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Sample records for isotope-coded affinity tags

  1. Fluorescent isotope-coded affinity tag 2: peptide labeling and affinity capture.

    PubMed

    Rivera-Monroy, Zuly; Bonn, Guenther K; Guttman, Andrs

    2009-04-01

    Fluorescent isotope-coded affinity tag (FCAT) is a novel reagent to label cysteine-containing peptides. The fluorescein group on the tag enables absolute quantification by fluorescence detection, also supporting affinity capture of the labeled peptides. In this paper we report the synthesis of the heavy isotopic form of the FCAT reagent and its use in labeling tryptic peptides. The heavy form of the reagent exhibited the same reactivity and chromatographic behavior that of the light version. Effective labeling of tryptic peptides from alpha-lactalbumin, fetuin, BSA and phosphorylase b was attained by using both the heavy and light FCAT tags. Selective capture of the FCAT-labeled peptides was easily performed by pipette tips containing either anti-FITC antibody or iminodiacetic-acid-coated beads. The differently labeled peptides were separated by RP HPLC and analyzed by MALDI-TOF MS. PMID:19288590

  2. Mass spectrometry in coupling with affinity capture-release and isotope-coded affinity tags for quantitative protein analysis.

    PubMed

    Turecek, Frantisek

    2002-01-01

    Affinity capture-release electrospray ionization mass spectrometry (ACESIMS) and isotope-coded affinity tags (ICAT) are two recently introduced techniques for the quantitation of protein activity and content with applications to clinical enzymology and functional proteomics, respectively. One common feature of these methods is that they use biotinylated tags that function as molecular handles for highly selective and reversible affinity capture of conjugates from complex biological mixtures such as cell homogenates and sub-cellular organelles. ACESIMS uses synthetic substrate conjugates specifically to target cellular enzymes that, when deficient, are the cause of genetic diseases. Multiplex determination of enzyme activities is used for the diagnosis of lysosomal storage diseases. The ICAT method relies on selective conjugation of cysteine thiol groups in proteins, followed by enzymatic digestion and quantitative analysis of peptide conjugates by mass spectrometry. Another common feature of the ACESIMS and ICAT approaches is that both use conjugates labeled with stable heavy isotopes as internal standards for quantitation. Selected applications of the ACESIMS and ICAT techniques are presented that include molecular-level diagnosis of genetic diseases in children and quantitative determination of protein expression in cells. PMID:11813306

  3. Proteolytic Affinity Tag Cleavage.

    PubMed

    Block, Helena; Maertens, Barbara; Spriestersbach, Anne; Kubicek, Jan; Schfer, Frank

    2015-01-01

    Here, we present protocols describing the use of the dipeptidyl-aminopeptidase-1 (DPP1, DAPase) exoprotease-based TAGZyme system and the endoprotease, Factor Xa. Both enable the recovery of proteins free of any amino acids encoded by the vector and/or protease recognition site. They also provide the possibility of removing the proteases from the preparation of the target protein by a simple subtractive chromatography step. TAGZyme enzymes contain an uncleavable His tag for removal by Immobilized Metal Ion Affinity Chromatography (IMAC). Factor Xa can be removed using Xa Removal Resin. PMID:26096504

  4. Overview of affinity tags for protein purification.

    PubMed

    Kimple, Michelle E; Brill, Allison L; Pasker, Renee L

    2013-01-01

    Addition of an affinity tag is a useful method for differentiating recombinant proteins expressed in bacterial and eukaryotic expression systems from the background of total cellular proteins, as well as for detecting protein-protein interactions. This overview describes the historical basis for the development of affinity tags, affinity tags that are commonly used today, how to choose an appropriate affinity tag for a particular purpose, and several recently developed affinity tag technologies that may prove useful in the near future. PMID:24510596

  5. Overview of affinity tags for protein purification.

    PubMed

    Kimple, Michelle E; Sondek, John

    2004-09-01

    Addition of an affinity tag is a useful method for differentiating recombinant proteins expressed in bacterial and eukaryotic expression systems from the background of total cellular proteins, and for detecting protein-protein interactions. This overview describes the historical basis for the development of affinity tags, affinity tags that are commonly used today, how to choose an appropriate affinity tag for a particular purpose, and several recently developed affinity tag technologies that may prove useful in the near future. PMID:18429272

  6. Mapping peptide thiol accessibility in membranes using a quaternary ammonium isotope-coded mass tag (ICMT)

    PubMed Central

    Su, Chiao-Yung; London, Erwin; Sampson, Nicole S.

    2013-01-01

    The plasma membrane contains a diverse array of proteins, including receptors, channels, and signaling complexes, that serve as decision-making centers. Investigation of membrane protein topology is important for understanding the function of these types of protein. Here, we report a method to determine protein topology in the membrane that utilizes labeling of cysteine with isotope-coded mass tags. The mass tags contain a thiol reactive moiety, linker, and a quaternary ammonium group to aid ionization in the mass spectrometer and were synthesizes as both light and heavy (deuterated) forms. The probes were found to be membrane impermeable when applied to lipid vesicles. To assess the utility of the probes for mapping peptide thiol topology, we employed a two-step labeling procedure. Vesicles containing ?-helical transmembrane peptides were labeled with heavy (or light) probe, solubilized by detergent, and then labeled by an excess of the complementary probe. Peptide for which the cysteine was oriented in the center of the lipid bilayer was not labeled until the lipid vesicles were lysed with detergent, consistent with the membrane impermeability of the probes and reduced ionization of the thiol in the hydrophobic membrane. Peptide for which the cysteine was positioned in the head group zone of the lipid bilayer was labeled rapidly. Peptide for which the cysteine was positioned below the head group abutting the hydrocarbon region was labeled at a reduced rate compared to the fully accessible cysteine. Moreover, the effect of lipid bilayer structure on the kinetics of peptide and lipid flipping in the bilayer was readily measured with our two-step labeling method. The small sample size required, the ease and rapidity of sample preparation, and the amenability of MALDI-TOF mass spectral to analysis in the presence of lipids will enable future facile investigation of membrane proteins in a cellular context. PMID:23725486

  7. Affinity tags in protein purification and peptide enrichment: an overview.

    PubMed

    Pina, Ana Sofia; Batalha, Iris L; Roque, Ana Cecília A

    2014-01-01

    The reversible interaction between an affinity ligand and a complementary receptor has been widely explored in purification systems for several biomolecules. The development of tailored affinity ligands highly specific towards particular target biomolecules is one of the options in affinity purification systems. However, both genetic and chemical modifications on proteins and peptides widen the application of affinity ligand-tag receptor pairs towards universal capture and purification strategies. In particular, this chapter will focus on two case studies highly relevant for biotechnology and biomedical areas, namely, the affinity tags and receptors employed on the production of recombinant fusion proteins and the chemical modification of phosphate groups on proteins and peptides and the subsequent specific capture and enrichment, a mandatory step before further proteomic analysis. PMID:24648075

  8. Orthogonal protein purification facilitated by a small bispecific affinity tag.

    PubMed

    Nilvebrant, Johan; Alm, Tove; Hober, Sophia

    2012-01-01

    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

  9. Affinity Purification of Protein Complexes Using TAP Tags

    PubMed Central

    Gerace, Erica; Moazed, Danesh

    2016-01-01

    This protocol is used for the isolation and analysis of protein complexes using the tandem affinity purification (TAP) tag system. The protocol describes the purification of a protein fused to a TAP tag comprised of two protein A domains and the calmodulin binding peptide separated by a TEV cleavage site. This is a powerful technique for rapid purification of protein complexes and the analysis of their stoichiometric composition, posttranslational modifications, structure, and functional activities. PMID:26096502

  10. Commonly used tag combinations for tandem affinity purification.

    PubMed

    Li, Yifeng

    2010-02-01

    TAP (tandem affinity purification) allows rapid and clean isolation of a tagged protein along with its interacting partners from cell lysates. Initially developed in yeast, the TAP method has subsequently been adapted to other cells and organisms. In combination with MS analysis, this method has become an indispensable tool for systematic identification of target-associated protein complexes. The key feature of TAP is the use of a dual-affinity tag, which is fused to the protein of interest. The original TAP tag consisted of two IgG-binding units of Protein A of Staphylococcus aureus and the calmodulin-binding peptide. As the technique has been widely exploited, a number of alternative TAP tags based on other affinity handles have been developed. The present review gives an overview of the various tag combinations for TAP with a highlight on those alternatives that result in improved yields or unique features. The information provided should assist in the selection and development of TAP tags for specific applications. PMID:20156193

  11. Protein Affinity Purification using Intein/Chitin Binding Protein Tags.

    PubMed

    Mitchell, Sarah F; Lorsch, Jon R

    2015-01-01

    Isolation of highly purified recombinant protein is essential for a wide range of biochemical and biophysical assays. Affinity purification in which a tag is fused to the desired protein and then specifically bound to an affinity column is a widely used method for obtaining protein of high purity. Many of these methods have the drawbacks of either leaving the recombinant tag attached to the protein or requiring the addition of a protease which then must be removed by further chromatographic steps. The fusion of a self-cleaving intein sequence followed by a chitin-binding domain (CBD) allows for one-step chromatographic purification of an untagged protein through the thiol-catalyzed cleavage of the intein sequence from the desired protein. The affinity purification is highly specific and can yield pure protein without any undesired N- or C-terminal extensions. This protocol is based on the IMPACT-System (intein mediated purification with an affinity chitin-binding tag) marketed by New England Biolabs. PMID:26096506

  12. Recombinant protein purification using complementary peptides as affinity tags.

    PubMed

    Martnez-Ceron, Mara C; Targovnik, Alexandra M; Urtasun, Nicols; Cascone, Osvaldo; Miranda, Mara V; Camperi, Silvia A

    2012-01-15

    Affinity tags have become highly popular tools for purifying recombinant proteins from crude extracts by affinity chromatography. Besides, short peptides are excellent ligands for affinity chromatography, as they are not likely to cause an immune response in case of leakage into the product, they are more stable than antibodies to elution and cleaning conditions and they usually have very acceptable selectivity. Hydropathically complementary peptides designed de novo show enough selectivity to be used successfully as peptide ligands for protein purification from crude extracts. Recognition specificity and selectivity in the interaction between the complementary peptide pair His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu and Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe have been demonstrated by other authors. In this work, we designed a recombinant protein purification method using a peptide affinity tag that binds to a peptide-binding partner immobilized on a chromatographic matrix. The enhanced green fluorescent protein expressed (EGFP) in Escherichia coli was used as the model. The peptide Gly-Gly-Gly-His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu was synthesized by solid phase using the Fmoc chemistry and immobilized in NHS-Sepharose (PC-Sepharose). Gly residues were added as a spacer arm at the N terminus. The EGFP was expressed either with the fusion tag Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe on the C terminus (EGFP-CPTag) or without any fusion tag. After cell disruption, the extract was directly applied to the PC-Sepharose column equilibrated with 20mM sodium phosphate buffer, pH 7.0. The adsorbed EGFP-CPTag was then eluted with 1M Tris. The yield was 98% and the purification factor 4.6. By contrast, EGFP without tag pass through without interacting with the PC-Sepharose column. The method designed can be applied for the purification of other recombinant proteins. PMID:21664994

  13. A metal-coded affinity tag approach to quantitative proteomics.

    PubMed

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

    2007-11-01

    The quantitative analysis of protein mixtures is pivotal for the understanding of variations in the proteome of living systems. Therefore, approaches have been recently devised that generally allow the relative quantitative analysis of peptides and proteins. Here we present proof of concept of the new metal-coded affinity tag (MeCAT) technique, which allowed the quantitative determination of peptides and proteins. A macrocyclic metal chelate complex (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)) loaded with different lanthanides (metal(III) ions) was the essential part of the tag. The combination of DOTA with an affinity anchor for purification and a reactive group for reaction with amino acids constituted a reagent that allowed quantification of peptides and proteins in an absolute fashion. For the quantitative determination, the tagged peptides and proteins were analyzed using flow injection inductively coupled plasma MS, a technique that allowed detection of metals with high precision and low detection limits. The metal chelate complexes were attached to the cysteine residues, and the course of the labeling reaction was followed using SDS-PAGE and MALDI-TOF MS, ESI MS, and inductively coupled plasma MS. To limit the width in isotopic signal spread and to increase the sensitivity for ESI analysis, we used the monoisotopic lanthanide macrocycle complexes. Peptides tagged with the reagent loaded with different metals coelute in liquid chromatography. In first applications with proteins, the calculated detection limit for bovine serum albumin for example was 110 amol, and we have used MeCAT to analyze proteins of the Sus scrofa eye lens as a model system. These data showed that MeCAT allowed quantification not only of peptides but also of proteins in an absolute fashion at low concentrations and in complex mixtures. PMID:17627934

  14. Purification of a Recombinant Polyhistidine-Tagged Glucosyltransferase Using Immobilized Metal-Affinity Chromatography (IMAC).

    PubMed

    de Costa, Fernanda; Barber, Carla J S; Pujara, Pareshkumar T; Reed, Darwin W; Covello, Patrick S

    2016-01-01

    Short peptide tags genetically fused to recombinant proteins have been widely used to facilitate detection or purification without the need to develop specific procedures. In general, an ideal affinity tag would allow the efficient purification of tagged proteins in high yield, without affecting its function. Here, we describe the purification steps to purify a recombinant polyhistidine-tagged glucosyltransferase from Centella asiatica using immobilized metal affinity chromatography. PMID:26843168

  15. PROTEIN-PROTEIN INTERACTIONS OF TANDEM AFFINITY PURIFICATION (TAP)-TAGGED PROTEIN KINASES IN RICE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Forty-one rice cDNAs encoding protein kinases were fused to the tandem affinity purification (TAP) tag and expressed in transgenic rice plants. The TAP-tagged kinases and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by mass spectrometry. Ninet...

  16. N-terminal processing of affinity-tagged recombinant proteins purified by IMAC procedures.

    PubMed

    Mooney, Jane T; Fredericks, Dale P; Christensen, Thorkild; Bruun Schidt, Christine; Hearn, Milton T W

    2015-07-01

    The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S-transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine-containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7-triazacyclononane (tacn). The use of this tag-tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli-expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP-1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI-TOF MS analysis of the cleaved products from the DAP-1 digestion of the recombinant N-terminally tagged proteins confirmed the complete removal of the tag within 4-12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn-based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli-expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes. PMID:25727088

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

    SciTech Connect

    Giannone, Richard J; McDonald, W Hayes; Hurst, Gregory {Greg} B; Huang, Ying; Wu, Jun; Liu, Yie; Wang, Yisong

    2007-01-01

    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.

  18. An overview of enzymatic reagents for the removal of affinity tags.

    PubMed

    Waugh, David S

    2011-12-01

    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. PMID:21871965

  19. Batch affinity adsorption of His-tagged proteins with EDTA-based chitosan.

    PubMed

    Hua, Weiwei; Lou, Yimin; Xu, Weiyuan; Cheng, Zhixian; Gong, Xingwen; Huang, Jianying

    2016-01-01

    Affinity adsorption purification of hexahistidine-tagged (His-tagged) proteins using EDTA-chitosan-based adsorption was designed and carried out. Chitosan was elaborated with ethylenediaminetetraacetic acid (EDTA), and the resulting polymer was characterized by FTIR, TGA, and TEM. Different metals including Ni(2+), Cu(2+), and Zn(2+) were immobilized with EDTA-chitosan, and their capability to the specific adsorption of His-tagged proteins were then investigated. The results showed that Ni(2+)-EDTA-chitosan and Zn(2+)-EDTA-chitosan had high affinity toward the His-tagged proteins, thus isolating them from protein mixture. The target fluorescent-labeled hexahistidine protein remained its fluorescent characteristic throughout the purification procedure when Zn(2+)-EDTA-chitosan was used as a sorbent, wherein the real-time monitor was performed to examine the immigration of fluorescent-labeled His-tagged protein. Comparatively, Zn(2+)-EDTA-chitosan showed more specific binding ability for the target protein, but with less binding capacity. It was further proved that this purification system could be recovered and reused at least for 5 times and could run on large scales. The presented M(2+)-EDTA-chitosan system, with the capability to specifically bind His-tagged proteins, make the purification of His-tagged proteins easy to handle, leaving out fussy preliminary treatment, and with the possibility of continuous processing and a reduction in operational cost in relation to the costs of conventional processes. PMID:26585443

  20. High-throughput purification of affinity-tagged recombinant proteins.

    PubMed

    Wiesler, Simone C; Weinzierl, Robert O J

    2012-01-01

    X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity. In order to attribute the different phenotypes to the nature of the mutation--rather than to fluctuating experimental conditions--it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates. Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex. We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase. Altogether, we generated, purified and analysed 381 mutants--a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results. This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins. PMID:22952005

  1. Reversible immobilization of proteins with streptavidin affinity tags on a surface plasmon resonance biosensor chip.

    PubMed

    Li, Yong-Jin; Bi, Li-Jun; Zhang, Xian-En; Zhou, Ya-Feng; Zhang, Ji-Bin; Chen, Yuan-Yuan; Li, Wei; Zhang, Zhi-Ping

    2006-11-01

    Dissociation of biotin from streptavidin is very difficult due to their high binding affinity. The re-use of streptavidin-modified surfaces is therefore almost impossible, making devices containing them (e.g. surface plasmon resonance (SPR) sensor chips) expensive. This paper describes a new protocol for reversible and site-directed immobilization of proteins with streptavidin affinity tags on the streptavidin-coated SPR biosensor chip (SA chip). Two streptavidin affinity tags, nano-tag and streptavidin-binding peptide (SBP tag), were applied. They both can specifically interact with streptavidin but have weaker binding force compared to the biotin-streptavidin system, thus allowing association and dissociation under controlled conditions. The SA chip surface could be regenerated repeatedly without loss of activity by injection of 50 mM NaOH solution. The fusion construct of a SBP tag and a single-chain antibody to mature bovine prion protein (scFv-Z186-SBP) interacts with the SA chip, resulting in a single-chain-antibody-modified surface. The chip showed kinetic response to the prion antigen with equilibrium dissociation constant K (D) approximately equal to 4.01 x 10(-7). All results indicated that the capture activity of the SA chip has no irreversible loss after repeated immobilization and regeneration cycles. The method should be of great benefit to various biosensors, biochips and immunoassay applications based on the streptavidin capture surface. PMID:17006676

  2. Crystallographic structure of Ni-Co coating on the affinity adsorption of histidine-tagged protein.

    PubMed

    Chang, Yaw-Jen; Chen, Sheng-Zheng; Ho, Ching-Yuan

    2015-04-01

    The principle of immobilized metal affinity chromatography (IMAC) has been recently implemented for protein microarrays for the study of protein abundance and function. Ni-Co film fabricated by electrodeposition is a novel microarray surface in an alloy type for immobilizing histidine-tagged proteins based on IMAC. In this paper, the effects of crystallographic structures and surface properties of Ni-Co coatings, with and without the annealing process, on the immobilization of histidine-tagged proteins were systematically investigated. The experimental results reveal that the stronger hcp texture, due to a higher Co content, results in better affinity adsorption for histidine-tagged biotin. Nevertheless, the allotropic phase transformation from hcp to fcc, due to the annealing process, leads to the decrease of affinity adsorption. The wettability property and the surface roughness of Ni-Co coating are, however, not important factors. Obviously, the crystallographic structure of Ni-Co coating is the dominant factor for the specific affinity adsorption of histidine-tagged protein. PMID:25731093

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

    SciTech Connect

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

    2009-01-01

    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.

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

    NASA Astrophysics Data System (ADS)

    Giannone, Richard J.; Liu, Yie; Wang, Yisong

    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 uses 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 demonstrate 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.

