrieved from MALDITOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the -factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.

Lee, Byung-Kwon [University of Tennessee, Knoxville (UTK); Jung, Kyung-Sik [University of Tennessee, Knoxville (UTK); Son, Cagdas D [ORNL; Kim, Heejung [University of Tennessee, Knoxville (UTK); Verberkmoes, Nathan C [ORNL; Arshava, Boris [College of Staten Island; Naider, Fred [College of Staten Island; Becker, Jeffrey Marvin [ORNL

2007-01-01

441

The affinity purification and characterization of ATP synthase complexes from mitochondria.  

PubMed

The mitochondrial F?-ATPase inhibitor protein, IF?, inhibits the hydrolytic, but not the synthetic activity of the F-ATP synthase, and requires the hydrolysis of ATP to form the inhibited complex. In this complex, the ?-helical inhibitory region of the bound IF? occupies a deep cleft in one of the three catalytic interfaces of the enzyme. Its N-terminal region penetrates into the central aqueous cavity of the enzyme and interacts with the ?-subunit in the enzyme's rotor. The intricacy of forming this complex and the binding mode of the inhibitor endow IF? with high specificity. This property has been exploited in the development of a highly selective affinity procedure for purifying the intact F-ATP synthase complex from mitochondria in a single chromatographic step by using inhibitor proteins with a C-terminal affinity tag. The inhibited complex was recovered with residues 1-60 of bovine IF? with a C-terminal green fluorescent protein followed by a His-tag, and the active enzyme with the same inhibitor with a C-terminal glutathione-S-transferase domain. The wide applicability of the procedure has been demonstrated by purifying the enzyme complex from bovine, ovine, porcine and yeast mitochondria. The subunit compositions of these complexes have been characterized. The catalytic properties of the bovine enzyme have been studied in detail. Its hydrolytic activity is sensitive to inhibition by oligomycin, and the enzyme is capable of synthesizing ATP in vesicles in which the proton-motive force is generated from light by bacteriorhodopsin. The coupled enzyme has been compared by limited trypsinolysis with uncoupled enzyme prepared by affinity chromatography. In the uncoupled enzyme, subunits of the enzyme's stator are degraded more rapidly than in the coupled enzyme, indicating that uncoupling involves significant structural changes in the stator region. PMID:23407638

Runswick, Michael J; Bason, John V; Montgomery, Martin G; Robinson, Graham C; Fearnley, Ian M; Walker, John E

2013-02-13

442

Chemistry meets proteomics: the use of chemical tagging reactions for MS-based proteomics.  

PubMed

As proteomics matures from a purely descriptive to a function-oriented discipline of the life sciences, there is strong demand for novel methodologies that increase the depth of information that can be obtained from proteomic studies. MS has long played a central role for protein identification and characterization, often in combination with dedicated chemical modification reactions. Today, chemistry is helping to advance the field of proteomics in numerous ways. In this review, we focus on those methodologies that have a significant impact for the large-scale study of proteins and peptides. This includes approaches that allow the introduction of affinity tags for the enrichment of subclasses of peptides or proteins and strategies for in vitro stable isotope labeling for quantification purposes, among others. Particular attention is given to the study of PTMs where recent advancements have been promising, but many interesting targets are not yet being addressed. PMID:16972287

Leitner, Alexander; Lindner, Wolfgang

2006-10-01

443

Phos-tag beads as an immunoblotting enhancer for selective detection of phosphoproteins in cell lysates.  

PubMed

The low specificity of anti-phosphoprotein antibodies is often a problem in immunoblotting analyses. We introduce a simple pretreatment procedure for cell lysates to give more specific detection of phosphoproteins in immunoblotting. Cellular phosphoproteins were preferentially trapped on Phos-tag agarose phosphate-affinity beads in a homemade spin-centrifuge microtube unit, and nonphosphorylated proteins were excluded in the filtrate. The phosphoprotein-bound beads suspended in a sample-loading dye solution were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western blotting. We demonstrated improved detection of phosphorylated Shc and mitogen-activated protein kinase isoforms in A431 cell lysates by this new technique. PMID:19318084

Kinoshita-Kikuta, Emiko; Kinoshita, Eiji; Koike, Tohru

2009-03-24

444

Molecular cloning, purification and functional implications of recombinant GST tagged hGMCSF cytokine.  