  5. A tandem affinity purification tag of TGA2 for isolation of interacting proteins in Arabidopsis thaliana.

    PubMed

    Stotz, Henrik U; Findling, Simone; Nukarinen, Ella; Weckwerth, Wolfram; Mueller, Martin J; Berger, Susanne

    2014-01-01

    Tandem affinity purification (TAP) tagging provides a powerful tool for isolating interacting proteins in vivo. TAP-tag purification offers particular advantages for the identification of stimulus-induced protein interactions. Type II bZIP transcription factors (TGA2, TGA5 and TGA6) play key roles in pathways that control salicylic acid, ethylene, xenobiotic and reactive oxylipin signaling. Although proteins interacting with these transcription factors have been identified through genetic and yeast 2-hybrid screening, others are still elusive. We have therefore generated a C-terminal TAP-tag of TGA2 to isolate additional proteins that interact with this transcription factor. Three lines most highly expressing TAP-tagged TGA2 were functional in that they partially complemented reactive oxylipin-responsive gene expression in a tga2 tga5 tga6 triple mutant. TAP-tagged TGA2 in the most strongly overexpressing line was proteolytically less stable than in the other 2 lines. Only this overexpressing line could be used in a 2-step purification process, resulting in isolation of co-purifying bands of larger molecular weight than TGA2. TAP-tagged TGA2 was used to pull down NPR1, a protein known to interact with this transcription factor. Mass spectrometry was used to identify peptides that co-purified with TAP-tagged TGA2. Having generated this TGA2 TAP-tag line will therefore be an asset to researchers interested in stimulus-induced signal transduction processes. PMID:25482810

  6. Design of affinity tags for one-step protein purification from immobilized zinc columns

    SciTech Connect

    Pasquinelli, R.S.; Shepherd, R.E.; Koepsel, R.R.; Zhao, A.; Ataai, M.M.

    2000-02-01

    Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to e superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. for example, almost all Escherichia coli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper the authors have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions.

  7. A Chimeric Affinity Tag for Efficient Expression and Chromatographic Purification of Heterologous Proteins from Plants

    PubMed Central

    Sainsbury, Frank; Jutras, Philippe V.; Vorster, Juan; Goulet, Marie-Claire; Michaud, Dominique

    2016-01-01

    The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study, we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC) of plant-made proteins based on the protein-stabilizing fusion partner SlCYS8. We show that the Cysta-tag may be used to readily purify proteins under native conditions, and then be removed enzymatically to isolate the protein of interest. We also show that commonly used protease recognition sites for linking purification tags are differentially stable in leaves of the commonly used expression host Nicotiana benthamiana, with those linkers susceptible to cysteine proteases being less stable then serine protease-cleavable linkers. As an example, we describe a Cysta-tag experimental scheme for the one-step purification of a clinically useful protein, human α1-antitrypsin, transiently expressed in N. benthamiana. With potential applicability to the variety of chromatography formats commercially available for IMAC-based protein purification, the Cysta-tag provides a convenient means for the efficient and cost-effective purification of recombinant proteins from plant tissues. PMID:26913045

  8. A Chimeric Affinity Tag for Efficient Expression and Chromatographic Purification of Heterologous Proteins from Plants.

    PubMed

    Sainsbury, Frank; Jutras, Philippe V; Vorster, Juan; Goulet, Marie-Claire; Michaud, Dominique

    2016-01-01

    The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study, we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC) of plant-made proteins based on the protein-stabilizing fusion partner SlCYS8. We show that the Cysta-tag may be used to readily purify proteins under native conditions, and then be removed enzymatically to isolate the protein of interest. We also show that commonly used protease recognition sites for linking purification tags are differentially stable in leaves of the commonly used expression host Nicotiana benthamiana, with those linkers susceptible to cysteine proteases being less stable then serine protease-cleavable linkers. As an example, we describe a Cysta-tag experimental scheme for the one-step purification of a clinically useful protein, human α1-antitrypsin, transiently expressed in N. benthamiana. With potential applicability to the variety of chromatography formats commercially available for IMAC-based protein purification, the Cysta-tag provides a convenient means for the efficient and cost-effective purification of recombinant proteins from plant tissues. PMID:26913045

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

    PubMed

    Schäffer, Ursula; Schlosser, Andreas; Müller, Kristian M; Schäfer, Angelika; Katava, Nenad; Baumeister, Ralf; Schulze, Ekkehard

    2010-04-01

    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. PMID:20047968

  10. Three Recombinant Engineered Antibodies against Recombinant Tags with High Affinity and Specificity

    PubMed Central

    Zhao, Hongyu; Shen, Ao; Xiang, Yang K.; Corey, David P.

    2016-01-01

    We describe three recombinant engineered antibodies against three recombinant epitope tags, constructed with divalent binding arms to recognize divalent epitopes and so achieve high affinity and specificity. In two versions, an epitope is inserted in tandem into a protein of interest, and a homodimeric antibody is constructed by fusing a high-affinity epitope-binding domain to a human or mouse Fc domain. In a third, a heterodimeric antibody is constructed by fusing two different epitope-binding domains which target two different binding sites in GFP, to polarized Fc fragments. These antibody/epitope pairs have affinities in the low picomolar range and are useful tools for many antibody-based applications. PMID:26943906

  11. The elution of certain protein affinity tags with millimolar concentrations of diclofenac.

    PubMed

    Baliova, Martina; Juhasova, Anna; Jursky, Frantisek

    2015-12-01

    Diclofenac (2-[(2, 6-dichlorophenyl)amino] benzeneacetic acid) is a sparingly soluble, nonsteroidal anti-inflammatory drug therapeutically acting at low micromolar concentrations. In pH range from 8 to 11, its aqueous solubility can be increased up to 200 times by the presence of counter ions such as sodium. Our protein interaction studies revealed that a millimolar concentration of sodium diclofenac is able to elute glutathione S-transferase (GST), cellulose binding protein (CBD), and maltose binding protein (MBP) but not histidine-tagged or PDZ-tagged proteins from their affinity resins. The elution efficiency of diclofenac is comparable with the eluting agents normally used at similar concentrations. Native gel electrophoresis of sodium diclofenac-treated proteins showed that the interaction is non-covalent and non-denaturing. These results suggest that sodium diclofenac, in addition to its pharmaceutical applications, can also be exploited as a lead for the development of new proteomics reagents. PMID:26551210

  12. A set of baculovirus transfer vectors for screening of affinity tags and parallel expression strategies.

    PubMed

    Abdulrahman, Wassim; Uhring, Muriel; Kolb-Cheynel, Isabelle; Garnier, Jean-Marie; Moras, Dino; Rochel, Natacha; Busso, Didier; Poterszman, Arnaud

    2009-02-15

    We report a set of baculovirus transfer vectors for parallel expression of proteins in fusion with a panel of affinity tags including GST, protein A, thioredoxin, CBP, and FLAG. This suite includes vectors to generate recombinant baculovirus by homologous recombination in insect cells or using the Bac-to-Bac technology. An application of the vector suite approach to the vitamin D receptor (VDR), a protein mainly expressed as inclusion bodies in Escherichia coli, is presented. We found that expression in fusion with GST and protein A provided an efficient compromise of excellent purification with acceptable yields and costs. PMID:19061853

  13. Robotic high-throughput purification of affinity-tagged recombinant proteins.

    PubMed

    Wiesler, Simone C; Weinzierl, Robert O J

    2015-01-01

    Affinity purification of recombinant proteins has become the method of choice to obtain good quantities and qualities of proteins for a variety of downstream biochemical applications. While manual or FPLC-assisted purification techniques are generally time-consuming and labor-intensive, the advent of high-throughput technologies and liquid handling robotics has simplified and accelerated this process significantly. Additionally, without the human factor as a potential source of error, automated purification protocols allow for the generation of large numbers of proteins simultaneously and under directly comparable conditions. The delivered material is ideal for activity comparisons of different variants of the same protein. Here, we present our strategy for the simultaneous purification of up to 24 affinity-tagged proteins for activity measurements in biochemical assays. The protocol described is suitable for the scale typically required in individual research laboratories. PMID:25749949

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

    PubMed

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

    2012-01-01

    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

  15. Purification and functional characterization of a Camelid-like single-domain antimycotic antibody by engineering in affinity tag.

    PubMed

    Kabir, M Enamul; Krishnaswamy, Senthilkumar; Miyamoto, Masahiko; Furuichi, Yasuhiro; Komiyama, Tadazumi

    2010-07-01

    Single-domain single-chain variable fragment (scFv) antibody is sometimes critical for purification using affinity tagging strategy. We failed in our initial effort to purify a prematurely developed Camelid-like E-tagged short scFv-K2 antibody that contained a complete variable region of the heavy chain and partial region of the light chain by using an anti-E-tag affinity column. To expedite the purification of this altered but interesting antimycotic agent, we replaced a long and large E-tag by a short and hydrophilic 6x-Histidine (His(6)) affinity tag by polymerase chain reaction. The short and compact His(6)-tag was placed on the previously constructed expression vector pCANTAB 5 E that contained the large affinity E-tag sequence (13 amino acids) by PCR-based mutagenesis and was expressed in Escherichia coli. The recombinant protein can then be purified by immobilized metal affinity chromatography (IMAC) and be used for biochemical and other functional characterization. This His(6)-tagged short scFv-K2 antibody (20 kDa) had strong cytocidal activity against Saccharomyces and Candida species with a IC(50) value of 0.44x10(-6)M and 1.10 x 10(-6)M, respectively. Tag replacement facilitates the purification of a Camelid-like single-domain scFv antibody and after that meets its different functional characteristics. The present study reflects that the V(H) domain of the scFv antibody is mainly responsible for its biological activity and single-domain scFv antibody may acts as a potent antimicrobial agent. PMID:20060473

  16. Characterization of a dockerin-based affinity tag: application for purification of a broad variety of target proteins.

    PubMed

    Demishtein, Alik; Karpol, Alon; Barak, Yoav; Lamed, Raphael; Bayer, Edward A

    2010-01-01

    Cellulose, a major component of plant matter, is degraded by a cell surface multiprotein complex called the cellulosome produced by several anaerobic bacteria. This complex coordinates the assembly of different glycoside hydrolases, via a high-affinity Ca(2+)-dependent interaction between the enzyme-borne dockerin and the scaffoldin-borne cohesin modules. In this study, we characterized a new protein affinity tag, ?Doc, a truncated version (48 residues) of the Clostridium thermocellum Cel48S dockerin. The truncated dockerin tag has a binding affinity (K(A)) of 7.7 10(8)M(-1), calculated by a competitive enzyme-linked assay system. In order to examine whether the tag can be used for general application in affinity chromatography, it was fused to a range of target proteins, including Aequorea victoria green fluorescent protein (GFP), C. thermocellum ?-glucosidase, Escherichia coli thioesterase/protease I (TEP1), and the antibody-binding ZZ-domain from Staphylococcus aureus protein A. The results of this study significantly extend initial studies performed using the Geobacillus stearothermophilus xylanase T-6 as a model system. In addition, the enzymatic activity of a C. thermocellum ?-glucosidase, purified using this approach, was tested and found to be similar to that of a ?-glucosidase preparation (without the ?Doc tag) purified using the standard His-tag. The truncated dockerin derivative functioned as an effective affinity tag through specific interaction with a cognate cohesin, and highly purified target proteins were obtained in a single step directly from crude cell extracts. The relatively inexpensive beaded cellulose-based affinity column was reusable and maintained high capacity after each cycle. This study demonstrates that deletion into the first Ca(2+)-binding loop of the dockerin module results in an efficient and robust affinity tag that can be generally applied for protein purification. PMID:21038354

  17. Adsorptive refolding of histidine-tagged glutathione S-transferase using metal affinity chromatography.

    PubMed

    Hutchinson, Matthew H; Chase, Howard A

    2006-09-22

    Column-based protein refolding strategies are often advantageous due to their ease of integration with purification operations, improved refolding yields, and the high concentrations at which the refolded protein can be recovered. His6-tagged glutathione S-transferase (GST-(His6)) was refolded while it was adsorbed in a metal affinity chromatography column. The redox environment could be controlled during the refolding reaction by the addition of reduced and oxidized glutathione without reducing the immobilized nickel metal ions. Adsorptive refolding limited the interaction of refolding intermediates at elevated protein concentrations, and thus improved the yield compared to experiments performed using dilution refolding techniques. The protein concentration during refolding was increased by a factor of 6.8 without reducing the yield achieved compared to dilution refolding. The ability of GST-(His6) to refold to the correct tertiary structure was not significantly affected by the interaction between the poly-histidine-tag and the adsorbent. Decreased refolding yields were achieved at elevated adsorbed protein concentrations, which indicated that at high concentrations the refolding intermediates aggregated despite immobilization. Following adsorptive refolding it was observed that only correctly folded protein could be eluted with imidazole, while the misfolded and aggregated proteins were retained in the column via non-specific interactions with the adsorbent matrix. An iterative refolding strategy was therefore used to re-denature the retained proteins and repeat the adsorptive refolding step, which increased the adsorptive refolding yield that could be achieved at elevated protein concentrations. The yield of correctly folded GST-(His6) from an iterative refolding process was comparable to dilution refolding performed at a 10-fold lower protein concentration. Selective elution and iterative refolding is likely to improve the yields achieved for other poly-histidine-tagged proteins refolded in metal affinity chromatography columns. PMID:16842804

  18. Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

    PubMed

    Kikot, Pamela; Polat, Aise; Achilli, Estefania; Fernandez Lahore, Marcelo; Grasselli, Mariano

    2014-11-01

    Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13. PMID:25277090

  19. Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

    PubMed Central

    Ma, Jingqun; Weake, Vikki Marie

    2014-01-01

    Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection. PMID:24686501

  20. Refolding and purification of histidine-tagged protein by artificial chaperone-assisted metal affinity chromatography.

    PubMed

    Dong, Xiao-Yan; Chen, Li-Jun; Sun, Yan

    2009-07-01

    This article has proposed an artificial chaperone-assisted immobilized metal affinity chromatography (AC-IMAC) for on-column refolding and purification of histidine-tagged proteins. Hexahistidine-tagged enhanced green fluorescent protein (EGFP) was overexpressed in Escherichia coli, and refolded and purified from urea-solubilized inclusion bodies by the strategy. The artificial chaperone system was composed of cetyltrimethylammonium bromide (CTAB) and beta-cyclodextrin (beta-CD). In the refolding process, denatured protein was mixed with CTAB to form a protein-CTAB complex. The mixture was then loaded to IMAC column and the complex was bound via metal chelating to the histidine tag. This was followed by washing with a refolding buffer containing beta-CD that removed CTAB from the bound protein and initiated on-column refolding. The effect of the washing time (i.e., on-column refolding time) on mass and fluorescence recoveries was examined. Extensive studies by comparison with other related refolding techniques have proved the advantages of AC-IMAC. In the on-column refolding, the artificial chaperone system suppressed protein interactions and facilitated protein folding to its native structure. So, the on-column refolding by AC-IMAC led to 99% pure EGFP with a fluorescence recovery of 80%. By comparison at a similar final EGFP concentration (0.6-0.8 mg/mL), this fluorescence recovery value was not only much higher than direct dilution (14%) and AC-assisted refolding (26%) in bulk solutions, but also superior to its partner, IMAC (60%). The operating conditions would be further optimized to improve the refolding efficiency. PMID:19473661

  1. Evaluation of Affinity-Tagged Protein Expression Strategies using Local and Global Isotope Ratio Measurements

    SciTech Connect

    Hervey, IV, William Judson; Khalsa-Moyers, Gurusahai K; Lankford, Patricia K; Owens, Elizabeth T; McKeown, Catherine K; Lu, Tse-Yuan S; Foote, Linda J; Morrell-Falvey, Jennifer L; McDonald, W Hayes; Pelletier, Dale A; Hurst, Gregory {Greg} B

    2009-01-01

    Protein enrichments of engineered, affinity-tagged (or bait ) fusion proteins with interaction partners are often laden with background, non-specific proteins, due to interactions that occur in vitro as an artifact of the technique. Furthermore, the in vivo expression of the bait protein may itself affect physiology or metabolism. In this study, intrinsic affinity purification challenges were investigated in a model protein complex, DNA-dependent RNA polymerase (RNAP), encompassing chromosome- and plasmid-encoding strategies for bait proteins in two different microbial species: Escherichia coli and Rhodopseudomonas palustris. Isotope ratio measurements of bait protein expression strains relative to native, wild-type strains were performed by liquid chromatography tandem mass spectrometry (LC-MS-MS) to assess bait protein expression strategies in each species. Authentic interacting proteins of RNAP were successfully discerned from artifactual co-isolating proteins by the isotopic differentiation of interactions as random or targeted (I-DIRT) method (A. J. Tackett et al. J. Proteome Res. 2005, 4 (5), 1752-1756). To investigate broader effects of bait protein production in the bacteria, we compared proteomes from strains harboring a plasmid that encodes an affinity-tagged subunit (RpoA) of the RNAP complex with the corresponding wild-type strains using stable isotope metabolic labeling. The ratio of RpoA abundance in plasmid strains versus wild type was 0.8 for R. palustris and 1.7 for E. coli. While most other proteins showed no appreciable difference, proteins significantly increased in abundance in plasmid-encoded bait-expressing strains of both species included the plasmid encoded antibiotic resistance protein, GenR and proteins involved in amino acid biosynthesis. Together, these local, complex-specific and more global, whole proteome isotopic abundance ratio measurements provided a tool for evaluating both in vivo and in vitro effects of plasmid-encoding strategies for bait protein expression. This approach has the potential for enabling discovery of protein-protein interactions among the growing number of sequenced microbial species without the need for development of chromosomal insertion systems.

  2. One-step purification of recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, the SBP-Tag.

    PubMed

    Keefe, A D; Wilson, D S; Seelig, B; Szostak, J W

    2001-12-01

    We describe the use of the SBP-tag, a new streptavidin-binding peptide, for both the one-step purification and the detection of recombinant proteins. The SBP-tag sequence is 38 amino acids long and binds to streptavidin with an equilibrium dissociation constant of 2.5 nM. We demonstrate that a single-step purification of SBP-tagged proteins from bacterial extract yields samples that are more pure than those purified using maltose-binding protein or the His-tag. The capacity of the immobilized streptavidin used to purify SBP-tagged proteins is about 0.5 mg per milliliter of matrix, which is high enough to isolate large quantities of proteins for further study. Also, the elution conditions from the streptavidin column are very mild and specific, consisting of the wash buffer plus biotin. This combination of high-affinity, high-yield, mild elution conditions, and simplicity of use makes the SBP-tag suitable for high-throughput protein expression/purification procedures, including robotically manipulated protocols with microtiter plates. Additionally, the SBP-tag can be used for detection since a wide variety of streptavidin-conjugated fluorescent and enzymatic systems are commercially available. We also present a new, rapid, method for the measurement of protein-protein, protein-peptide, or protein-small molecule equilibrium dissociation constants that require as little as 1 fmol of labeled protein. We call this method the spin-filter binding inhibition assay. PMID:11722181

  3. Two-step metal affinity purification of double-tagged (NusA-His6) fusion proteins.

    PubMed

    de Marco, Ario

    2006-01-01

    The present purification protocol applies to target proteins that are fused to a double tag, such as NusA-His6, through a linker that includes a protease-recognition sequence. It involves two steps of immobilized metal ion affinity chromatography (IMAC). NusA stabilizes the passenger protein during translation, whereas the His-tag enables affinity purification of the fusion. The eluate resulting from the first IMAC is buffer-exchanged to remove the imidazole and to achieve optimal conditions for the enzymatic cleavage performed by a His-tagged recombinant protease. The digested sample is loaded directly for a second IMAC step and the target protein is selectively recovered in the flow-through. The resin binds residual non-digested fusion protein, double-tagged moiety, protease and any contaminant that bound the affinity resin and was eluted from the first IMAC. The purity of the target protein usually makes a further purification step unnecessary for most of the lab applications. It takes less than 5 hours to purify the protein from a 5 g pellet. PMID:17406446

  4. The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins.

    PubMed

    Schmidt, Thomas G M; Skerra, Arne

    2007-01-01

    The Strep-tag II is an eight-residue minimal peptide sequence (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) that exhibits intrinsic affinity toward streptavidin and can be fused to recombinant proteins in various fashions. We describe a protocol that enables quick and mild purification of corresponding Strep-tag II fusion proteins--including their complexes with interacting partners--both from bacterial and eukaryotic cell lysates using affinity chromatography on a matrix carrying an engineered streptavidin (Strep-Tactin), which can be accomplished within 1 h. A high-affinity monoclonal antibody (StrepMAB-Immo) permits stable immobilization of Strep-tag II fusion proteins to solid surfaces, for example, for surface plasmon resonance analysis. Selective and sensitive detection on western blots is achieved with Strep-Tactin/enzyme conjugates or another monoclonal antibody (StrepMAB-Classic). Thus, the Strep-tag II, which is short, biologically inert, proteolytically stable and does not interfere with membrane translocation or protein folding, offers a versatile tool both for the rapid isolation of a functional gene product and for its detection or molecular interaction analysis. PMID:17571060

  5. Affinity purification of in vitro transcribed RNA with homogeneous ends using a 3'-ARiBo tag.

    PubMed

    Di Tomasso, Genevive; Salvail-Lacoste, Alix; Bouvette, Jonathan; Omichinski, James G; Legault, Pascale

    2014-01-01

    Common approaches for purification of RNAs synthesized in vitro by the T7 RNA polymerase often denature the RNA and produce RNAs with chemically heterogeneous 5'- and 3'-ends. Thus, native affinity purification strategies that incorporate 5' and 3' trimming technologies provide a solution to two main disadvantages that arise from standard approaches for RNA purification. This chapter describes procedures for nondenaturing affinity purification of in vitro transcribed RNA using a 3'-ARiBo tag, which yield RNAs with a homogeneous 3'-end. The applicability of the method to RNAs of different sequences, secondary structures, and sizes (29-614 nucleotides) is described, including suggestions for troubleshooting common problems. In addition, this chapter presents three complementary approaches to producing 5'-homogeneity of the affinity-purified RNA: (1) selection of the starting sequence; (2) Cse3 endoribonuclease cleavage of a 5'-CRISPR tag; or (3) self-cleavage of a 5'-hammerhead ribozyme tag. The additional steps to express and purify the Cse3 endonuclease are detailed. In light of recent results, the advantages and limitations of current approaches to achieve 5'-homogeneity of affinity-purified RNA are discussed, such that one can select a suitable strategy to purify the RNA of interest. PMID:25432744

  6. Recombinant enterokinase light chain with affinity tag: expression from Saccharomyces cerevisiae and its utilities in fusion protein technology.