PubMed

We report the cloning, purification and cell proliferative activity of a novel recombinant GST tagged human granulocyte macrophage colony stimulating factor (GST-hGMCSF). The hGMCSF gene was PCR amplified from the cDNA of ACHN renal carcinoma cells and was cloned into the bacterial expression vector. The GST-hGMCSF was purified to homogeneity using glutathione agarose affinity chromatography and subsequently characterized by Western blot, circular dichroism (CD) and MALDI TOF-TOF analysis. Homology modelling studies revealed the possible binding domains of the recombinant cytokine with cognate receptor. The proliferation of THP-1, Raw 264.7, MCF-7 and U87MG cells upon GST-hGMCSF addition was found to be dose dependent. Hence, this functionally active recombinant cytokine has potential application in cancer therapy for stimulating facile growth recovery of normal cell population. PMID:23334834

Chaubey, Nidhi; Ghosh, Siddhartha Sankar

2013-01-20

445

IgG purification using affinity filtration with sulfamethazine-affinity carriers.  

PubMed

Immunoglobulin G (IgG) antibodies are used extensively for analytical, diagnostic, and therapeutic applications. However, there are some disadvantages to purify IgG antibodies by protein A and G affinity chromatography. Therefore, it is necessary to find an effective alternative and nonchromatographic method to purify IgG. Dextran microparticles were activated and coupled with sulfamethazine to form sulfamethazine-affinity carriers. Then the carriers were used to purify IgG by affinity filtration. Quantitative and qualitative determination proved that sulfamethazine would successfully bond to the surface of dextran microparticles with a density of 85.5 ?mol/g (wet). Affinity carriers were proved to withstand high shear force and reveal rare sulfamethazine leakage under filtration conditions between pH 3 to 11. The maximum IgG-binding capacity of affinity carriers was 8.03 mg IgG/g (wet). The affinity filtration process obtained a recovery yield above 80% and purity above 90%. Thus, this work involved in both the advantages of membrane filtration and affinity purification. The results, for the first time, proved that it is possible to use the small ligand sulfamethazine for affinity filtration of IgG. It is an attractive alternative to conventional protein A or G affinity chromatography. PMID:23030470

Yi, Yu; Zhu, Li; Mei, Jianfeng; Chen, Jianshu; Ying, Guoqing

2012-01-01

446

An Examination of the Effectiveness of Social Tagging for Resource Discovery  

Microsoft Academic Search

Social tagging allows users to assign keywords (tags) to resources facilitating their future access by the tag creator, and possibly by other users. In terms of its support for resource discovery, social tagging has both proponents and critics. The goal of this paper investigates if tags are an effective means for helping users locate useful resources. Adopting techniques from text

Dion Hoe-Lian Goh; Chei Sian Lee; Alton Y. K. Chua; Khasfariyati Razikin

2008-01-01

447

@toread and Cool: Subjec- tive, Affective and Associa- tive Factors in Tagging  

Microsoft Academic Search

This paper examines the use of non subject related tags in social bookmarking tools. Previous studies of tag- ging determined that many common tags are not directly subject related but are in fact affective tags dwelling on a user's emotional response to a document or are time and task related tags related to a users current projects or ac- tivities.

Margaret E. I. Kipp

448

The loss rates of web tags applied to day-old Anas and Aythya ducklings  

USGS Publications Warehouse

Researchers studied the loss rate of web tags on Anas and Aythya ducklings by double marking day-old ducklings of five species with web tags and plasticine-filled rings. Tag loss was examined over three-month, one-year, and three-year periods. Web tag loss was greatest for Anas and occurred mostly in the first three months following tagging.

Blums, P.; Mednis, A.; Bauga, I.; Nichols, J.D.; Hines, J.E.

1997-01-01

449

Design of an UHF RFID tag antenna for paper money management system  

Microsoft Academic Search

In this paper, an UHF dipole tag antenna with a short stub and meander lines is proposed for RFID-based paper money management system. The short stub and meander lines in tag antenna are used to improve the impedance matching between tag antenna and tag IC. The proposed RFID tag antenna can be used effectively to band wads of paper money

Taeik Kim; Uisheon Kim; Gyubong Jung; Jaehoon Choi

2009-01-01

450

Effect of T-Bar Anchor Tags on Growth of Black Crappies  

Microsoft Academic Search

T-bar anchor tags provide a means for identifying individual fish in tagging experiments. Despite prior use, no information is available regarding the effect of these tags on the growth of black crappies Pomoxis nigromaculatus. We compared the growth of black crappies larger than 180 mm total length that were tagged with t-bar anchor tags with the growth of untagged members

Bradford G. Parsons; Jeffrey R. Reed

2005-01-01

451

Gas-phase nitronium ion affinities.  