    PubMed

    Choi, S I; Song, H W; Moon, J W; Seong, B L

    2001-12-20

    Enterokinase and recombinant enterokinase light chain (rEK(L)) have been used widely to cleave fusion proteins with the target sequence of (Asp)(4)-Lys. In this work, we show that their utility as a site-specific cleavage agent is compromised by sporadic cleavage at other sites, albeit at low levels. Further degradation of the fusion protein in cleavage reaction is due to an intrinsic broad specificity of the enzyme rather than to the presence of contaminating proteases. To offer facilitated purification from fermentation broth and efficient removal of rEK(L) after cleavage reaction, thus minimizing unwanted cleavage of target protein, histidine affinity tag was introduced into rEK(L). Utilizing the secretion enhancer peptide derived from the human interleukin 1 beta, the recombinant EK(L) was expressed in Saccharomyces cerevisiae and efficiently secreted into culture medium. The C-terminal His-tagged EK(L) was purified in a single-step procedure on nickel affinity chromatography. It retained full enzymatic activity similar to that of EK(L), whereas the N-terminal His-tagged EK(L) was neither efficiently purified nor had any enzymatic activity. After cleavage reaction of fusion protein, the C-terminal His-tagged EK(L) was efficiently removed from the reaction mixture by a single passage through nickel-NTA spin column. The simple affinity tag renders rEK(L) extremely useful for purification, post-cleavage removal, recovery, and recycling and will broaden the utility and the versatility of the enterokinase for the production of recombinant proteins. PMID:11745150

  7. The bioactivity and receptor affinity of recombinant tagged EGF designed for tissue engineering applications is defined by the nature and position of the tags.

    PubMed

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

    2008-12-01

    For tissue engineering applications, growth factor immobilization on cell culture scaffolds bears the potential to stimulate cell proliferation while minimizing costs associated to soluble growth factor supply. In order to evaluate the potential of a de novo-designed heterodimerization peptide pair, namely the E and K coils, for epidermal growth factor (EGF) grafting on various scaffolds, production of coil-tagged EGF chimeras using a mammalian cell expression system as well as their purification have been performed. The influence of the type of coil (E or K) upon EGF bioactivity, assessed in an in vitro cell assay, was compared to that of the fragment crystallizable (Fc) domain of immunoglobulin G by monitoring phosphorylation of EGF receptor (EGFR) upon chimeric EGF exposure. Our results demonstrate that the type and the location of the tag have a strong impact on growth factor bioactivity (EC50 ranging from 5.5 to 63 nM). Additional surface plasmon resonance-based biosensor experiments were conducted to test the ability of captured chimeric EGF to bind to their receptor ectodomain in vitro. These experiments indicated that the oriented coiled-coil-mediated immobilization of EGF was significantly more efficient than a random approach as coil-tagged EGF displayed enhanced affinities for artificially dimerized EGFR ectodomain when compared to Fc-tagged EGF (apparent KD of 5 pM vs. 16 nM). Altogether, our results highly suggest that coil-tagged chimeras represent an attractive avenue for the oriented immobilization of growth factors for tissue engineering applications and that HEK293 cells offer a robust platform for their expression in a bioactive form. PMID:18652537

  8. Engineering Streptavidin and a Streptavidin-Binding Peptide with Infinite Binding Affinity and Reversible Binding Capability: Purification of a Tagged Recombinant Protein to High Purity via Affinity-Driven Thiol Coupling

    PubMed Central

    Fogen, Dawson; Wu, Sau-Ching; Ng, Kenneth Kai-Sing; Wong, Sui-Lam

    2015-01-01

    To extend and improve the utility of the streptavidin-binding peptide tag (SBP-tag) in applications ranging from affinity purification to the reversible immobilization of recombinant proteins, a cysteine residue was introduced to the streptavidin mutein SAVSBPM18 and the SBP-tag to generate SAVSBPM32 and SBP(A18C), respectively. This pair of derivatives is capable of forming a disulfide bond through the newly introduced cysteine residues. SAVSBPM32 binds SBP-tag and biotin with binding affinities (Kd ~ 10-8M) that are similar to SAVSBPM18. Although SBP(A18C) binds to SAVSBPM32 more weakly than SBP-tag, the binding affinity is sufficient to bring the two binding partners together efficiently before they are locked together via disulfide bond formation–a phenomenon we have named affinity-driven thiol coupling. Under the condition with SBP(A18C) tags in excess, two SBP(A18C) tags can be captured by a tetrameric SAVSBPM32. The stoichiometry of the disulfide-bonded SAVSBPM32-SBP(A18C) complex was determined using a novel two-dimensional electrophoresis method which has general applications for analyzing the composition of disulfide-bonded protein complexes. To illustrate the application of this reversible immobilization technology, optimized conditions were established to use the SAVSBPM32-affinity matrix for the purification of a SBP(A18C)-tagged reporter protein to high purity. Furthermore, we show that the SAVSBPM32-affinity matrix can also be applied to purify a biotinylated protein and a reporter protein tagged with the unmodified SBP-tag. The dual (covalent and non-covalent) binding modes possible in this system offer great flexibility to many different applications which need reversible immobilization capability. PMID:26406477

  9. Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5' and 3' Noncoding Regions of Genomic RNAs.

    PubMed

    Flather, Dylan; Cathcart, Andrea L; Cruz, Casey; Baggs, Eric; Ngo, Tuan; Gershon, Paul D; Semler, Bert L

    2016-01-01

    Despite being intensely studied for more than 50 years, a complete understanding of the enterovirus replication cycle remains elusive. Specifically, only a handful of cellular proteins have been shown to be involved in the RNA replication cycle of these viruses. In an effort to isolate and identify additional cellular proteins that function in enteroviral RNA replication, we have generated multiple recombinant polioviruses containing RNA affinity tags within the 3' or 5' noncoding region of the genome. These recombinant viruses retained RNA affinity sequences within the genome while remaining viable and infectious over multiple passages in cell culture. Further characterization of these viruses demonstrated that viral protein production and growth kinetics were unchanged or only slightly altered relative to wild type poliovirus. However, attempts to isolate these genetically-tagged viral genomes from infected cells have been hindered by high levels of co-purification of nonspecific proteins and the limited matrix-binding efficiency of RNA affinity sequences. Regardless, these recombinant viruses represent a step toward more thorough characterization of enterovirus ribonucleoprotein complexes involved in RNA replication. PMID:26861382

  10. Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs

    PubMed Central

    Flather, Dylan; Cathcart, Andrea L.; Cruz, Casey; Baggs, Eric; Ngo, Tuan; Gershon, Paul D.; Semler, Bert L.

    2016-01-01

    Despite being intensely studied for more than 50 years, a complete understanding of the enterovirus replication cycle remains elusive. Specifically, only a handful of cellular proteins have been shown to be involved in the RNA replication cycle of these viruses. In an effort to isolate and identify additional cellular proteins that function in enteroviral RNA replication, we have generated multiple recombinant polioviruses containing RNA affinity tags within the 3′ or 5′ noncoding region of the genome. These recombinant viruses retained RNA affinity sequences within the genome while remaining viable and infectious over multiple passages in cell culture. Further characterization of these viruses demonstrated that viral protein production and growth kinetics were unchanged or only slightly altered relative to wild type poliovirus. However, attempts to isolate these genetically-tagged viral genomes from infected cells have been hindered by high levels of co-purification of nonspecific proteins and the limited matrix-binding efficiency of RNA affinity sequences. Regardless, these recombinant viruses represent a step toward more thorough characterization of enterovirus ribonucleoprotein complexes involved in RNA replication. PMID:26861382

  11. High-affinity gold nanoparticle pin to label and localize histidine-tagged protein in macromolecular assemblies

    PubMed Central

    Anthony, Kelsey C.; You, Changjiang; Piehler, Jacob; Pomeranz Krummel, Daniel A.

    2014-01-01

    SUMMARY There is significant demand for experimental approaches to aid protein localization in electron microscopy micrographs and ultimately in three-dimensional reconstructions of macromolecular assemblies. We report preparation and use of a reagent consisting of tris-nitrilotriacetic acid (tris-NTA) conjugated with a monofunctional gold nanoparticle (AuNPtris-NTA) for site-specific, non-covalent labeling of protein termini fused to a histidine-tag (His-tag). Multivalent binding of tris-NTA to a His-tag via complexed Ni(II) ions results in subnanomolar affinity and a defined 1:1 stoichiometry. Precise localization of AuNPtris-NTA labeled proteins by electron microscopy is further ensured by the reagent’s short conformationally restricted linker. We have employed AuNPtris-NTA to localize His-tagged proteins in an oligomeric ATPase and in the bacterial 50S ribosomal subunit. AuNPtris-NTA can specifically bind to the target proteins in these assemblies and is clearly discernible. Our new labeling reagent should find broad application in non-covalent site-specific labeling of protein termini to pinpoint their location in macromolecular assemblies. PMID:24560806

  12. Application of a New Dual Localization-Affinity Purification Tag Reveals Novel Aspects of Protein Kinase Biology in Aspergillus nidulans

    PubMed Central

    De Souza, Colin P.; Hashmi, Shahr B.; Osmani, Aysha H.; Osmani, Stephen A.

    2014-01-01

    Filamentous fungi occupy critical environmental niches and have numerous beneficial industrial applications but devastating effects as pathogens and agents of food spoilage. As regulators of essentially all biological processes protein kinases have been intensively studied but how they regulate the often unique biology of filamentous fungi is not completely understood. Significant understanding of filamentous fungal biology has come from the study of the model organism Aspergillus nidulans using a combination of molecular genetics, biochemistry, cell biology and genomic approaches. Here we describe dual localization-affinity purification (DLAP) tags enabling endogenous N or C-terminal protein tagging for localization and biochemical studies in A. nidulans. To establish DLAP tag utility we endogenously tagged 17 protein kinases for analysis by live cell imaging and affinity purification. Proteomic analysis of purifications by mass spectrometry confirmed association of the CotA and NimXCdk1 kinases with known binding partners and verified a predicted interaction of the SldABub1/R1 spindle assembly checkpoint kinase with SldBBub3. We demonstrate that the single TOR kinase of A. nidulans locates to vacuoles and vesicles, suggesting that the function of endomembranes as major TOR cellular hubs is conserved in filamentous fungi. Comparative analysis revealed 7 kinases with mitotic specific locations including An-Cdc7 which unexpectedly located to mitotic spindle pole bodies (SPBs), the first such localization described for this family of DNA replication kinases. We show that the SepH septation kinase locates to SPBs specifically in the basal region of apical cells in a biphasic manner during mitosis and again during septation. This results in gradients of SepH between G1 SPBs which shift along hyphae as each septum forms. We propose that SepH regulates the septation initiation network (SIN) specifically at SPBs in the basal region of G1 cells and that localized gradients of SIN activity promote asymmetric septation. PMID:24599037

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

    PubMed

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

    1999-12-24

    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

  14. C-Terminally fused affinity Strep-tag II is removed by proteolysis from recombinant human erythropoietin expressed in transgenic tobacco plants

    PubMed Central

    Kittur, Farooqahmed S.; Lalgondar, Mallikarjun; Hung, Chiu-Yueh; Sane, David C.

    2014-01-01

    Asialo-erythropoietin (asialo-EPO), a desialylated form of EPO, is a potent tissue-protective agent. Recently, we and others have exploited a low cost plant-based expression system to produce recombinant human asialo-EPO (asialo-rhuEPOP). To facilitate purification from plant extracts, Strep-tag II was engineered at the C-terminus of EPO. Although asialo-rhuEPOP was efficiently expressed in transgenic tobacco plants, affinity purification based on Strep-tag II did not result in the recovery of the protein. In this study, we investigated the stability of Strep-tag II tagged asialo-rhuEPOP expressed in tobacco plants to understand whether this fused tag is cleaved or inaccessible. Sequencing RT-PCR products confirmed that fused DNA sequences encoding Strep-tag II were properly transcribed, and three-dimensional protein structure model revealed that the tag must be fully accessible. However, Western blot analysis of leaf extracts and purified asialo-rhuEPOP revealed that the Strep-tag II was absent on the protein. Additionally, no peptide fragment containing Strep-tag II was identified in the LC-MS/MS analysis of purified protein further supporting that the affinity tag was absent on asialo-rhuEPOP. However, Strep-tag II was detected on asialo-rhuEPOP that was retained in the endoplasmic reticulum, suggesting that the Strep-tag II is removed during protein secretion or extraction. These findings together with recent reports that C-terminally fused Strep-tag II or IgG Fc domain are also removed from EPO in tobacco plants, suggest that its C-terminus may be highly susceptible to proteolysis in tobacco plants. Therefore, direct fusion of purification tags at the C-terminus of EPO should be avoided while expressing it in tobacco plants. PMID:25504272

  15. Development of a one-step ELISA method using an affinity peptide tag specific to a hydrophilic polystyrene surface.

    PubMed

    Kumada, Yoichi; Katoh, Shigeo; Imanaka, Hiroyuki; Imamura, Koreyoshi; Nakanishi, Kazuhiro

    2007-01-01

    Glutathione S-transferase genetically fused with an affinity peptide tag, PS19 (RAFIASRRIKRP) having a specific affinity for a hydrophilic polystyrene (PS) surface, was preferentially immobilized on a hydrophilic PS (phi-PS) plate without suffering from interference by coexisting protein molecules. Furthermore, rabbit IgG chemically conjugated with a peptide, KPS19R10, in which (10)Lys in PS19 was replaced with Arg and one Lys residue was added at the N-terminus as a coupling site for glutaraldehyde, showed a higher immobilization affinity to the phi-PS plate than that conjugated with the PS19 peptide. On the basis of these findings, the use of a phi-PS plate and peptide tag-linked ligand proteins permitted a one-step or two-step enzyme-linked immunosorbent assay (ELISA) to be achieved, resulting in a substantial reduction in operational time compared with the conventional ELISA method using a hydrophobic PS (pho-PS) plate, while maintaining a high sensitivity. Furthermore, the sensitivity was increased to a greater extent compared to the conventional ELISA meihod when the one-step ELISA was applied to the detection of bovine insulin in a sandwich mode, due to the reduced number of washing and incubation steps. The method proposed here would be a versatile method for use in various ELISA techniques such as sandwich and competitive ELISAs using an antigen, an antibody and streptavidin that are genetically fused or chemically conjugated with the PS-specific affinity peptide as the ligand protein. PMID:16950537

  16. A photo-cleavable biotin affinity tag for the facile release of a photo-crosslinked carbohydrate-binding protein.

    PubMed

    Chang, Tsung-Che; Adak, Avijit K; Lin, Ting-Wei; Li, Pei-Jhen; Chen, Yi-Ju; Lai, Chain-Hui; Liang, Chien-Fu; Chen, Yu-Ju; Lin, Chun-Cheng

    2016-03-15

    The use of photo-crosslinking glycoprobes represents a powerful strategy for the covalent capture of labile protein complexes and allows detailed characterization of carbohydrate-mediated interactions. The selective release of target proteins from solid support is a key step in functional proteomics. We envisaged that light activation can be exploited for releasing labeled protein in a dual photo-affinity probe-based strategy. To investigate this possibility, we designed a trifunctional, galactose-based, multivalent glycoprobe for affinity labeling of carbohydrate-binding proteins. The resulting covalent protein-probe adduct is attached to a photo-cleavable biotin affinity tag; the biotin moiety enables specific presentation of the conjugate on streptavidin-coated beads, and the photolabile linker allows the release of the labeled proteins. This dual probe promotes both the labeling and the facile cleavage of the target protein complexes from the solid surfaces and the remainder of the cell lysate in a completely unaltered form, thus eliminating many of the common pitfalls associated with traditional affinity-based purification methods. PMID:26857483

  17. Construction of a dual-tag system for gene expression, protein affinity purification and fusion protein processing.

    PubMed

    Motejadded, Hassan; Altenbuchner, Josef

    2009-04-01

    An E. coli vector system was constructed which allows the expression of fusion genes via a L: -rhamnose-inducible promotor. The corresponding fusion proteins consist of the maltose-binding protein and a His-tag sequence for affinity purification, the Saccharomyces cerevisiae Smt3 protein for protein processing by proteolytic cleavage and the protein of interest. The Smt3 gene was codon-optimized for expression in E. coli. In a second rhamnose-inducible vector, the S. cerevisiae Ulp1 protease gene for processing Smt3 fusion proteins was fused in the same way to maltose-binding protein and His-tag sequence but without the Smt3 gene. The enhanced green fluorescent protein (eGFP) was used as reporter and protein of interest. Both fusion proteins (MalE-6xHis-Smt3-eGFP and MalE-6xHis-Ulp1) were efficiently produced in E. coli and separately purified by amylose resin. After proteolytic cleavage the products were applied to a Ni-NTA column to remove protease and tags. Pure eGFP protein was obtained in the flow-through of the column in a yield of around 35% of the crude cell extract. PMID:19127343

  18. DOTA-M8: An extremely rigid, high-affinity lanthanide chelating tag for PCS NMR spectroscopy.

    PubMed

    Häussinger, Daniel; Huang, Jie-rong; Grzesiek, Stephan

    2009-10-21

    A new lanthanide chelating tag (M8) for paramagnetic labeling of biomolecules is presented, which is based on an eight-fold, stereoselectively methyl-substituted DOTA that can be covalently linked to the host molecule by a single disulfide bond. The steric overcrowding of the DOTA scaffold leads to an extremely rigid, kinetically and chemically inert lanthanide chelator. Its steric bulk restricts the motion of the tag relative to the host molecule. These properties result in very large pseudocontact shifts (>5 ppm) and residual dipolar couplings (>20 Hz) for Dy-M8 linked to ubiquitin, which are unprecedented for a small, single-point-attachment tag. Such large pseudocontact shifts should be well detectable even for larger proteins and distances beyond approximately 50 A. Due to its exceptionally high stability and lanthanide affinity M8 can be used under extreme chemical or physical conditions, such as those applied for protein denaturation, or when it is undesirable that buffer or protein react with excess lanthanide ions. PMID:19785413

  19. Synthesis of an azido-tagged low affinity ratiometric calcium sensor

    PubMed Central

    Caldwell, Stuart T.; Cairns, Andrew G.; Olson, Marnie; Chalmers, Susan; Sandison, Mairi; Mullen, William; McCarron, John G.; Hartley, Richard C.

    2015-01-01

    Changes in high localised concentrations of Ca2+ ions are fundamental to cell signalling. The synthesis of a dual excitation, ratiometric calcium ion sensor with a Kd of 90 μM, is described. It is tagged with an azido group for bioconjugation, and absorbs in the blue/green and emits in the red region of the visible spectrum with a large Stokes shift. The binding modulating nitro group is introduced to the BAPTA core prior to construction of a benzofuran-2-yl carboxaldehyde by an allylation–oxidation–cyclisation sequence, which is followed by condensation with an azido-tagged thiohydantoin. The thiohydantoin unit has to be protected with an acetoxymethyl (AM) caging group to allow CuAAC click reaction and incorporation of the KDEL peptide endoplasmic reticulum (ER) retention sequence. PMID:26709317

  20. A generic protocol for the purification and characterization of water-soluble complexes of affinity-tagged proteins and lipids.

    PubMed

    Maeda, Kenji; Poletto, Mattia; Chiapparino, Antonella; Gavin, Anne-Claude

    2014-09-01

    Interactions between lipids and proteins in the aqueous phases of cells contribute to many aspects of cell physiology. Here we describe a detailed protocol to systematically characterize in vivo-assembled complexes of soluble proteins and lipids. Saccharomyces cerevisiae strains expressing physiological amounts of a protein of interest fused to the tandem-affinity purification (TAP) tag are first lysed in the absence of detergent to capture intact protein-lipid complexes. The affinity-purified complexes (typically 30-50 kDa) are subjected to analytical size-exclusion chromatography (SEC) to remove contaminating lipids that elute at the void volume (>600 kDa), in order to achieve sufficient signal-to-background lipid ratios. Proteins in the SEC fractions are then analyzed by denaturing gel electrophoresis. Lipidomics techniques such as high-performance thin-layer chromatography or gas or liquid chromatography-mass spectrometry can then be applied to measure the elution profiles of lipids and to pinpoint the true interactors co-eluting with the TAP fusions. The procedure (starting from cell lysis) requires 2 d, and it can easily be adapted to other organisms. PMID:25167057

  1. Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner.

    PubMed

    Li, Xiao-Jing; Liu, Jin-Ling; Gao, Dong-Sheng; Wan, Wen-Yan; Yang, Xia; Li, Yong-Tao; Chang, Hong-Tao; Chen, Lu; Wang, Chuan-Qing; Zhao, Jun

    2016-03-01

    Previous research showed that a lectin from the mushroom Laetiporus sulphureus, designed LSL, bound to Sepharose and could be eluted by lactose. In this study, by taking advantage of the strong affinity of LSL-tag for Sepharose, we developed a single-step purification method for LSL-tagged fusion proteins. We utilized unmodified Sepharose-4B as a specific adsorbent and 0.2 M lactose solution as an elution buffer. Fusion proteins of LSL-tag and porcine circovirus capsid protein, designated LSL-Cap was recovered with purity of 90 ± 4%, and yield of 87 ± 3% from crude extract of recombinant Escherichia coli. To enable the remove of LSL-tag, tobacco etch virus (TEV) protease recognition sequence was placed downstream of LSL-tag in the expression vector, and LSL-tagged TEV protease, designated LSL-TEV, was also expressed in E. coli., and was recovered with purity of 82 ± 5%, and yield of 85 ± 2% from crude extract of recombinant E. coli. After digestion of LSL-tagged recombinant proteins with LSL-TEV, the LSL tag and LSL-TEV can be easily removed by passing the digested products through the Sepharose column. It is of worthy noting that the Sepharose can be reused after washing with PBS. The LSL affinity purification method enables rapid and inexpensive purification of LSL-tagged fusion proteins and scale-up production of native proteins. PMID:26616099

  2. Selective Metabolite and Peptide Capture/Mass Detection using Fluorous Affinity Tags

    PubMed Central

    Go, Eden P.; Uritboonthai, Wilasinee; Apon, Junefredo V.; Trauger, Sunia A.; Nordstrom, Anders; O'Maille, Grace; Brittain, Scott M.; Peters, Eric C.; Siuzdak, Gary

    2008-01-01

    A new and general methodology is described for the targeted enrichment and subsequent direct mass spectrometric characterization of sample subsets bearing various chemical functionalities from highly complex mixtures of biological origin. Specifically, sample components containing a chemical moiety of interest are first selectively labeled with perfluoroalkyl groups, and the entire sample is then applied to a perfluoroalkyl-silylated porous silicon (pSi) surface. Due to the unique hydrophobic and lipophobic nature of the perfluorinated tags, unlabeled sample components are readily removed using simple surface washes, and the enriched sample fraction can then directly be analyzed by desorption/ionization on silicon mass spectrometry (DIOS-MS). Importantly, this fluorous-based enrichment methodology provides a single platform that is equally applicable to both peptide as well as small molecule focused applications. The utility of this technique is demonstrated by the enrichment and mass spectrometric analysis of both various peptide subsets from protein digests as well as amino acids from serum. PMID:17343404

  3. Protein Delivery System Containing a Nickel-Immobilized Polymer for Multimerization of Affinity-Purified His-Tagged Proteins Enhances Cytosolic Transfer.

    PubMed

    Postupalenko, Viktoriia; Desplancq, Dominique; Orlov, Igor; Arntz, Youri; Spehner, Danile; Mely, Yves; Klaholz, Bruno P; Schultz, Patrick; Weiss, Etienne; Zuber, Guy

    2015-09-01

    Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea-grafted polyethylenimine (?PEI) with affinity-purified His-tagged proteins pre-organized onto a nickel-immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His-tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2?nm His-tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single-chain antibodies or caspase?3, into the cytosol of living cells. Transduction of the protease caspase?3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions. PMID:26230624

  4. Affinity-tagged green fluorescent protein (GFP) extraction from a clarified E. coli cell lysate using a two-phase aqueous micellar system.

    PubMed

    Mazzola, Priscila G; Lam, Henry; Kavoosi, Mojgan; Haynes, Charles A; Pessoa, Adalberto; Penna, Thereza Christina Vessoni; Wang, Daniel I C; Blankschtein, Daniel

    2006-04-01

    Green fluorescent protein (GFP) has been proposed as an ideal choice for a protein-based biological indicator for use in the validation of decontamination or disinfection treatments. In this article, we present a potentially scalable and cost-effective way to purify recombinant GFP, produced by fermentation in Escherichia coli, by affinity-enhanced extraction in a two-phase aqueous micellar system. Affinity-enhanced partitioning, which improves the specificity and yield of the target protein by specific bioaffinity interactions, has been demonstrated. A novel affinity tag, family 9 carbohydrate-binding module (CBM9) is fused to GFP, and the resulting fusion protein is affinity-extracted in a decyl beta-D-glucopyranoside (C10G1) two-phase aqueous micellar system. In this system, C10G1 acts as phase forming and as affinity surfactant. We will further demonstrate the implementation of this concept to attain partial recovery of affinity-tagged GFP from a clarified E. coli cell lysate, including the simultaneous removal of other contaminating proteins. The cell lysate was partitioned at three levels of dilution (5x, 10x, and 40x). Irrespective of the dilution level, CBM9-GFP was found to partition preferentially to the micelle-rich phase, with the same partition coefficient value as that found in the absence of the cell lysate. The host cell proteins from the cell lysate were found to partition preferentially to the micelle-poor phase, where they experience less excluded-volume interactions. The demonstration of proof-of-principle of the direct affinity-enhanced extraction of CBM9-GFP from the cell lysate represents an important first step towards developing a cost-effective separation method for GFP, and more generally, for other proteins of interest. PMID:16470873

  5. A New Versatile Immobilization Tag Based on the Ultra High Affinity and Reversibility of the Calmodulin-Calmodulin Binding Peptide Interaction.

    PubMed

    Mukherjee, Somnath; Ura, Marcin; Hoey, Robert J; Kossiakoff, Anthony A

    2015-08-14

    Reversible, high-affinity immobilization tags are critical tools for myriad biological applications. However, inherent issues are associated with a number of the current methods of immobilization. Particularly, a critical element in phage display sorting is functional immobilization of target proteins. To circumvent these problems, we have used a mutant (N5A) of calmodulin binding peptide (CBP) as an immobilization tag in phage display sorting. The immobilization relies on the ultra high affinity of calmodulin to N5A mutant CBP (RWKKNFIAVSAANRFKKIS) in presence of calcium (KD~2 pM), which can be reversed by EDTA allowing controlled "capture and release" of the specific binders. To evaluate the capabilities of this system, we chose eight targets, some of which were difficult to overexpress and purify with other tags and some had failed in sorting experiments. In all cases, specific binders were generated using a Fab phage display library with CBP-fused constructs. KD values of the Fabs were in subnanomolar to low nanomolar (nM) ranges and were successfully used to selectively recognize antigens in cell-based experiments. Some of these targets were problematic even without any tag; thus, the fact that all led to successful selection endpoints means that borderline cases can be worked on with a high probability of a positive outcome. Taken together with examples of successful case specific, high-level applications like generation of conformation-, epitope- and domain-specific Fabs, we feel that the CBP tag embodies all the attributes of covalent immobilization tags but does not suffer from some of their well-documented drawbacks. PMID:26159704

  6. Characterization of the diatomite binding domain in the ribosomal protein L2 from E. coli and functions as an affinity tag.

    PubMed

    Li, Junhua; Zhang, Yang; Yang, Yanjun

    2013-03-01

    The ribosomal protein L2, a constituent protein of the 50S large ribosomal subunit, can be used as Si-tag using silica particles for the immobilization and purification of recombinant proteins (Ikeda et al. (Protein Expr Purif 71:91-95, 2010); Taniguchi et al. (Biotechnol Bioeng 96:1023-1029, 2007)). We applied a diatomite powder, a sedimentary rock mainly composed with diatoms silica, as an affinity solid phase and small ubiquitin-like modifier (SUMO) technology to release a target protein from the solid phase. The L2 (203-273) was the sufficient region for the adsorption of ribosomal protein L2 on diatomite. We comparatively analyzed the different adsorption properties of the two deleted proteins of L2 (L2 (1-60, 203-273) and L2 (203-273)) on diatomite. The time required to reach adsorption equilibrium of L2 (203-273) fusion protein on diatomite was shorter than that of L2 (1-60, 203-273) fusion protein. The maximum adsorption capacity of L2 (203-273) fusion protein was larger than that of L2 (1-60, 203-273) fusion protein. In order to study whether the L2 (203-273) can function as an affinity purification tag, SUMO was introduced as one specific protease cleavage site between the target protein and the purification tags. The L2 (203-273) and SUMO fusion protein purification method was tested using enhanced green fluorescent protein as a model protein; the result shows that the purification performance of this affinity purification method was good. The strong adsorption characteristic of L2 (203-273) on diatomite also provides a potential protein fusion tag for the immobilization of enzyme. PMID:22926644

  7. Investigation of differences in the binding affinities of two analogous ligands for untagged and tagged p38 kinase using thermodynamic integration MD simulation.

    PubMed

    Sun, Ying-Chieh; Hsu, Wen-Chi; Hsu, Chia-Jen; Chang, Chia-Ming; Cheng, Kai-Hsiang

    2015-11-01

    Thermodynamic integration (TI) molecular dynamics (MD) simulations for the binding of a pair of a reference ("ref") ligand and an analogous ("analog") ligand to either tagged (with six extra residues at the N-terminus) or untagged p38 kinase proteins were carried out in order to probe how the binding affinity is influenced by the presence or absence of the peptide tag in p38 kinase. This possible effect of protein length on the binding affinity of a ligand-which is seldom addressed in the literature-is important because, even when two labs claim to have performed experiments with the same protein, they may actually have studied variants of the same protein with different lengths because they applied different protein expression conditions/procedures. Thus, if we wanted to compare ligand binding affinities measured in the two labs, it would be necessary to account for any variation in ligand binding affinity with protein length. The pair of ligand-p38 kinase complexes examined in this work (pdb codes: 3d7z and 3lhj, respectively) were ideal for investigating this effect. The experimentally determined binding energy for the ref ligand with the untagged p38 kinase was -10.9 kcalmol(-1), while that for the analog ligand with the tagged p38 kinase was -11.9 kcalmol(-1). The presentTI-MD simulation of the mutation of the ref ligand into the analog ligand while the ligand is bound to the untagged p38 kinase predicted that the binding affinity of the analog ligand is 2.0kcalmol(-1) greater than that of the ref ligand. A similar simulation also indicated that the same was true for ligand binding to the tagged protein, but in this case the binding affinity for the analog ligand is 2.5kcalmol(-1) larger than that for the ref ligand. These results therefore suggest that the presence of the peptide tag on p38 kinase increased the difference in the binding energies of the ligands by a small amount of 0.5 kcalmol(-1). This result supports the assumption that the presence of a peptide tag has only a minor effect on ?G values. The errorbars in the computed ?G values were then estimated via confidence interval analysis and a time autocorrelation function for the quantity dV/d?. The estimated correlation time was ~0.5 ps and the error bar in the ?G values estimated using nanosecond-scale simulations was 0.3 kcalmol(-1) at a confidence level of 95%. These predicted results can be verified in future experiments and should prove useful in subsequent similar studies. Graphical Abstract Thermodynamic cycles for binding of two analogous ligands with untagged and tagged p38 kinases and associated Gibbs free energy. PMID:26450350

  8. A generic method for the production of recombinant proteins in Escherichia coli using a dual hexahistidine-maltose-binding protein affinity tag.

    PubMed

    Tropea, Joseph E; Cherry, Scott; Nallamsetty, Sreedevi; Bignon, Christophe; Waugh, David S

    2007-01-01

    A generic protocol that utilizes a dual hexahistidine-maltose-binding protein (His6-MBP) affinity tag has been developed for the production of recombinant proteins in Escherichia coli. The MBP moiety improves the yield and enhances the solubility of the passenger protein while the His-tag facilitates its purification. The fusion protein (His6-MBP-passenger) is purified by immobilized metal affinity chromatography (IMAC) on nickel-nitrilotriacetic acid (Ni-NTA) resin and then cleaved in vitro with His6-tobacco etch virus protease to separate the His6-MBP from the passenger protein. In the final step, the unwanted byproducts of the digest are absorbed by a second round of IMAC, leaving nothing but the pure passenger protein in the flow-through fraction. Endogenous proteins that bind to the Ni-NTA resin during the first IMAC step also do so during the second round of IMAC. Hence, the application of two successive IMAC steps, rather than just one, is the key to obtaining crystallization-grade protein with a single affinity technique. PMID:17272834

  9. A dual affinity-tag strategy for the expression and purification of human linker histone H1.4 in Escherichia coli.

    PubMed

    Ryan, Daniel P; Tremethick, David J

    2016-04-01

    Linker histones are an abundant and critical component of the eukaryotic chromatin landscape. They play key roles in regulating the higher order structure of chromatin and many genetic processes. Higher eukaryotes possess a number of different linker histone subtypes and new data are consistently emerging that indicate these subtypes are functionally distinct. We were interested in studying one of the most abundant human linker histone subtypes, H1.4. We have produced recombinant full-length H1.4 in Escherichia coli. An N-terminal Glutathione-S-Transferase tag was used to promote soluble expression and was combined with a C-terminal hexahistidine tag to facilitate a simple non-denaturing two-step affinity chromatography procedure that results in highly pure full-length H1.4. The purified H1.4 was shown to be functional via invitro chromatin assembly experiments and remains active after extended storage at-80C. PMID:26739785

  10. Affinity purification and partial characterization of a yeast multiprotein complex for nucleotide excision repair using histidine-tagged Rad14 protein.

    PubMed

    Rodriguez, K; Talamantez, J; Huang, W; Reed, S H; Wang, Z; Chen, L; Feaver, W J; Friedberg, E C; Tomkinson, A E

    1998-12-18

    The nucleotide excision repair (NER) pathway of eukaryotes involves approximately 30 polypeptides. Reconstitution of this pathway with purified components is consistent with the sequential assembly of NER proteins at the DNA lesion. However, recent studies have suggested that NER proteins may be pre-assembled in a high molecular weight complex in the absence of DNA damage. To examine this model further, we have constructed a histidine-tagged version of the yeast DNA damage recognition protein Rad14. Affinity purification of this protein from yeast nuclear extracts resulted in the co-purification of Rad1, Rad7, Rad10, Rad16, Rad23, RPA, RPB1, and TFIIH proteins, whereas none of these proteins bound to the affinity resin in the absence of recombinant Rad14. Furthermore, many of the co-purifying proteins were present in approximately equimolar amounts. Co-elution of these proteins was also observed when the nuclear extract was fractionated by gel filtration, indicating that the NER proteins were associated in a complex with a molecular mass of >1000 kDa prior to affinity chromatography. The affinity purified NER complex catalyzed the incision of UV-irradiated DNA in an ATP-dependent reaction. We conclude that active high molecular weight complexes of NER proteins exist in undamaged yeast cells. PMID:9852079

  11. Ni(2+)-zeolite/ferrosphere and Ni(2+)-silica/ferrosphere beads for magnetic affinity separation of histidine-tagged proteins.

    PubMed

    Vereshchagina, T A; Fedorchak, M A; Sharonova, O M; Fomenko, E V; Shishkina, N N; Zhizhaev, A M; Kudryavtsev, A N; Frank, L A; Anshits, A G

    2016-01-19

    Magnetic Ni(2+)-zeolite/ferrosphere and Ni(2+)-silica/ferrosphere beads (Ni-ferrosphere beads - NFB) of a core-shell structure were synthesized starting from coal fly ash ferrospheres having diameters in the range of 0.063-0.050 mm. The strategy of NFB fabrication is an oriented chemical modification of the outer surface preserving the magnetic core of parent beads with the formation of micro-mesoporous coverings. Two routes of ferrosphere modification were realized, such as (i) hydrothermal treatment in an alkaline medium resulting in a NaP zeolite layer and (ii) synthesis of micro-mesoporous silica on the glass surface using conventional methods. Immobilization of Ni(2+) ions in the siliceous porous shell of the magnetic beads was carried out via (i) the ion exchange of Na(+) for Ni(2+) in the zeolite layer or (ii) deposition of NiO clusters in the zeolite and silica pores. The final NFB were tested for affinity in magnetic separation of the histidine-tagged green fluorescent protein (GFP) directly from a cell lysate. Results pointed to the high affinity of the magnetic beads towards the protein in the presence of 10 mM EDTA. The sorption capacity of the ferrosphere-based Ni-beads with respect to GFP was in the range 1.5-5.7 mg cm(-3). PMID:26688000

  12. Affinity of aptamers binding 33-mer gliadin peptide and gluten proteins: Influence of immobilization and labeling tags.

    PubMed

    Amaya-Gonzlez, Sonia; Lpez-Lpez, Laura; Miranda-Castro, Rebeca; de-los-Santos-lvarez, Noem; Miranda-Ordieres, Arturo Jos; Lobo-Castan, Mara Jess

    2015-05-11

    Aptamers are starting to increase the reagents tool box to develop more sensitive and reliable methods for food allergens. In most of these assays, aptamers have to be modified for detection and/or immobilization purposes. To take full advantage of their affinity, which decisively influence the detectability, these modifications must be faced rationally. In this work, a recently developed aptamer for an immunotoxic peptide of gliadin associated to celiac disease is used in different configurations and modified with various markers and anchored groups to evaluate the influence of such modifications on the real affinity. The interaction in solution with the peptide is strong for a relatively small molecule (Kd = 45 10 nM, 17 C) and slightly stronger than that for the immobilized intact protein due to a cooperative binding effect. Comparatively, while only minor differences were found when the peptide or the aptamer were immobilized, labeling with a biotin resulted preferable over fluorescein (Kd = 102 11 vs 208 54 nM, 25 C). These findings are of prime importance for the design of an aptamer-based analytical method for gluten quantification. PMID:25911431

  13. Efficient production and purification of recombinant human interleukin-12 (IL-12) overexpressed in mammalian cells without affinity tag

    PubMed Central

    Jayanthi, Srinivas; Koppolu, Bhanu prasanth; Smith, Sean G.; Jalah, Rashmi; Bear, Jenifer; Rosati, Margherita; Pavlakis, George N.; Felber, Barbara K.; Zaharoff, David A.; Kumar, Thallapuranam Krishnaswamy Suresh

    2014-01-01

    Interleukin-12 is a heterodimeric, pro-inflammatory cytokine that is a key driver of cell-mediated immunity. Clinical interest in IL-12 is significant due to its potent anti-tumor activity and efficacy in controlling certain infectious diseases such as Leishmaniasis and Listeria infection. For clinical applications, the ease of production and purification of IL-12 and the associated cost continues to be a consideration. In this context, we report a simple and effective heparin-affinity based purification of recombinant human IL-12 (hIL-12) from the serum-free supernatants of stable IL-12-transduced HEK293 cells. Fractionation of culture supernatants on heparin Sepharose columns revealed that hIL-12 elutes as a single peak in 500 mM NaCl. Coomassie staining and Western blot analysis showed that hIL-12 eluted in 500 mM NaCl is homogeneous.Purity of hIL-12 was ascertained by RP-HPLC and ESI-MS analysis, and found to be ~98%. Western blot analysis, using monoclonal antibodies, demonstrated that the crucial inter-subunit disulfide bond linking the p35 and p40 subunits is intact in the purified hIL-12. Results of far UV circular dichrosim, steady-state tryptophan fluorescence, and differential scanning calorimetry experiments suggest that purified hIL-12 is in its stable native conformation. Enzyme linked immunosorbent assays (ELISAs) and bioactivity studies demonstrate that hIL-12 is obtained in high yields (0.31 0.05 mg/ mL of the culture medium) and is also fully bioactive. Isothermal titration calorimetry data show that IL-12 exhibits a moderate binding affinity (Kd(app) = 69 1 ?M) to heparin. The purification method described in this study is expected to provide greater impetus for research on the role of heparin in the regulation of the function of IL-12. In addition, the results of this study provide an avenue to obtain high amounts of IL-12 required for structural studies which are aimed at the development of novel IL-12-based therapeutics. PMID:25123642

  14. Efficient production and purification of recombinant human interleukin-12 (IL-12) overexpressed in mammalian cells without affinity tag.