PubMed Central

Evaluation of nitronium ion-transfer equilibria, L1NO2+ + L2 = L2NO2+ + L1 (where L1 and L2 are ligands 1 and 2, respectively) by Fourier-transform ion cyclotron resonance mass spectrometry and application of the kinetic method, based on the metastable fragmentation of L1(NO2+)L2 nitronium ion-bound dimers led to a scale of relative gas-phase nitronium ion affinities. This scale, calibrated to a recent literature value for the NO2+ affinity of water, led for 18 ligands, including methanol, ammonia, representative ketones, nitriles, and nitroalkanes, to absolute NO2+ affinities, that fit a reasonably linear general correlation when plotted vs. the corresponding proton affinities (PAs). The slope of the plot depends to a certain extent on the specific nature of the ligands and, hence, the correlations between the NO2+ affinities, and the PAs of a given class of compounds display a better linearity than the general correlation and may afford a useful tool for predicting the NO2+ affinity of a molecule based on its PA. The NO2+ binding energies are considerably lower than the corresponding PAs and well below the binding energies of related polyatomic cations, such as NO+, a trend consistent with the available theoretical results on the structure and the stability of simple NO2+ complexes. The present study reports an example of extension of the kinetic method to dimers, such as L1(NO2+)L2, bound by polyatomic ions, which may considerably widen its scope. Finally, measurement of the NO2+ affinity of ammonia allowed evaluation of the otherwise inaccessible PA of the amino group of nitramide and, hence, direct experimental verification of previous theoretical estimates.

Cacace, F; de Petris, G; Pepi, F; Angelelli, F

1995-01-01

452

Generation and analysis of recombinant Bunyamwera orthobunyaviruses expressing V5 epitope-tagged L proteins.  

PubMed

The L protein of Bunyamwera virus (BUNV; family Bunyaviridae) is an RNA-dependent RNA polymerase, 2238 aa in length, that catalyses transcription and replication of the negative-sense, tripartite RNA genome. To learn more about the molecular interactions of the L protein and to monitor its intracellular distribution we inserted a 14 aa V5 epitope derived from parainfluenza virus type 5, against which high-affinity antibodies are available, into different regions of the protein. Insertion of the epitope at positions 1935 or 2046 resulted in recombinant L proteins that retained functionality in a minireplicon assay. Two viable recombinant viruses, rBUNL4V5 and rBUNL5V5, expressing the tagged L protein were rescued by reverse genetics, and characterized with respect to their plaque size, growth kinetics and protein synthesis profile. The recombinant viruses behaved similarly to wild-type (wt) BUNV in BHK-21 cells, but formed smaller plaques and grew to lower titres in Vero E6 cells compared with wt BUNV. Immunofluorescent staining of infected cells showed the L protein to have a punctate to reticular distribution in the cytoplasm, and cell fractionation studies indicated that the L protein was present in both soluble and microsomal fractions. Co-immunoprecipitation and confocal microscopic assays confirmed an interaction between BUNV L and N proteins. The recombinant viruses expressing tagged L protein will be highly valuable reagents for the detailed dissection of the role of the BUNV L protein in virus replication. PMID:19141438

Shi, Xiaohong; Elliott, Richard M

2009-02-01

453

A novel method for high-level production of TEV protease by superfolder GFP tag.  

PubMed

Because of its stringent sequence specificity, tobacco etch virus (TEV) protease is widely used to remove fusion tags from recombinant proteins. Due to the poor solubility of TEV protease, many strategies have been employed to increase the expression level of this enzyme. In our work, we introduced a novel method to produce TEV protease by using visible superfolder green fluorescent protein (sfGFP) as the fusion tag. The soluble production and catalytic activity of six variants of sfGFP-TEV was examined, and then the best variant was selected for large-scale production. After purified by Ni-NTA affinity chromatography and Q anion exchange chromatography, the best variant of sfGFP-TEV fusion protease was obtained with purity of over 98% and yield of over 320 mg per liter culture. The sfGFP-TEV had a similar catalytic activity to that of the original TEV protease. Our research showed a novel method of large-scale production of visible and functional TEV protease for structural genomics research and other applications. PMID:20182554