    PubMed

    Jayanthi, Srinivas; Koppolu, Bhanu prasanth; Smith, Sean G; Jalah, Rashmi; Bear, Jenifer; Rosati, Margherita; Pavlakis, George N; Felber, Barbara K; Zaharoff, David A; Kumar, Thallapuranam Krishnaswamy Suresh

    2014-10-01

    Interleukin-12 is a heterodimeric, pro-inflammatory cytokine that is a key driver of cell-mediated immunity. Clinical interest in IL-12 is significant due to its potent anti-tumor activity and efficacy in controlling certain infectious diseases such as Leishmaniasis and Listeria infection. For clinical applications, the ease of production and purification of IL-12 and the associated cost continues to be a consideration. In this context, we report a simple and effective heparin-affinity based purification of recombinant human IL-12 (hIL-12) from the serum-free supernatants of stable IL-12-transduced HEK293 cells. Fractionation of culture supernatants on heparin Sepharose columns revealed that hIL-12 elutes as a single peak in 500 mM NaCl. Coomassie staining and Western blot analysis showed that hIL-12 eluted in 500 mM NaCl is homogeneous. Purity of hIL-12 was ascertained by RP-HPLC and ESI-MS analysis, and found to be ∼98%. Western blot analysis, using monoclonal antibodies, demonstrated that the crucial inter-subunit disulfide bond linking the p35 and p40 subunits is intact in the purified hIL-12. Results of far UV circular dichroism, steady-state tryptophan fluorescence, and differential scanning calorimetry experiments suggest that purified hIL-12 is in its stable native conformation. Enzyme linked immunosorbent assays (ELISAs) and bioactivity studies demonstrate that hIL-12 is obtained in high yields (0.31±0.05 mg/mL of the culture medium) and is also fully bioactive. Isothermal titration calorimetry data show that IL-12 exhibits a moderate binding affinity (Kd(app)=69±1 μM) to heparin. The purification method described in this study is expected to provide greater impetus for research on the role of heparin in the regulation of the function of IL-12. In addition, the results of this study provide an avenue to obtain high amounts of IL-12 required for structural studies which are aimed at the development of novel IL-12-based therapeutics. PMID:25123642

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

    PubMed

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

    2002-10-01

    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

  16. 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

    Gretes, Michael C; Karplus, P Andrew

    2013-10-01

    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

  17. Isotope Coded Protein Labeling Coupled Immunoprecipitation (ICPL-IP): A Novel Approach for Quantitative Protein Complex Analysis From Native Tissue*

    PubMed Central

    Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

    2013-01-01

    High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organismsincluding humansare hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)1 with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated ?-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method. PMID:23268931

  18. 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

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

    2005-08-25

    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.

  19. Expression platforms for producing eukaryotic proteins: a comparison of E. coli cell-based and wheat germ cell-free synthesis, affinity and solubility tags, and cloning strategies.

    PubMed

    Aceti, David J; Bingman, Craig A; Wrobel, Russell L; Frederick, Ronnie O; Makino, Shin-Ichi; Nichols, Karl W; Sahu, Sarata C; Bergeman, Lai F; Blommel, Paul G; Cornilescu, Claudia C; Gromek, Katarzyna A; Seder, Kory D; Hwang, Soyoon; Primm, John G; Sabat, Grzegorz; Vojtik, Frank C; Volkman, Brian F; Zolnai, Zsolt; Phillips, George N; Markley, John L; Fox, Brian G

    2015-06-01

    Vectors designed for protein production in Escherichia coli and by wheat germ cell-free translation were tested using 21 well-characterized eukaryotic proteins chosen to serve as controls within the context of a structural genomics pipeline. The controls were carried through cloning, small-scale expression trials, large-scale growth or synthesis, and purification. Successfully purified proteins were also subjected to either crystallization trials or (1)H-(15)N HSQC NMR analyses. Experiments evaluated: (1) the relative efficacy of restriction/ligation and recombinational cloning systems; (2) the value of maltose-binding protein (MBP) as a solubility enhancement tag; (3) the consequences of in vivo proteolysis of the MBP fusion as an alternative to post-purification proteolysis; (4) the effect of the level of LacI repressor on the yields of protein obtained from E. coli using autoinduction; (5) the consequences of removing the His tag from proteins produced by the cell-free system; and (6) the comparative performance of E. coli cells or wheat germ cell-free translation. Optimal promoter/repressor and fusion tag configurations for each expression system are discussed. PMID:25854603

  20. Semi-quantitative measurement of a specific glycoform using a DNA-tagged antibody and lectin affinity chromatography for glyco-biomarker development.

    PubMed

    Lee, Ju Hee; Cho, Chang Hee; Kim, Sun Hee; Kang, Jeong Gu; Yoo, Jong Shin; Chang, Chulhun Ludgerus; Ko, Jeong-Heon; Kim, Yong-Sam

    2015-03-01

    Aberrant glycosylation-targeted disease biomarker development is based on cumulative evidence that certain glycoforms are mass-produced in a disease-specific manner. However, the development process has been hampered by the absence of an efficient validation method based on a sensitive and multiplexed platform. In particular, ELISA-based analytical tools are not adequate for this purpose, mainly because of the presence of a pair of N-glycans of IgG-type antibodies. To overcome the associated hurdles in this study, antibodies were tagged with oligonucleotides with T7 promoter and then allowed to form a complex with corresponding antigens. An antibody-bound specific glycoform was isolated by lectin chromatography and quantitatively measured on a DNA microarray chip following production of fluorescent RNA by T7-trascription. This tool ensured measurement of targeted glycoforms of multiple biomarkers with high sensitivity and multiplexity. This analytical method was applied to an in vitro diagnostic multivariate index assay where a panel of hepatocellular carcinoma (HCC) biomarkers comprising alpha-fetoprotein, hemopexin, and alpha-2-macroglobulin (A2M) was examined in terms of the serum level and their fuco-fractions. The results indicated that the tests using the multiplexed fuco-biomarkers provided improved discriminatory power between non- hepatocellular carcinoma and hepatocellular carcinoma subjects compared with the alpha-fetoprotein level or fuco-alpha-fetoprotein test alone. The developed method is expected to facilitate the validation of disease-specific glycan biomarker candidates. PMID:25525205

  1. Semi-quantitative Measurement of a Specific Glycoform Using a DNA-tagged Antibody and Lectin Affinity Chromatography for Glyco-biomarker Development*

    PubMed Central

    Lee, Ju Hee; Cho, Chang Hee; Kim, Sun Hee; Kang, Jeong Gu; Yoo, Jong Shin; Chang, Chulhun Ludgerus; Ko, Jeong-Heon; Kim, Yong-Sam

    2015-01-01

    Aberrant glycosylation-targeted disease biomarker development is based on cumulative evidence that certain glycoforms are mass-produced in a disease-specific manner. However, the development process has been hampered by the absence of an efficient validation method based on a sensitive and multiplexed platform. In particular, ELISA-based analytical tools are not adequate for this purpose, mainly because of the presence of a pair of N-glycans of IgG-type antibodies. To overcome the associated hurdles in this study, antibodies were tagged with oligonucleotides with T7 promoter and then allowed to form a complex with corresponding antigens. An antibody-bound specific glycoform was isolated by lectin chromatography and quantitatively measured on a DNA microarray chip following production of fluorescent RNA by T7-trascription. This tool ensured measurement of targeted glycoforms of multiple biomarkers with high sensitivity and multiplexity. This analytical method was applied to an in vitro diagnostic multivariate index assay where a panel of hepatocellular carcinoma (HCC) biomarkers comprising alpha-fetoprotein, hemopexin, and alpha-2-macroglobulin (A2M) was examined in terms of the serum level and their fuco-fractions. The results indicated that the tests using the multiplexed fuco-biomarkers provided improved discriminatory power between non- hepatocellular carcinoma and hepatocellular carcinoma subjects compared with the alpha-fetoprotein level or fuco-alpha-fetoprotein test alone. The developed method is expected to facilitate the validation of disease-specific glycan biomarker candidates. PMID:25525205

  2. NLS-tagging: an alternative strategy to tag nuclear proteins.

    PubMed

    Giraud, Guillaume; Stadhouders, Ralph; Conidi, Andrea; Dekkers, Dick H W; Huylebroeck, Danny; Demmers, Jeroen A A; Soler, Eric; Grosveld, Frank G

    2014-12-01

    The characterization of transcription factor complexes and their binding sites in the genome by affinity purification has yielded tremendous new insights into how genes are regulated. The affinity purification requires either the use of antibodies raised against the factor of interest itself or by high-affinity binding of a C- or N-terminally added tag sequence to the factor. Unfortunately, fusing extra amino acids to the termini of a factor can interfere with its biological function or the tag may be inaccessible inside the protein. Here, we describe an effective solution to that problem by integrating the 'tag' close to the nuclear localization sequence domain of the factor. We demonstrate the effectiveness of this approach with the transcription factors Fli-1 and Irf2bp2, which cannot be tagged at their extremities without loss of function. This resulted in the identification of novel proteins partners and a new hypothesis on the contribution of Fli-1 to hematopoiesis. PMID:25260593

  3. Brain quantitative proteomics combining GeLC-MS and isotope-coded protein labeling (ICPL).

    PubMed

    Maccarrone, Giuseppina; Lebar, Maria; Martins-de-Souza, Daniel

    2014-01-01

    Proteomics has been revolutionized by the rapid advance of mass spectrometric instrumentations and techniques. Parallel methodologies for the quantification of proteomes also evolved, including in vitro stable isotope labeling. Here, we present a protocol for employing isotope-coded protein labeling (ICPL) as part of a shotgun proteomics workflow denoting its advantages and disadvantages. This protocol is suitable to studying any proteome of interest, only requiring a specific sample preparation and protein identification. Given our expertise, descriptions here are centered on the study of brain disorders. PMID:24791988

  4. Simplified Protein Purification Using an Autoprocessing, Inducible Enzyme Tag

    PubMed Central

    Shen, Aimee

    2015-01-01

    Summary The development of affinity tags has greatly simplified protein purification procedures. A variety of affinity tags are now available to improve expression, solubility, and/or tag removal. In this chapter, we describe a method for purifying recombinant proteins expressed in Escherichia coli that uses a highly specific, inducible, C-terminal autoprocessing protease tag. This method streamlines affinity purification, cleavage, and tag separation into a one-step purification procedure, avoiding the need to remove fusion tags from target proteins with exogenous proteases. In addition to accelerating protein purification, we show that this method can enhance the expression, stability, and solubility of select proteins. PMID:24943314

  5. Quantitative Serum Proteomics Using Dual Stable Isotope Coding and nano LC-MS/MSMS

    PubMed Central

    Wang, Hong; Wong, Chee-Hong; Chin, Alice; Kennedy, Jacob; Zhang, Qing; Hanash, Samir

    2015-01-01

    Stable isotope coding technique in combination with mass spectrometry has emerged as a powerful tool to accurately identify and differentially quantify proteins within complex protein mixtures. We present a novel methodology to increase the yield of quantified proteins while maintaining a high stable-isotopic labeling efficacy. With this approach, intact proteins in complex biological sample such as sera are labeled with the designated dual stable isotope coding (DSIC) systems. In brief, intact proteins are coded sequentially with acrylamide to label Cysteine residues (Cys) and with succinic anhydride to label Lysine residues (Lys). Protein samples coded with this dual stable isotope are subjected to an online 2D-HPLC fractionation. The resolved protein fractions are individually digested with trypsin and analyzed with nano LC-MS/MSMS. Our results show that the DSIC labeling efficiency is 100% for Cysteine (Cys) labeled with acrylamide and 98% for Lysine (Lys) labeled with succinic anhydride. A comparative analysis of DSIC labeling and single labeling of Cysteine residues was made. Analysis of an entire anion-exchange chromatography subfraction of sera yielded 165 identified proteins (criteria: error rate <5% and unique peptides ?2), 104 of which were quantified using the single labeling method (i.e., Cysteine acrylamide labeling only). In contrast, using same criteria for identification, a total 185 proteins were identified and 174 proteins were quantified using the DSIC labeling technique. PMID:19817497

  6. Expression of cold-adapted ?-1,3-xylanase as a fusion protein with a ProS2 tag and purification using immobilized metal affinity chromatography with a high concentration of ArgHCl.

    PubMed

    Kudou, Motonori; Okazaki, Fumiyoshi; Asai-Nakashima, Nanami; Ogino, Chiaki; Kondo, Akihiko

    2015-01-01

    Cold-adapted ?-1,3-xylanase (P.t.Xyn26A) from the psychrotrophic bacterium, Psychroflexus torquis, was expressed as a fusion protein with tandem repeats of the N-terminal domain of Protein S from Myxocuccus xanthus (ProS2) in Escherichia coli. After cell lysis in phosphate buffer, most of the ProS2-P.t.Xyn26A was located in the insoluble fraction and aggregated during purification. Arginine hydrochloride (ArgHCl) efficiently solubilized the ProS2-P.t.Xyn26A. The solubilized ProS2-P.t.Xyn26A was purified using immobilized metal affinity chromatography (IMAC) with 500 mM ArgHCl. After cleavage of ProS2-P.t.Xyn26A by human rhinovirus 3C protease, we confirmed that recombinant P.t.Xyn26A maintained its native fold. This is the first report of the expression of a cold-adapted enzyme fused with a ProS2 tag under IMAC purification using a high concentration of ArgHCl. These insights into the expression and purification should be useful during the handling of cold-adapted enzymes. PMID:25214227

  7. Preparation of core-shell structure Fe3 O4 @SiO2 superparamagnetic microspheres immoblized with iminodiacetic acid as immobilized metal ion affinity adsorbents for His-tag protein purification.

    PubMed

    Ni, Qian; Chen, Bing; Dong, Shaohua; Tian, Lei; Bai, Quan

    2016-04-01

    The core-shell structure Fe3 O4 /SiO2 magnetic microspheres were prepared by a sol-gel method, and immobiled with iminodiacetic acid (IDA) as metal ion affinity ligands for protein adsorption. The size, morphology, magnetic properties and surface modification of magnetic silica nanospheres were characterized by various modern analytical instruments. It was shown that the magnetic silica nanospheres exhibited superparamagnetism with saturation magnetization values of up to 58.1 emu/g. Three divalent metal ions, Cu(2+) , Ni(2+) and Zn(2+) , were chelated on the Fe3 O4 @SiO2 -IDA magnetic microspheres to adsorb lysozyme. The results indicated that Ni(2+) -chelating magnetic microspheres had the maximum adsorption capacity for lysozyme of 51.0 mg/g, adsorption equilibrium could be achieved within 60 min and the adsorbed protein could be easily eluted. Furthermore, the synthesized Fe3 O4 @SiO2 -IDA-Ni(2+) magnetic microspheres were successfully applied for selective enrichment lysozyme from egg white and His-tag recombinant Homer 1a from the inclusion extraction expressed in Escherichia coli. The result indicated that the magnetic microspheres showed unique characteristics of high selective separation behavior of protein mixture, low nonspecific adsorption, and easy handling. This demonstrates that the magnetic silica microspheres can be used efficiently in protein separation or purification and show great potential in the pretreatment of the biological sample. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26268650

  8. Tagging Walruses

    USGS Multimedia Gallery

    A transmitter tag (left) is being deployed by a USGS Wildlife Biologist (far right). Transmitter tags are deployed on the back of walruses where their skin is thickest and where their data transmissions may be received from passing satellites. Tag deployment happens in the blink of an eye with the ...

  9. Challenges and opportunities in the purification of recombinant tagged proteins.

    PubMed

    Pina, Ana Sofia; Lowe, Christopher R; Roque, Ana Cecília A

    2014-01-01

    The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs "tag-ligand" combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established "tag-ligand" systems available for fusion protein purification and also explores current unconventional strategies under development. PMID:24334194

  10. English Declarative Tags, Intonation Tags, and Tag Questions. Volume 10.

    ERIC Educational Resources Information Center

    Armagost, James L.

    This paper seeks to discover the rules active in the formation of tags (intonation tags, declarative tags, and tag questions) in English. The author discusses former analyses of these constructions and presents his own thoughts with many examples, concluding that English has at least two tag formation rules: one that accounts (perhaps

  11. Affinity Chromatography.

    ERIC Educational Resources Information Center

    Gray, Gary R.

    1980-01-01

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

  12. Detection of protein-protein interactions using tandem affinity purification.

    PubMed

    Goodfellow, Ian; Bailey, Dalan

    2014-01-01

    Tandem affinity purification (TAP) is an invaluable technique for identifying interaction partners for an affinity tagged bait protein. The approach relies on the fusion of dual tags to the bait before separate rounds of affinity purification and precipitation. Frequently two specific elution steps are also performed to increase the specificity of the overall technique. In the method detailed here, the two tags used are protein G and a short streptavidin binding peptide; however, many variations can be employed. In our example the tags are separated by a cleavable tobacco etch virus protease target sequence, allowing for specific elution after the first round of affinity purification. Proteins isolated after the final elution step in this process are concentrated before being identified by mass spectrometry. The use of dual affinity tags and specific elution in this technique dramatically increases both the specificity and stringency of the pull-downs, ensuring a low level of background nonspecific interactions. PMID:24943319

  13. Inntags: small self-structured epitopes for innocuous protein tagging.

    PubMed

    Georgieva, Maya V; Yahya, Galal; Cod, Laia; Ortiz, Ral; Teixid, Laura; Claros, Jos; Jara, Ricardo; Jara, Mnica; Iborra, Antoni; Gelp, Josep Llus; Gallego, Carme; Orozco, Modesto; Aldea, Mart

    2015-10-01

    Protein tagging is widely used in approaches ranging from affinity purification to fluorescence-based detection in live cells. However, an intrinsic limitation of tagging is that the native function of the protein may be compromised or even abolished by the presence of the tag. Here we describe and characterize a set of small, innocuous protein tags (inntags) that we anticipate will find application in a variety of biological techniques. PMID:26322837

  14. The tandem affinity purification technology: an overview.

    PubMed

    Li, Yifeng

    2011-08-01

    Tandem affinity purification (TAP) is a methodology for the isolation of protein complexes from endogenous sources. It involves incorporation of a dual-affinity tag into the protein of interest and introduction of the construct into desired cell lines or organisms. Using the two affinity handles, the protein complex assembled under physiological conditions, which contains the tagged target protein and its interacting partners, can be isolated by a sequential purification scheme. Compared with single-step purification, TAP greatly reduces non-specific background and isolates protein complexes with higher purity. TAP-based protein retrieval plus mass spectrometry-based analysis has become a standard approach for identification and characterization of multi-protein complexes. The present article gives an overview of the TAP method, with a focus on its key feature-the dual-affinity tag. In addition, the application of this technology in various systems is briefly discussed. PMID:21424840

  15. NLS-tagging: an alternative strategy to tag nuclear proteins

    PubMed Central

    Giraud, Guillaume; Stadhouders, Ralph; Conidi, Andrea; Dekkers, Dick H.W.; Huylebroeck, Danny; Demmers, Jeroen A.A.; Soler, Eric; Grosveld, Frank G.

    2014-01-01

    The characterization of transcription factor complexes and their binding sites in the genome by affinity purification has yielded tremendous new insights into how genes are regulated. The affinity purification requires either the use of antibodies raised against the factor of interest itself or by high-affinity binding of a C- or N-terminally added tag sequence to the factor. Unfortunately, fusing extra amino acids to the termini of a factor can interfere with its biological function or the tag may be inaccessible inside the protein. Here, we describe an effective solution to that problem by integrating the ‘tag’ close to the nuclear localization sequence domain of the factor. We demonstrate the effectiveness of this approach with the transcription factors Fli-1 and Irf2bp2, which cannot be tagged at their extremities without loss of function. This resulted in the identification of novel proteins partners and a new hypothesis on the contribution of Fli-1 to hematopoiesis. PMID:25260593

  16. Use of Tandem Affinity Chromatography for Purification of Cannabinoid Receptor CB2

    PubMed Central

    Locatelli-Hoops, Silvia C.; Yeliseev, Alexei A.

    2016-01-01

    Tandem affinity purification has been increasingly applied to isolation of recombinant proteins. It relies on two consecutive chromatographic steps that take advantage of the affinity tags placed at opposing ends of the target protein. This allows for efficient removal of contaminating proteins, including products of proteolytic degradation of the fusion that lack either N- or C-terminal tags. Here, we describe the use of two small affinity tags, a poly-histidine tag and a Strep-tag for expression and purification of the human cannabinoid receptor CB2, an integral membrane G protein-coupled receptor. PMID:24943318

  17. Shark Tagging Activities.

    ERIC Educational Resources Information Center

    Current: The Journal of Marine Education, 1998

    1998-01-01

    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

  18. Identification of Candidate Angiogenic Inhibitors Processed by Matrix Metalloproteinase 2 (MMP-2) in Cell-Based Proteomic Screens: Disruption of Vascular Endothelial Growth Factor (VEGF)/Heparin Affin Regulatory Peptide (Pleiotrophin) and VEGF/Connective Tissue Growth Factor Angiogenic Inhibitory Complexes by MMP-2 Proteolysis?

    PubMed Central

    Dean, Richard A.; Butler, Georgina S.; Hamma-Kourbali, Yamina; Delb, Jean; Brigstock, David R.; Courty, Jos; Overall, Christopher M.

    2007-01-01

    Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2?/? mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2?/? cells and those rescued by MMP-2 transfection. Proteome signatures that are hallmarks of proteolysis revealed cleavage of many known MMP-2 substrates in the cellular context. Proteomic evidence of MMP-2 processing of novel substrates was found. Insulin-like growth factor binding protein 6, follistatin-like 1, and cystatin C protein cleavage by MMP-2 was biochemically confirmed, and the cleavage sites in heparin affin regulatory peptide (HARP; pleiotrophin) and connective tissue growth factor (CTGF) were sequenced by matrix-assisted laser desorption ionization-time of flight mass spectrometry. MMP-2 processing of HARP and CTGF released vascular endothelial growth factor (VEGF) from angiogenic inhibitory complexes. The cleaved HARP N-terminal domain increased HARP-induced cell proliferation, whereas the HARP C-terminal domain was antagonistic and decreased cell proliferation and migration. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis. PMID:17908800

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

    PubMed Central

    Rauniyar, Navin; Prokai, Laszlo

    2011-01-01

    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 chromatographymass 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. PMID:22012663

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

    PubMed

    Kuo, Wen-Hui K; Chase, Howard A

    2010-12-01

    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 tagged species are adsorbed to the immobilized metal affinity chromatography (IMAC) adsorbent. Adsorptive detagging was performed in the presence of 50mM imidazole in order to allow the cleavage reaction by the hexa-histidine tagged DAPase to occur. The progress of batch and adsorptive detagging by DAPase of maltose binding protein (MBP) tagged with two variants of hexa-histidine fusion tag was successfully monitored using cationic exchange chromatography. A single-step, column-based detagging strategy was then optimized to maximize the recovery of native MBP. The kinetics of batch and on-column digestion for both HT6 and HT15 fusion tags were investigated. The process involved the sequential removal of dipeptides during the digestion of full-length fusion protein down to its fully detagged native form. During the course of tag digestion, 4 and 7 different intermediates were detected for HT6 and HT15 tagged MBP respectively. The characteristics of on-column cleavage of poly-histidine fusion tags by DAPase as a function of incubation temperature and amount of protease activity used were examined. It was found that the influence of fusion tag design on the batch and column-based detagging yield and efficiency was substantial. In addition, the structural difference of fusion tags affects the binding strength of the fusion protein, which can influence the resulting product purity. Despite being a longer tag, HT15 fusion tag was the preferred sequence for shortening the time needed for on-column detagging. These results can be applied to the wider use of the proposed platform protocol for the on-column cleavage of poly-histidine tagged proteins using exopeptidases. PMID:21055759

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

    PubMed Central

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

    2013-01-01

    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. PMID:22668517

  2. Donor Tag Game

    MedlinePLUS

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

  3. 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. Some skin tags are as long as ...

  4. Paramagnetic Ligand Tagging To Identify Protein Binding Sites

    PubMed Central

    2015-01-01

    Transient biomolecular interactions are the cornerstones of the cellular machinery. The identification of the binding sites for low affinity molecular encounters is essential for the development of high affinity pharmaceuticals from weakly binding leads but is hindered by the lack of robust methodologies for characterization of weakly binding complexes. We introduce a paramagnetic ligand tagging approach that enables localization of low affinity protein–ligand binding clefts by detection and analysis of intermolecular protein NMR pseudocontact shifts, which are invoked by the covalent attachment of a paramagnetic lanthanoid chelating tag to the ligand of interest. The methodology is corroborated by identification of the low millimolar volatile anesthetic interaction site of the calcium sensor protein calmodulin. It presents an efficient route to binding site localization for low affinity complexes and is applicable to rapid screening of protein–ligand systems with varying binding affinity. PMID:26289584

  5. Extracting Tag Hierarchies

    PubMed Central

    Tibly, Gergely; Pollner, Pter; Vicsek, Tams; Palla, Gergely

    2013-01-01

    Tagging items with descriptive annotations or keywords is a very natural way to compress and highlight information about the properties of the given entity. Over the years several methods have been proposed for extracting a hierarchy between the tags for systems with a "flat", egalitarian organization of the tags, which is very common when the tags correspond to free words given by numerous independent people. Here we present a complete framework for automated tag hierarchy extraction based on tag occurrence statistics. Along with proposing new algorithms, we are also introducing different quality measures enabling the detailed comparison of competing approaches from different aspects. Furthermore, we set up a synthetic, computer generated benchmark providing a versatile tool for testing, with a couple of tunable parameters capable of generating a wide range of test beds. Beside the computer generated input we also use real data in our studies, including a biological example with a pre-defined hierarchy between the tags. The encouraging similarity between the pre-defined and reconstructed hierarchy, as well as the seemingly meaningful hierarchies obtained for other real systems indicate that tag hierarchy extraction is a very promising direction for further research with a great potential for practical applications. Tags have become very prevalent nowadays in various online platforms ranging from blogs through scientific publications to protein databases. Furthermore, tagging systems dedicated for voluntary tagging of photos, films, books, etc. with free words are also becoming popular. The emerging large collections of tags associated with different objects are often referred to as folksonomies, highlighting their collaborative origin and the flat organization of the tags opposed to traditional hierarchical categorization. Adding a tag hierarchy corresponding to a given folksonomy can very effectively help narrowing or broadening the scope of search. Moreover, recommendation systems could also benefit from a tag hierarchy. PMID:24391901

  6. Extracting tag hierarchies.

    PubMed

    Tibly, Gergely; Pollner, Pter; Vicsek, Tams; Palla, Gergely

    2013-01-01

    Tagging items with descriptive annotations or keywords is a very natural way to compress and highlight information about the properties of the given entity. Over the years several methods have been proposed for extracting a hierarchy between the tags for systems with a "flat", egalitarian organization of the tags, which is very common when the tags correspond to free words given by numerous independent people. Here we present a complete framework for automated tag hierarchy extraction based on tag occurrence statistics. Along with proposing new algorithms, we are also introducing different quality measures enabling the detailed comparison of competing approaches from different aspects. Furthermore, we set up a synthetic, computer generated benchmark providing a versatile tool for testing, with a couple of tunable parameters capable of generating a wide range of test beds. Beside the computer generated input we also use real data in our studies, including a biological example with a pre-defined hierarchy between the tags. The encouraging similarity between the pre-defined and reconstructed hierarchy, as well as the seemingly meaningful hierarchies obtained for other real systems indicate that tag hierarchy extraction is a very promising direction for further research with a great potential for practical applications. Tags have become very prevalent nowadays in various online platforms ranging from blogs through scientific publications to protein databases. Furthermore, tagging systems dedicated for voluntary tagging of photos, films, books, etc. with free words are also becoming popular. The emerging large collections of tags associated with different objects are often referred to as folksonomies, highlighting their collaborative origin and the "flat" organization of the tags opposed to traditional hierarchical categorization. Adding a tag hierarchy corresponding to a given folksonomy can very effectively help narrowing or broadening the scope of search. Moreover, recommendation systems could also benefit from a tag hierarchy. PMID:24391901

  7. Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Walters, Rodney R.

    1985-01-01

    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)

  8. DESIGN, SYNTHESIS, AND APPLICATION OF THE TRIMETHOPRIM-BASED CHEMICAL TAG FOR LIVE CELL IMAGING

    PubMed Central

    Jing, Chaoran; Cornish, Virginia W.

    2013-01-01

    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 dihydrofolatereductase and the antibiotic trimethoprim and 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. Alternative protocols for synthesizing and using the covalent and the fluorogenic TMP-tags are also included. PMID:23839994

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

    PubMed

    Terpe, K

    2003-01-01

    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 proteins to facilitate purification and detection of recombinant proteins are well-recognized. Nevertheless, it is difficult to choose the right purification system for a specific protein of interest. This review gives an overview of the most frequently used and interesting systems: Arg-tag, calmodulin-binding peptide, cellulose-binding domain, DsbA, c-myc-tag, glutathione S-transferase, FLAG-tag, HAT-tag, His-tag, maltose-binding protein, NusA, S-tag, SBP-tag, Strep-tag, and thioredoxin. PMID:12536251

  10. Identification of Predictive Markers for Response to Neoadjuvant Chemoradiation in Rectal Carcinomas by Proteomic Isotope Coded Protein Label (ICPL) Analysis

    PubMed Central

    Croner, Roland S.; Sevim, Müzeyyen; Metodiev, Metodi V.; Jo, Peter; Ghadimi, Michael; Schellerer, Vera; Brunner, Maximillian; Geppert, Carol; Rau, Tilman; Stürzl, Michael; Naschberger, Elisabeth; Matzel, Klaus E.; Hohenberger, Werner; Lottspeich, Friedrich; Kellermann, Josef

    2016-01-01

    Neoadjuvant chemoradiation (nCRT) is an established procedure in stage union internationale contre le cancer (UICC) II/III rectal carcinomas. Around 53% of the tumours present with good tumor regression after nCRT, and 8%–15% are complete responders. Reliable selection markers would allow the identification of poor or non-responders prior to therapy. Tumor biopsies were harvested from 20 patients with rectal carcinomas, and stored in liquid nitrogen prior to therapy after obtaining patients’ informed consent (Erlangen-No.3784). Patients received standardized nCRT with 5-Fluoruracil (nCRT I) or 5-Fluoruracil ± Oxaliplatin (nCRT II) according to the CAO/ARO/AIO-04 protocol. After surgery, regression grading (Dworak) of the tumors was performed during histopathological examination of the specimens. Tumors were classified as poor (Dworak 1 + 2) or good (Dworak 3 + 4) responders. Laser capture microdissection (LCM) for tumor enrichment was performed on preoperative biopsies. Differences in expressed proteins between poor and good responders to nCRT I and II were identified by proteomic analysis (Isotope Coded Protein Label, ICPL™) and selected markers were validated by immunohistochemistry. Tumors of 10 patients were classified as histopathologically poor (Dworak 1 or 2) and the other 10 tumor samples as histopathologically good (Dworak 3 or 4) responders to nCRT after surgery. Sufficient material in good quality was harvested for ICPL analysis by LCM from all biopsies. We identified 140 differentially regulated proteins regarding the selection criteria and the response to nCRT. Fourteen of these proteins were synchronously up-regulated at least 1.5-fold after nCRT I or nCRT II (e.g., FLNB, TKT, PKM2, SERINB1, IGHG2). Thirty-five proteins showed a complete reciprocal regulation (up or down) after nCRT I or nCRT II and the rest was regulated either according to nCRT I or II. The protein expression of regulated proteins such as PLEC1, TKT, HADHA and TAGLN was validated successfully by immunohistochemistry. ICPL is a valid method to identify differentially expressed proteins in rectal carcinoma tissue between poor vs. good responders to nCRT. The identified protein markers may act as selection criteria for nCRT in the future, but our preliminary findings must be reproduced and validated in a prospective cohort. PMID:26861291

  11. Identification of Predictive Markers for Response to Neoadjuvant Chemoradiation in Rectal Carcinomas by Proteomic Isotope Coded Protein Label (ICPL) Analysis.

    PubMed

    Croner, Roland S; Sevim, Müzeyyen; Metodiev, Metodi V; Jo, Peter; Ghadimi, Michael; Schellerer, Vera; Brunner, Maximillian; Geppert, Carol; Rau, Tilman; Stürzl, Michael; Naschberger, Elisabeth; Matzel, Klaus E; Hohenberger, Werner; Lottspeich, Friedrich; Kellermann, Josef

    2016-01-01

    Neoadjuvant chemoradiation (nCRT) is an established procedure in stage union internationale contre le cancer (UICC) II/III rectal carcinomas. Around 53% of the tumours present with good tumor regression after nCRT, and 8%-15% are complete responders. Reliable selection markers would allow the identification of poor or non-responders prior to therapy. Tumor biopsies were harvested from 20 patients with rectal carcinomas, and stored in liquid nitrogen prior to therapy after obtaining patients' informed consent (Erlangen-No.3784). Patients received standardized nCRT with 5-Fluoruracil (nCRT I) or 5-Fluoruracil ± Oxaliplatin (nCRT II) according to the CAO/ARO/AIO-04 protocol. After surgery, regression grading (Dworak) of the tumors was performed during histopathological examination of the specimens. Tumors were classified as poor (Dworak 1 + 2) or good (Dworak 3 + 4) responders. Laser capture microdissection (LCM) for tumor enrichment was performed on preoperative biopsies. Differences in expressed proteins between poor and good responders to nCRT I and II were identified by proteomic analysis (Isotope Coded Protein Label, ICPL™) and selected markers were validated by immunohistochemistry. Tumors of 10 patients were classified as histopathologically poor (Dworak 1 or 2) and the other 10 tumor samples as histopathologically good (Dworak 3 or 4) responders to nCRT after surgery. Sufficient material in good quality was harvested for ICPL analysis by LCM from all biopsies. We identified 140 differentially regulated proteins regarding the selection criteria and the response to nCRT. Fourteen of these proteins were synchronously up-regulated at least 1.5-fold after nCRT I or nCRT II (e.g., FLNB, TKT, PKM2, SERINB1, IGHG2). Thirty-five proteins showed a complete reciprocal regulation (up or down) after nCRT I or nCRT II and the rest was regulated either according to nCRT I or II. The protein expression of regulated proteins such as PLEC1, TKT, HADHA and TAGLN was validated successfully by immunohistochemistry. ICPL is a valid method to identify differentially expressed proteins in rectal carcinoma tissue between poor vs. good responders to nCRT. The identified protein markers may act as selection criteria for nCRT in the future, but our preliminary findings must be reproduced and validated in a prospective cohort. PMID:26861291

  12. HaloTag Technology: A Versatile Platform for Biomedical Applications

    PubMed Central

    2015-01-01

    Exploration of protein function and interaction is critical for discovering links among genomics, proteomics, and disease state; yet, the immense complexity of proteomics found in biological systems currently limits our investigational capacity. Although affinity and autofluorescent tags are widely employed for protein analysis, these methods have been met with limited success because they lack specificity and require multiple fusion tags and genetic constructs. As an alternative approach, the innovative HaloTag protein fusion platform allows protein function and interaction to be comprehensively analyzed using a single genetic construct with multiple capabilities. This is accomplished using a simplified process, in which a variable HaloTag ligand binds rapidly to the HaloTag protein (usually linked to the protein of interest) with high affinity and specificity. In this review, we examine all current applications of the HaloTag technology platform for biomedical applications, such as the study of protein isolation and purification, protein function, protein–protein and protein–DNA interactions, biological assays, in vitro cellular imaging, and in vivo molecular imaging. In addition, novel uses of the HaloTag platform are briefly discussed along with potential future applications. PMID:25974629

  13. Tagged Prairie Dog

    USGS Multimedia Gallery

    This wild prairie dog has been tagged by scientists in an effort to study the efficacy of a USGS-developed oral sylvatic plague vaccine (SPV) to help immunize prairie dogs against plague. It was released after being tagged and after scientists took hair, whisker, and blood samples. I...

  14. Rapid analysis of biogenic amines from rice wine with isotope-coded derivatization followed by high performance liquid chromatography-tandem mass spectrometry.

    PubMed

    Cai, Yiping; Sun, Zhiwei; Chen, Guang; Liu, Xiaomei; You, Jinmao; Zhang, Caiqing

    2016-02-01

    A pair of isotope-coded derivatization reagents, d0-10-methyl-acridone-2-sulfonyl chloride (d0-MASC, light form) and d3-10-methyl-acridone-2-sulfonyl chloride (d3-MASC, heavy form), were used for labeling biogenic amines (BAs). On basis of the isotope-coded derivatization, a global isotope internal standard quantitative method for determining seven BAs by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed. The d0-MASC and d3-MASC can easily label BAs under mild conditions within 15 min at 50 C. The obtained light and heavy labeled BAs were monitored by the transitions of [M+H](+) ? 208 and [M+H](+) ? 211, respectively. Relative quantification of BAs was achieved by calculation of the peak area ratios of d0-MASC/d3-MASC labeled derivatives. Excellent linear responses for relative quantification were observed in the range of 1/10-10/1. The developed method has been successfully applied to the quantification of BAs in Chinese rice wine with recoveries ranging from 94.9% to 104.5%. PMID:26304364

  15. Protein-protein interactions of TAP-tagged protein kinases in rice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Eighty-eight rice (Oryza sativa) cDNAs encoding leaf expressed protein kinases (RLePKs) were fused to a Tandem Affinity Purification tag (TAPtag) and expressed in transgenic rice plants. The TAP-tagged RLePKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants a...

  16. TAG Advertisement Hardware

    NASA Technical Reports Server (NTRS)

    1994-01-01

    LaRc SI Material Overall photograph showing the material specimens, the graphite composite, the gold composite and the molded gears on a black background. These photos were used for the TAG CO-OP Public Relations and promotions

  17. Multiple affinity domains for the detection, purification and immobilization of recombinant proteins.

    PubMed

    Nilsson, J; Larsson, M; Ståhl, S; Nygren, P A; Uhlén, M

    1996-01-01

    Affinity systems based on specific molecular recognition are valuable tools for detection, purification and immobilization of recombinant proteins. Here, novel multipartite affinity fusion vectors were assembled and investigated to allow flexible binding and elution conditions. The rationale for the assembly of different combinations of affinity domains was to take advantage of the wide variety of molecular interactions of these domains for purification, solubilization, detection and immobilization. In total, seven different affinity tags representing five different types of tag-ligand interactions were studied: (i) monoclonal antibodies-peptides (T7-tag and FLAG peptide); (ii) streptavidin-peptide (Strep-tag); (iii) hexahistidyl-metal ions (His6-tag; (iv) bacterial receptors-serum proteins (staphyloccal protein A-Fc and streptococcal protein G-serum albumin); (v) streptavidin-biotin (in vivo biotinylated peptide). Selected tags were evaluated for the production and purification of Escherichia coli DNA polymerase I (Klenow fragment). On the basis of the results, a vector (pAff2c) was assembled using a novel combination of affinity domains: (i) an in vivo biotinylated peptide; (ii) a His6 sequence, and (iii) a highly soluble serum albumin binding region. Using these three affinities, a wide variety of conditions can be employed for both the binding and the elution steps. PMID:9174944

  18. Facets: Ersatz, Resource and Tag

    ERIC Educational Resources Information Center

    Frick, Martin H.

    2013-01-01

    Introduction: Faceted classification appears to be of utmost importance. Ersatz facets, resource faceting and tag faceting: The distinctions are drawn between facets and ersatz facets, and between faceted resources and faceted tags. Single tag resource faceting and multiple tag information object faceting: The basic features are explored of single

  19. Quantitative analysis of aberrant fatty acid composition of zebrafish hepatic lipids induced by organochlorine pesticide using stable isotope-coded transmethylation and gas chromatography-mass spectrometry.