Wu, Xudong; Wu, Di; Lu, Zhisheng; Chen, Wentao; Hu, Xiaojian; Ding, Yu

2010-02-23

454

Development of stereoscopic molecular tagging velocimetry  

NASA Astrophysics Data System (ADS)

This paper describes the development of a stereoscopic molecular tagging velocimetry (sMTV) technique for measuring the instantaneous three-component velocity field over a plane in a flow. The calibration technique employed allows the stereo imaging parameters to be determined directly from a target image without the direct physical measurement of those parameters. Effects of index of refraction variations are accounted for approximately in the calibration method. Sensitivity analysis and target test experiments for a typical optical setup indicate maximum error levels for the in-plane components to be nominally the same as two-component MTV measurements. The error in the out-of-plane velocity component can be one to three times larger, depending on the stereo viewing angle. Experimental results are presented to validate the sMTV technique against other known measured results, along with an application to the highly three-dimensional flow field near the tip of a model propeller.

Bohl, D. G.; Koochesfahani, M. M.; Olson, B. J.

455

Reliable Food Traceability Using RFID Tagging  

NASA Astrophysics Data System (ADS)

Radio Frequency IDentification (RFID) technology has numerous potential applications in various industries. One important use is for complete traceability of a specific product with the added advantage of being able to verify that quality controls have been passed, with all the necessary steps complied with and for the time required. The aim of this work is to present a food traceability system using RFID tags with contents guaranteed secure by the use of public-key cryptography and at an affordable cost without the need for substantial investment in infrastructure. Aggregate signatures are used so that all the steps can be signed in a reduced memory space. This type of signature is a cryptographic primitive that "consolidates" several signatures into one in such a way that if n users sign n messages, all the signatures can be grouped into one single signature.

Azuara, Guillermo; Salazar, José L.; Tornos, José L.; Piles, Joan J.

456

Tandem affinity purification tagging of fatty acid biosynthetic enzymes in Synechocystis sp. PCC6803 and Arabidopsis thaliana  

Microsoft Academic Search

De novo fatty acid synthesis in plants occurs primarily in the plastids and is catalysed by a type-II fatty acid synthase (FAS) in which separate enzymes catalyse sequential reactions. Genes encoding all of the plant FAS components have been identified, following en- zyme purification or by homology to Escherichia coli genes, and the structure of a number of the individual

Adrian P. Brown; Valerie Affleck; Tony Fawcett; Antoni R. Slabas

2006-01-01

457

Purification of E. coli-expressed HIS-tagged hepatitis B core antigen by Ni 2+-chelate affinity chromatography  

Microsoft Academic Search

Hepatitis B virus is a major cause of human liver disease. In the case of chronic infection the virus can lead to liver cancer and cirrhosis. The virion consists of an outer envelope containing lipids of the endoplasmic reticulum and virally-encoded surface proteins. This lipoprotein shell encloses the nucleocapsid or core antigen (HBcAg), which contains the viral genome. The capsid

H. Wizemann; A. von Brunn

1999-01-01

458

ONE-STEP METAL-AFFINITY PURIFICATION OF HISTIDINE-TAGGED PROTEINS BY TEMPERATURE-TRIGGERED PRECIPITATION. (R829606)  

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

459

Collaborative Filtering Recommender Systems based on Popular Tags  

Microsoft Academic Search

The social tags in web 2.0 are becoming another important information source to profile users' interests and preferences for making personalized recommendations. However, the uncontrolled vocabulary causes a lot of problems to profile users accurately, such as ambiguity, synonyms, misspelling, low information sharing etc. To solve these problems, this paper proposes to use popular tags to represent the actual topics

Huizhi Liang; Yue Xu; Yuefeng Li; Richi Nayak

460

Paramagnetic tagging of diamagnetic proteins for solution NMR.  

PubMed

In this article, approaches towards the paramagnetic tagging of diamagnetic proteins are reviewed. Alignment can be achieved by adding paramagnetic fusion proteins or peptides to the C- or the N-terminus or by attaching paramagnetic tags to Cystein residues. Applications for the study of homodimer structures and protein/ligand interactions, as well as protein domain dynamics, are reviewed. PMID:16921533

Rodriguez-Castañeda, Fernando; Haberz, Peter; Leonov, Andrei; Griesinger, Christian

2006-07-01