    PubMed

    Zhong, Hongying; Dong, Linjie; Dong, Qingjian; Ke, Changshu; Fu, Jieying; Wang, Xiaoli; Liu, Cong; Dai, Ling

    2012-07-01

    Organochlorine pesticides have been extensively used worldwide for agricultural purposes. Due to their resistance to metabolism, a major public health concern has been raised. Aberrant hepatic lipid composition has been a hallmark of many liver diseases associated with exposure to various toxins and chemicals. And thus lots of efforts have been focused on the development of analytical techniques that can rapidly and quantitatively determine the changes in fatty acid composition of hepatic lipids. In this work, changes in fatty acid composition of hepatic lipids in response to DDT (dichlorodiphenyltrichloroethane) exposure were quantitatively analyzed by a gas chromatography-mass spectrometric approach based on stable isotope-coded transmethylation. It has been quantitatively demonstrated that polyunsaturated fatty acids including C20:3n3, C20:4n6, and C22:6n3 decrease in response to DDT exposure. However, saturated long chain fatty acids including C16:0, C18:0, as well as monounsaturated long chain fatty acid C18:1n9 consistently increase in a DDT-concentration-dependent manner. In particular, much higher changes in the level of hepatic C16:0 and C18:0 for male fish were observed than that for female fish. These experimental results are in accordance with qualitative histopathological analysis that revealed liver morphological alterations. The stable isotope-coded mass spectrometric approach provides a reliable means for investigating hepatotoxicity associated with fatty acid synthesis, desaturation, mitochondrial beta-oxidation, and lipid mobilization. It should be useful in elucidation of hepatotoxic mechanisms and safety assessment of environmental toxins. PMID:22648165

  20. Deciphering Systemic Wound Responses of the Pumpkin Extrafascicular Phloem by Metabolomics and Stable Isotope-Coded Protein Labeling1[C][W

    PubMed Central

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

    2012-01-01

    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

  1. Metal-chelate affinity precipitation of proteins using responsive polymers.

    PubMed

    Mattiasson, Bo; Kumar, Ashok; Ivanov, Alexander E; Galaev, Igor Y

    2007-01-01

    Affinity precipitation of proteins uses polymers capable of reversible soluble-insoluble transitions in response to small environmental changes (temperature, pH or solvent composition). Here we describe protocols for (i) the synthesis of responsive polymers with specific affinity to target proteins and (ii) the purification of proteins using these polymers. The purification is based on precipitation of the affinity complex between the protein and the polymer, which is induced by environmental changes. This separation strategy is simpler and more cost effective than conventional affinity column chromatography. Specifically, we describe the synthesis of thermoresponsive 1-vinylimidazole:N-isopropylacrylamide copolymers. The whole procedure takes 2-3 h when applied to purification of recombinant His-tag proteins or proteins with natural metal binding groups by means of metal chelate affinity precipitation. Optimization of the polymer composition and the type of chelating ions allows for target protein yields of 80% and higher. PMID:17401356

  2. Avoiding the ends: internal epitope tagging of proteins using transposon Tn7.

    PubMed

    Zordan, Rebecca E; Beliveau, Brian J; Trow, Jonathan A; Craig, Nancy L; Cormack, Brendan P

    2015-05-01

    Peptide tags fused to proteins are used in a variety of applications, including as affinity tags for purification, epitope tags for immunodetection, or fluorescent protein tags for visualization. However, the peptide tags can disrupt the target protein function. When function is disrupted by fusing a peptide to either the N or C terminus of the protein of interest, identifying alternative ways to create functional tagged fusion proteins can be difficult. Here, we describe a method to introduce protein tags internal to the coding sequence of a target protein. The method employs in vitro Tn7-transposon mutagenesis of plasmids for random introduction of the tag, followed by subsequent Gateway cloning steps to isolate alleles with mutations in the coding sequence of the target gene. The Tn7-epitope cassette is designed such that essentially all of the transposon is removed through restriction enzyme digestion, leaving only the protein tag at diverse sites internal to the ORF. We describe the use of this system to generate a panel of internally epitope-tagged versions of the Saccharomyces cerevisiae GPI-linked membrane protein Dcw1 and the Candida glabrata transcriptional regulator Sir3. This internal protein tagging system is, in principle, adaptable to tag proteins in any organism for which Gateway-adapted expression vectors exist. PMID:25745023

  3. Affine Toda Sutherland systems

    NASA Astrophysics Data System (ADS)

    Khare, Avinash; Loris, I.; Sasaki, R.

    2004-02-01

    A cross between two well-known integrable multi-particle dynamics, an affine Toda molecule and a Sutherland system, is introduced for any affine root system. Though it is not completely integrable but partially integrable, or quasi-exactly solvable, it inherits many remarkable properties from the parents. The equilibrium position is algebraic, i.e. proportional to the Weyl vector. The frequencies of small oscillations near equilibrium are proportional to the affine Toda masses, which are essential ingredients of the exact factorizable S-matrices of affine Toda field theories. Some lower lying frequencies are integer times a coupling constant for which the corresponding exact quantum eigenvalues and eigenfunctions are obtained. An affine Toda-Calogero system, with a corresponding rational potential, is also discussed.

  4. Prairie Dog Tagging

    USGS Multimedia Gallery

    An anaesthetized prairie dog is tagged in Wind Cave National Park.  Over 30 organizations and agencies are testing a USGS-developed oral vaccine to prevent the spread of plague in prairie dogs. If successful, the sylvatic plague vaccine could help protect endangered black-f...

  5. Project 8 Tags

    ERIC Educational Resources Information Center

    Ramos-Burrows, Michele

    2010-01-01

    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

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

    PubMed

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

    2013-11-01

    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-tagII 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-tagII is affinity engineered streptavidin, named Strep-Tactin. Strep-tagII 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-tagII 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-tagII moieties connected by a short linker. The resulting avidity effect, i.e., the combined synergistic binding of two Strep-tagII 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-tagII, 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

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

    PubMed Central

    N Peterson, Scott; Kwon, Keehwan

    2012-01-01

    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. PMID:23115610

  8. Special Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Parikh, Indu; Cuatrecasas, Pedro

    1985-01-01

    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)

  9. Quantitative isomer-specific N-glycan fingerprinting using isotope coded labeling and high performance liquid chromatography-electrospray ionization-mass spectrometry with graphitic carbon stationary phase.

    PubMed

    Michael, Claudia; Rizzi, Andreas M

    2015-02-27

    Glycan reductive isotope labeling (GRIL) using (12)C6-/(13)C6-aniline as labeling reagent is reported with the aim of quantitative N-glycan fingerprinting. Porous graphitized carbon (PGC) as stationary phase in capillary scale HPLC coupled to electrospray mass spectrometry with time of flight analyzer was applied for the determination of labeled N-glycans released from glycoproteins. The main benefit of using stable isotope-coding in the context of comparative glycomics lies in the improved accuracy and precision of the quantitative analysis in combined samples and in the potential of correcting for structure-dependent incomplete enzymatic release of oligosaccharides when comparing identical target proteins. The method was validated with respect to mobile phase parameters, reproducibility, accuracy, linearity and limit of detection/quantification (LOD/LOQ) using test glycoproteins. It is shown that the developed method is capable of determining relative amounts of N-glycans (including isomers) comparing two samples in one single HPLC-MS run. The analytical potential and usefulness of GRIL in combination with PGC-ESI-TOF-MS is demonstrated comparing glycosylation in human monoclonal antibodies produced in Chinese hamster ovary cells (CHO) and hybridoma cell lines. PMID:25638265

  10. Comparative analysis of S-fatty acylation of gel-separated proteins by stable isotope-coded fatty acid transmethylation and mass spectrometry.

    PubMed

    Dong, Linjie; Li, Jianjian; Li, Lun; Li, Tingting; Zhong, Hongying

    2011-09-01

    Covalent attachment of palmitic acid or other fatty acids to the thiol groups of cysteine residues of proteins through reversible thioester bonds has an important role in the regulation of diverse biological processes. We describe here the development of a mass spectrometry protocol based on stable isotope-coded fatty acid transmethylation (iFAT) for qualitative and comparative analysis of protein S-fatty acylation under different experimental conditions. In this approach, cellular proteins extracted from different cell states are separated by SDS-PAGE and then the gel is stained with either Coomassie blue or Nile red for improved sensitivity. Protein bands are excised and then an in-gel stable iFAT procedure is performed. The fatty acid methyl esters resulting from derivatization with d0- and d3-methanol are identified by mass spectrometry. By measuring the intensities of labeled and unlabeled fragment ion pairs of fatty acid methyl esters, the levels of S-fatty acylation in different cells or tissues can be compared. This approach has been applied to monitor the changes of S-fatty acylation of zebrafish liver proteome in response to environmental dichlorodiphenyltrichloroethane exposure. Compared with the approach using metabolic incorporation of radioactive fatty acid analogs, it is not only simple and effective but also eliminates the hazards of handling radioactive isotopes. PMID:21886103

  11. Differential proteomic analysis using isotope-coded protein-labeling strategies: comparison, improvements and application to simulated microgravity effect on Cupriavidus metallidurans CH34.

    PubMed

    Leroy, Baptiste; Rosier, Caroline; Erculisse, Vanessa; Leys, Natalie; Mergeay, Max; Wattiez, Ruddy

    2010-06-01

    Among differential proteomic methods based on stable isotopic labeling, isotope-coded protein labeling (ICPL) is a recent non-isobaric technique devised to label primary amines found in proteins. ICPL overcomes some of the disadvantages found in other chemical-labeling techniques, such as iTRAQ or ICAT. However, previous analyses revealed that more than 30% of the proteins identified in regular ICPL generally remain unquantified. In this study, we describe a modified version of ICPL, named Post-digest ICPL, that makes it possible to label and thus to quantify all the peptides in a sample (bottom-up approach). Optimization and validation of this Post-digest ICPL approach were performed using a standard protein mixture and complex protein samples. Using this strategy, the number of proteins that were identified and quantified was greatly increased in comparison with regular ICPL and cICAT approaches. The pros and cons of this improvement are discussed. This complementary approach to traditional ICPL was applied to the analysis of modification of protein abundances in the model bacterium Cupriavidus metallidurans CH34 after cultivation under simulated microgravity. In this context, two different systems - a 2-D clinorotation and 3-D random positioning device - were used and the results were compared and discussed. PMID:20391527

  12. Social Tagging Recommender Systems

    NASA Astrophysics Data System (ADS)

    Marinho, Leandro Balby; Nanopoulos, Alexandros; Schmidt-Thieme, Lars; Jschke, Robert; Hotho, Andreas; Stumme, Gerd; Symeonidis, Panagiotis

    The new generation of Web applications known as (STS) is successfully established and poised for continued growth. STS are open and inherently social; features that have been proven to encourage participation. But while STS bring new opportunities, they revive old problems, such as information overload. Recommender Systems are well known applications for increasing the level of relevant content over the "noise" that continuously grows as more and more content becomes available online. In STS however, we face new challenges. Users are interested in finding not only content, but also tags and even other users. Moreover, while traditional recommender systems usually operate over 2-way data arrays, STS data is represented as a third-order tensor or a hypergraph with hyperedges denoting (user, resource, tag) triples. In this chapter, we survey the most recent and state-of-the-art work about a whole new generation of recommender systems built to serve STS.We describe (a) novel facets of recommenders for STS, such as user, resource, and tag recommenders, (b) new approaches and algorithms for dealing with the ternary nature of STS data, and (c) recommender systems deployed in real world STS. Moreover, a concise comparison between existing works is presented, through which we identify and point out new research directions.

  13. Isolation of transcription factor complexes by in vivo biotinylation tagging and direct binding to streptavidin beads.

    PubMed

    Rodriguez, Patrick; Braun, Harald; Kolodziej, Katarzyna E; de Boer, Ernie; Campbell, Jennifer; Bonte, Edgar; Grosveld, Frank; Philipsen, Sjaak; Strouboulis, John

    2006-01-01

    Efficient tagging methodologies are an integral aspect of protein complex characterization by proteomic approaches. Owing to the very high affinity of biotin for avidin and streptavidin, biotinylation tagging offers an attractive approach for the efficient purification of protein complexes. The very high affinity of the biotin/(strept)avidin system also offers the potential for the single-step capture of lower abundance protein complexes, such as transcription factor complexes. The identification of short peptide tags that are efficiently biotinylated by the bacterial BirA biotin ligase led to an approach for the single-step purification of transcription factor complexes by specific in vivo biotinylation tagging. A short sequence tag fused N-terminally to the transcription factor of interest is very efficiently biotinylated by BirA coexpressed in the same cells, as was demonstrated by the tagging of the essential hematopoietic transcription factor GATA-1. The direct binding to streptavidin of biotinylated GATA-1 in nuclear extracts resulted in the single-step capture of the tagged factor and associated proteins, which were eluted and identified by mass spectrometry. This led to the characterization of several distinct GATA-1 complexes with other transcription factors and chromatin remodeling cofactors, which are involved in activation and repression of gene targets. Thus, BirA-mediated tagging is an efficient approach for the direct capture and characterization of transcription factor complexes. PMID:16888367

  14. His6 tag-assisted chemical protein synthesis

    NASA Astrophysics Data System (ADS)

    Bang, Duhee; Kent, Stephen B. H.

    2005-04-01

    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. affinity purification | native chemical ligation

  15. Social Tagging of Mission Data

    NASA Technical Reports Server (NTRS)

    Norris, Jeffrey S.; Wallick, Michael N.; Joswig, Joseph C.; Powell, Mark W.; Torres, Recaredo J.; Mittman, David S.; Abramyan, Lucy; Crockett, Thomas M.; Shams, Khawaja S.; Fox, Jason M.; Pyrzak, Guy; Vaughn, Michael B.

    2010-01-01

    Mars missions will generate a large amount of data in various forms, such as daily plans, images, and scientific information. Often, there is a semantic linkage between images that cannot be captured automatically. Software is needed that will provide a method for creating arbitrary tags for this mission data so that items with a similar tag can be related to each other. The tags should be visible and searchable for all users. A new routine was written to offer a new and more flexible search option over previous applications. This software allows users of the MSLICE program to apply any number of arbitrary tags to a piece of mission data through a MSLICE search interface. The application of tags creates relationships between data that did not previously exist. These tags can be easily removed and changed, and contain enough flexibility to be specifically configured for any mission. This gives users the ability to quickly recall or draw attention to particular pieces of mission data, for example: Give a semantic and meaningful description to mission data; for example, tag all images with a rock in them with the tag "rock." Rapidly recall specific and useful pieces of data; for example, tag a plan as"driving template." Call specific data to a user s attention; for example, tag a plan as "for:User." This software is part of the MSLICE release, which was written in Java. It will run on any current Windows, Macintosh, or Linux system.

  16. Application of Screening Experimental Designs to Assess Chromatographic Isotope Effect upon Isotope-Coded Derivatization for Quantitative Liquid ChromatographyMass Spectrometry

    PubMed Central

    2015-01-01

    Isotope effect may cause partial chromatographic separation of labeled (heavy) and unlabeled (light) isotopologue pairs. Together with a simultaneous matrix effect, this could lead to unacceptable accuracy in quantitative liquid chromatographymass spectrometry assays, especially when electrospray ionization is used. Four biologically relevant reactive aldehydes (acrolein, malondialdehyde, 4-hydroxy-2-nonenal, and 4-oxo-2-nonenal) were derivatized with light or heavy (d3-, 13C6-, 15N2-, or 15N4-labeled) 2,4-dinitrophenylhydrazine and used as model compounds to evaluate chromatographic isotope effects. For comprehensive assessment of retention time differences between light/heavy pairs under various gradient reversed-phase liquid chromatography conditions, major chromatographic parameters (stationary phase, mobile phase pH, temperature, organic solvent, and gradient slope) and different isotope labelings were addressed by multiple-factor screening using experimental designs that included both asymmetrical (Addelman) and PlackettBurman schemes followed by statistical evaluations. Results confirmed that the most effective approach to avoid chromatographic isotope effect is the use of 15N or 13C labeling instead of deuterium labeling, while chromatographic parameters had no general influence. Comparison of the alternate isotope-coded derivatization assay (AIDA) using deuterium versus 15N labeling gave unacceptable differences (>15%) upon quantifying some of the model aldehydes from biological matrixes. On the basis of our results, we recommend the modification of the AIDA protocol by replacing d3-2,4-dinitrophenylhydrazine with 15N- or 13C-labeled derivatizing reagent to avoid possible unfavorable consequences of chromatographic isotope effects. PMID:24922593

  17. Methods for differential and quantitative analyses of brain neurosteroid levels by LC/MS/MS with ESI-enhancing and isotope-coded derivatization.

    PubMed

    Higashi, Tatsuya; Aiba, Naoto; Tanaka, Tomoya; Yoshizawa, Kazumi; Ogawa, Shoujiro

    2016-01-01

    The analysis of changes in the brain neurosteroid (NS) levels due to various stimuli can contribute to the elucidation of their physiological roles, and the discovery and development of new antipsychotic agents targeting neurosteroidogenesis. We developed methods for the differential and quantitative analyses of the brain levels of allopregnanolene (AP) and its precursor, pregnenolone (PREG), using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) combined with derivatization using 2-hydrazino-1-methylpyridine (HMP) and its isotope-coded analogue, (2)H3-HMP (d-HMP). For the differential analysis, the brain sample of an untreated rat was derivatized with HMP, while the brain sample of a treated (stressed or drug-administered) rat was derivatized with d-HMP. The two derivatives were mixed and then subjected to LC/ESI-MS/MS. The stress- and drug (clozapine and fluoxetine)-evoked increases in the brain AP and PREG levels were accurately analyzed by the developed method. It was also possible to determine the absolute concentrations of the brain steroids when a deuterium-coded moiety was introduced to the standard steroids of known amounts by the derivatization and the resulting derivatives were used as internal standards. The HMP-derivatization enabled the highly sensitive detection and the use of d-HMP significantly improved the assay precision [the intra- (n=5) and inter-assay (n=5) relative standard deviations did not exceed 13.7%] and accuracy (analytical recovery ranged from 98.7 to 106.7%). PMID:26355769

  18. Antenna for passive RFID tags

    NASA Astrophysics Data System (ADS)

    Schiopu, Paul; Manea, Adrian; Cristea, Ionica; Grosu, Neculai; Vladescu, Marian; Craciun, Anca-Ileana; Craciun, Alexandru

    2015-02-01

    Minuscule devices, called RFID tags are attached to objects and persons and emit information which positioned readers may capture wirelessly. Many methods of identification have been used, but that of most common is to use a unique serial number for identification of person or object. RFID tags can be characterized as either active or passive [1,2]. Traditional passive tags are typically in "sleep" state until awakened by the reader's emitted field. In passive tags, the reader's field acts to charge the capacitor that powers the badge and this can be a combination of antenna and barcodes obtained with SAW( Surface Acoustic Wave) devices [1,2,3] . The antenna in an RFID tag is a conductive element that permits the tag to exchange data with the reader. The paper contribution are targeted to antenna for passive RFID tags. The electromagnetic field generated by the reader is somehow oriented by the reader antenna and power is induced in the tag only if the orientation of the tag antenna is appropriate. A tag placed orthogonal to the reader yield field will not be read. This is the reason that guided manufacturers to build circular polarized antenna capable of propagating a field that is alternatively polarized on all planes passing on the diffusion axis. Passive RFID tags are operated at the UHF frequencies of 868MHz (Europe) and 915MHz (USA) and at the microwave frequencies of 2,45 GHz and 5,8 GHz . Because the tags are small dimensions, in paper, we present the possibility to use circular polarization microstrip antenna with fractal edge [2].

  19. Review on SAW RFID tags.

    PubMed

    Plessky, Victor P; Reindl, Leonhard M

    2010-03-01

    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

  20. Buddy Tag CONOPS and Requirements.

    SciTech Connect

    Brotz, Jay Kristoffer; Deland, Sharon M.

    2015-12-01

    This document defines the concept of operations (CONOPS) and the requirements for the Buddy Tag, which is conceived and designed in collaboration between Sandia National Laboratories and Princeton University under the Department of State Key VerificationAssets Fund. The CONOPS describe how the tags are used to support verification of treaty limitations and is only defined to the extent necessary to support a tag design. The requirements define the necessary functions and desired non-functional features of the Buddy Tag at a high level

  1. A Highly Efficient Multifunctional Tandem Affinity Purification Approach Applicable to Diverse Organisms*

    PubMed Central

    Ma, Hanhui; McLean, Janel R.; Chao, Lucy Fang-I; Mana-Capelli, Sebastian; Paramasivam, Murugan; Hagstrom, Kirsten A.; Gould, Kathleen L.; McCollum, Dannel

    2012-01-01

    Determining the localization, binding partners, and secondary modifications of individual proteins is crucial for understanding protein function. Several tags have been constructed for protein localization or purification under either native or denaturing conditions, but few tags permit all three simultaneously. Here, we describe a multifunctional tandem affinity purification (MAP) method that is both highly efficient and enables protein visualization. The MAP tag utilizes affinity tags inserted into an exposed surface loop of mVenus offering two advantages: (1) mVenus fluorescence can be used for protein localization or FACS-based selection of cell lines; and (2) spatial separation of the affinity tags from the protein results in high recovery and reduced variability between proteins. MAP purification was highly efficient in multiple organisms for all proteins tested. As a test case, MAP combined with liquid chromatography-tandem MS identified known and new candidate binding partners and modifications of the kinase Plk1. Thus the MAP tag is a new powerful tool for determining protein modification, localization, and interactions. PMID:22474084

  2. Structure of classical affine and classical affine fractional W -algebras

    NASA Astrophysics Data System (ADS)

    Suh, Uhi Rinn

    2015-01-01

    We introduce a classical BRST complex (See Definition 3.2.) and show that one can construct a classical affine W -algebra via the complex. This definition clarifies that classical affine W -algebras can be considered as quasi-classical limits of quantum affine W -algebras. We also give a definition of a classical affine fractional W -algebra as a Poisson vertex algebra. As in the classical affine case, a classical affine fractional W -algebra has two compatible ?-brackets and is isomorphic to an algebra of differential polynomials as a differential algebra. When a classical affine fractional W -algebra is associated to a minimal nilpotent, we describe explicit forms of free generators and compute ?-brackets between them. Provided some assumptions on a classical affine fractional W -algebra, we find an infinite sequence of integrable systems related to the algebra, using the generalized Drinfel'd and Sokolov reduction.

  3. Affine fusion tadpoles

    NASA Astrophysics Data System (ADS)

    Urichuk, Andrew

    Fusion dimensions are integer-valued quantities equal to the dimensions of the spaces of conformal blocks, which describe the interactions of a conformal field theory (CFT). Our focus was on the Wess-Zumino-Witten models, a particularly interesting type of CFT, whose primary fields correspond to representations of affine Lie groups. Arguably, affine fusion tadpoles are the simplest g ? 1 fusion dimension, having only a single incoming field and g = 1. We study the symmetries of the SU(N) tadpole and Verlinde formula with the intention of finding a non-negative-integer decomposition. Such a decomposition might be indicative of a combinatorial atom for fusion, which could suggest a new combinatorial account of fusion dimensions. From produced tables we found that tadpole values appeared to be polynomial in the level k. Several conjectures were made and we sketch a method obtaining general forms of SU(N) tadpoles via dominant weight sums.

  4. Tandem Affinity Purification in Drosophila Heads and Ovaries

    PubMed Central

    Pepper, Anita; Bhogal, Balpreet; Jongens, Thomas

    2016-01-01

    Tandem affinity purification (TAP) (Pugi et al., 2001; Rigaut et al., 1999) is a method that uses a tagging approach of a target protein of interest for a two-step purification scheme in order to pull down protein complexes under native conditions and expression levels. The TAP tag consists of three components: a calmodulin-binding peptide, a Tobacco etch virus (TEV) protease cleavage site and Protein A which is an immunoglobulin G (IgG)-binding domain. This protocol was modified from the original methodology used in yeast cells (Pugi et al., 2001; Rigaut et al., 1999) for isolation of protein complexes from Drosophila heads and ovaries expressing a TAP tagged protein of interest. To determine in vivo binding partners of the Drosophila fragile X protein (dFMR1), we developed a transgenic strain of flies expressing a recombinant form of dFMR1 with a carboxy-terminal TAP tag (Tsai and Carstens, 2006). To ensure that the construct was expressed at wild-type levels, we engineered this form of the tagged protein in the context of a genomic rescue construct that rescued a mutant sterility phenotype. The purification process was performed using mild conditions to maintain native protein interactions. For TAP methods in Drosophila S2 cell culture, we have successfully used a protocol previously published by Tsai and Carstens (Tsai and Carstens, 2006; Bhogal et al., 2011).

  5. Overexpression of Soluble Recombinant Human Lysyl Oxidase by Using Solubility Tags: Effects on Activity and Solubility

    PubMed Central

    Smith, Madison A.; Gonzalez, Jesica; Hussain, Anjum; Oldfield, Rachel N.; Johnston, Kathryn A.; Lopez, Karlo M.

    2016-01-01

    Lysyl oxidase is an important extracellular matrix enzyme that has not been fully characterized due to its low solubility. In order to circumvent the low solubility of this enzyme, three solubility tags (Nus-A, Thioredoxin (Trx), and Glutathione-S-Transferase (GST)) were engineered on the N-terminus of mature lysyl oxidase. Total enzyme yields were determined to be 1.5 mg for the Nus-A tagged enzyme (0.75 mg/L of media), 7.84 mg for the Trx tagged enzyme (3.92 mg/L of media), and 9.33 mg for the GST tagged enzyme (4.67 mg/L of media). Enzymatic activity was calculated to be 0.11 U/mg for the Nus-A tagged enzyme and 0.032 U/mg for the Trx tagged enzyme, and no enzymatic activity was detected for the GST tagged enzyme. All three solubility-tagged forms of the enzyme incorporated copper; however, the GST tagged enzyme appears to bind adventitious copper with greater affinity than the other two forms. The catalytic cofactor, lysyl tyrosyl quinone (LTQ), was determined to be 92% for the Nus-A and Trx tagged lysyl oxidase using the previously reported extinction coefficient of 15.4 mM−1 cm−1. No LTQ was detected for the GST tagged lysyl oxidase. Given these data, it appears that Nus-A is the most suitable tag for obtaining soluble and active recombinant lysyl oxidase from E. coli culture. PMID:26942005

  6. Overexpression of Soluble Recombinant Human Lysyl Oxidase by Using Solubility Tags: Effects on Activity and Solubility.

    PubMed

    Smith, Madison A; Gonzalez, Jesica; Hussain, Anjum; Oldfield, Rachel N; Johnston, Kathryn A; Lopez, Karlo M

    2016-01-01

    Lysyl oxidase is an important extracellular matrix enzyme that has not been fully characterized due to its low solubility. In order to circumvent the low solubility of this enzyme, three solubility tags (Nus-A, Thioredoxin (Trx), and Glutathione-S-Transferase (GST)) were engineered on the N-terminus of mature lysyl oxidase. Total enzyme yields were determined to be 1.5?mg for the Nus-A tagged enzyme (0.75?mg/L of media), 7.84?mg for the Trx tagged enzyme (3.92?mg/L of media), and 9.33?mg for the GST tagged enzyme (4.67?mg/L of media). Enzymatic activity was calculated to be 0.11?U/mg for the Nus-A tagged enzyme and 0.032?U/mg for the Trx tagged enzyme, and no enzymatic activity was detected for the GST tagged enzyme. All three solubility-tagged forms of the enzyme incorporated copper; however, the GST tagged enzyme appears to bind adventitious copper with greater affinity than the other two forms. The catalytic cofactor, lysyl tyrosyl quinone (LTQ), was determined to be 92% for the Nus-A and Trx tagged lysyl oxidase using the previously reported extinction coefficient of 15.4?mM(-1?)cm(-1). No LTQ was detected for the GST tagged lysyl oxidase. Given these data, it appears that Nus-A is the most suitable tag for obtaining soluble and active recombinant lysyl oxidase from E. coli culture. PMID:26942005

  7. Expression and purification of soluble His(6)-tagged TEV protease.

    PubMed

    Tropea, Joseph E; Cherry, Scott; Waugh, David S

    2009-01-01

    This chapter describes a simple method for overproducing a soluble form of the tobacco etch virus (TEV) protease in Escherichia coli and purifying it to homogeneity so that it may be used as a reagent for removing affinity tags from recombinant proteins by site-specific endoproteolysis. The protease is initially produced as a fusion to the C-terminus of E. coli maltose binding protein (MBP), which causes it to accumulate in a soluble and active form rather than in inclusion bodies. The fusion protein subsequently cleaves itself in vivo to remove the MBP moiety, yielding a soluble TEV protease catalytic domain with an N-terminal polyhistidine tag. The His-tagged TEV protease can be purified in two steps using immobilized metal affinity chromatography (IMAC) followed by gel filtration. An S219V mutation in the protease reduces its rate of autolysis by approximately 100-fold and also gives rise to an enzyme with greater catalytic efficiency than the wild-type protease. PMID:18988033

  8. D-TAG: erasing the tag of gang membership.

    PubMed

    Gurke, B; Armstrong, M L

    1997-04-01

    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

  9. Tagging insulin in microgravity

    NASA Technical Reports Server (NTRS)

    Dobeck, Michael; Nelson, Ronald S.

    1992-01-01

    Knowing the exact subcellular sites of action of insulin in the body has the potential to give basic science investigators a basis from which a cause and cure for this disease can be approached. The goal of this project is to create a test reagent that can be used to visualize these subcellular sites. The unique microgravity environment of the Shuttle will allow the creation of a reagent that has the possibility of elucidating the subcellular sites of action of insulin. Several techniques have been used in an attempt to isolate the sites of action of items such as insulin. One of these is autoradiography in which the test item is obtained from animals fed radioactive materials. What is clearly needed is to visualize individual insulin molecules at their sites of action. The insulin tagging process to be used on G-399 involves the conjugation of insulin molecules with ferritin molecules to create a reagent that will be used back on Earth in an attempt to elucidate the sites of action of insulin.

  10. Quantum tagging for tags containing secret classical data

    SciTech Connect

    Kent, Adrian

    2011-08-15

    Various authors have considered schemes for quantum tagging, that is, authenticating the classical location of a classical tagging device by sending and receiving quantum signals from suitably located distant sites, in an environment controlled by an adversary whose quantum information processing and transmitting power is potentially unbounded. All of the schemes proposed elsewhere in the literature assume that the adversary is able to inspect the interior of the tagging device. All of these schemes have been shown to be breakable if the adversary has unbounded predistributed entanglement. We consider here the case in which the tagging device contains a finite key string shared with distant sites but kept secret from the adversary, and show this allows the location of the tagging device to be authenticated securely and indefinitely. Our protocol relies on quantum key distribution between the tagging device and at least one distant site, and demonstrates a new practical application of quantum key distribution. It also illustrates that the attainable security in position-based cryptography can depend crucially on apparently subtle details in the security scenario considered.

  11. A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae

    PubMed Central

    Carrick, Brian H.; Hao, Linxuan; Smaldino, Philip J.; Engelke, David R.

    2015-01-01

    Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs) using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant CelTag DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies. PMID:26715090

  12. Engineering foot-and-mouth disease virus serotype O IND R2/1975 for one-step purification by immobilized metal affinity chromatography.

    PubMed

    Biswal, Jitendra K; Bisht, Punam; Subramaniam, Saravanan; Ranjan, Rajeev; Sharma, Gaurav K; Pattnaik, Bramhadev

    2015-09-01

    Immobilized metal affinity chromatography (IMAC) allows for the efficient protein purification via metal affinity tag such as hexa-histidine (His6) sequence. To develop a new chromatography strategy for the purification and concentration of foot-and-mouth disease virus (FMDV) particles, we inserted the His6-tag at the earlier reported site in the VP1 G-H loop of the FMD virus serotype O vaccine strain IND R2/1975. Display of the His6-tag on the capsid surface, endowed the virus with an increased affinity for immobilized nickel ions. We demonstrated that the His6-tagged FMDV could be produced to high titre and purified from the infected BHK-21 cell lysates by IMAC efficiently. Further, a 1150-fold reduction in protein contaminant level and an 8400-fold reduction in DNA contaminant level were achieved in the IMAC purification of His6-tagged FMDV. Through various functional assays it has been found that the tagged virus retains its functionality and infectivity similar to the non-tagged virus. The affinity purification of the His6-tagged FMDV may offer a feasible, alternative approach to the current methods of FMDV antigen purification, concentration and process scalability. PMID:26123433

  13. TAG Based Skimming In ATLAS

    NASA Astrophysics Data System (ADS)

    Doherty, T.; Cranshaw, J.; Hrivnac, J.; Slater, M.; Nowak, M.; Quilty, D.; Zhang, Q.

    2012-12-01

    The ATLAS detector at the LHC takes data at 200-500 Hz for several months per year accumulating billions of events for hundreds of physics analyses. TAGs are event-level metadata allowing a quick search for interesting events based on selection criteria defined by the user. They are stored in a file-based format as well as in relational databases. The overall TAG system architecture encompasses a range of interconnected services that provide functionality for the required use cases such as event selection, display, extraction and skimming. Skimming can be used to navigate to any of the pre-TAG data products. The services described in this paper address use cases that range in scale from selecting a handful of interesting events for an analysis specific study to creating physics working group samples on the ATLAS production system. This paper will focus on the workflow aspects involved in creating pre and post TAG data products from a TAG selection using the Grid in the context of the overall TAG system architecture. The emphasis will be on the range of demands that the implemented use cases place on these workflows and on the infrastructure. The tradeoffs of various workflow strategies will be discussed including scalability issues and other concerns that occur when integrating with data management and production systems.

  14. Affinity Pull-Down of Proteins Using Anti-FLAG M2 Agarose Beads

    PubMed Central

    Gerace, Erica; Moazed, Danesh

    2016-01-01

    FLAG is an affinity tag widely used for rapid and highly specific one-step protein purification. Native elution of protein from anti-FLAG antibody resins allows the identification of protein and nucleic acid binding partners and functional analysis using biochemical activity assays. PMID:26096505

  15. Affinity chromatography using biocompatible and reusable biotinylated membranes.

    PubMed

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

    2007-11-01

    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

  16. Tunable Charge Tags for Electron-Based Methods of Peptide Sequencing: Design and Applications

    NASA Astrophysics Data System (ADS)

    Zimnicka, Magdalena; Moss, Christopher L.; Chung, Thomas W.; Hui, Renjie; Ture?ek, Frantiek

    2012-04-01

    Charge tags using basic auxiliary functional groups 6-aminoquinolinylcarboxamido, 4-aminopyrimidyl-1-methylcarboxamido, 2-aminobenzoimidazolyl-1-methylcarboxamido, and the fixed-charge 4-(dimethylamino)pyridyl-1-carboxamido moiety are evaluated as to their properties in electron transfer dissociation mass spectra of arginine C-terminated peptides. The neutral tags have proton affinities that are competitive with those of amino acid residues in peptides. Charge reduction by electron transfer from fluoranthene anion-radicals results in peptide backbone dissociations that improve sequence coverage by providing extensive series of N-terminal c-type fragments without impeding the formation of C-terminal z fragments. Comparison of ETD mass spectra of free and tagged peptides allows one to resolve ambiguities in fragment ion assignment through mass shifts of c ions. Simple chemical procedures are reported for N-terminal tagging of Arg-containing tryptic peptides.

  17. Dimerization Capacities of FGF2 Purified with or without Heparin-Affinity Chromatography

    PubMed Central

    Chiu, Liang-Yuan; Taouji, Said; Moroni, Elisabetta; Colombo, Giorgio; Chevet, Eric; Sue, Shih-Che; Bikfalvi, Andreas

    2014-01-01

    Fibroblast growth factor-2 (FGF2) is a pleiotropic growth factor exhibiting a variety of biological activities. In this article, we studied the capacity of FGF2 purified with or without heparin affinity chromatography to self-associate. Analyzing the NMR HSQC spectra for different FGF2 concentrations, heparin-affinity purified FGF2 showed perturbations that indicate dimerization and are a higher-order oligomerization state. HSQC perturbation observed with different FGF2 concentrations revealed a heparin-binding site and two dimer interfaces. Thus, with increasing protein concentrations, FGF2 monomers make contacts with each other and form dimers or higher order oligomers. On the contrary, FGF2 purified with ion-exchange chromatography did not show similar perturbation indicating that self-association of FGF2 is eliminated if purification is done without heparin-affinity chromatography. The HSQC spectra of heparin-affinity purified FGF2 can be reproduced to some extent by adding heparin tetra-saccharide to ion exchange chromatography purified FGF2. Heparin-affinity purified FGF2 bound to acceptor and donor beads in a tagged form using His-tagged or GST-tagged proteins, also dimerized in the AlphaScreen assay. This assay was further validated using different experimental conditions and competitors. The assay constitutes an interesting tool to study dimerization of other FGF forms as well. PMID:25299071

  18. Dimerization capacities of FGF2 purified with or without heparin-affinity chromatography.

    PubMed

    Platonova, Natalia; Miquel, Graldine; Chiu, Liang-Yuan; Taouji, Said; Moroni, Elisabetta; Colombo, Giorgio; Chevet, Eric; Sue, Shih-Che; Bikfalvi, Andreas

    2014-01-01

    Fibroblast growth factor-2 (FGF2) is a pleiotropic growth factor exhibiting a variety of biological activities. In this article, we studied the capacity of FGF2 purified with or without heparin affinity chromatography to self-associate. Analyzing the NMR HSQC spectra for different FGF2 concentrations, heparin-affinity purified FGF2 showed perturbations that indicate dimerization and are a higher-order oligomerization state. HSQC perturbation observed with different FGF2 concentrations revealed a heparin-binding site and two dimer interfaces. Thus, with increasing protein concentrations, FGF2 monomers make contacts with each other and form dimers or higher order oligomers. On the contrary, FGF2 purified with ion-exchange chromatography did not show similar perturbation indicating that self-association of FGF2 is eliminated if purification is done without heparin-affinity chromatography. The HSQC spectra of heparin-affinity purified FGF2 can be reproduced to some extent by adding heparin tetra-saccharide to ion exchange chromatography purified FGF2. Heparin-affinity purified FGF2 bound to acceptor and donor beads in a tagged form using His-tagged or GST-tagged proteins, also dimerized in the AlphaScreen assay. This assay was further validated using different experimental conditions and competitors. The assay constitutes an interesting tool to study dimerization of other FGF forms as well. PMID:25299071

  19. Collaborative Video Annotation by Sharing Tag Clouds

    NASA Astrophysics Data System (ADS)

    Yamamoto, Daisuke; Masuda, Tomoki; Ohira, Shigeki; Nagao, Katashi

    In this paper, we propose a video scene annotation method based on tag clouds. First, user comments associated with a video are collected from existing video sharing services. Next, a tag cloud is generated from these user comments. The tag cloud is displayed on the video window of the Web browser. When users click on a tag included in the tag cloud while watching the video, the tag gets associated with the time point of the video. Users can share the information on the tags that have already been clicked. We confirmed that the coverage of annotations generated by this method is higher than that of the existing methods, and users are motivated to add tags by sharing tag clouds. This method will contribute to advanced video applications.

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

    PubMed

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

    2011-09-01

    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

  1. Immobilized-metal affinity chromatography (IMAC): a review.

    PubMed

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

    2009-01-01

    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:19892187

  2. Synaptic Tagging During Memory Allocation

    PubMed Central

    Rogerson, Thomas; Cai, Denise; Frank, Adam; Sano, Yoshitake; Shobe, Justin; Aranda, Manuel L.; Silva, Alcino J.

    2014-01-01

    There is now compelling evidence that the allocation of memory to specific neurons (neuronal allocation) and synapses (synaptic allocation) in a neurocircuit is not random and that instead specific mechanisms, such as increases in neuronal excitability and synaptic tagging and capture, determine the exact sites where memories are stored. We propose an integrated view of these processes, such that neuronal allocation, synaptic tagging and capture, spine clustering and metaplasticity reflect related aspects of memory allocation mechanisms. Importantly, the properties of these mechanisms suggest a set of rules that profoundly affect how memories are stored and recalled. PMID:24496410

  3. WebTag: Web Browsing into Sensor Tags over NFC

    PubMed Central

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

    2012-01-01

    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. PMID:23012511

  4. WebTag: Web browsing into sensor tags over NFC.

    PubMed

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

    2012-01-01

    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. PMID:23012511

  5. Electron Affinity Calculations for Thioethers

    NASA Technical Reports Server (NTRS)

    Sulton, Deley L.; Boothe, Michael; Ball, David W.; Morales, Wilfredo

    1997-01-01

    Previous work indicated that polyphenyl thioethers possessed chemical properties, related to their electron affinities, which could allow them to function as vapor phase lubricants (VPL). Indeed, preliminary tribological tests revealed that the thioethers could function as vapor phase lubricants but not over a wide temperature and hertzian pressure range. Increasing the electron affinity of the thioethers may improve their VPL properties over this range. Adding a substituent group to the thioether will alter its electron affinity in many cases. Molecular orbital calculations were undertaken to determine the effect of five different substituent groups on the electron affinity of polyphenyl thioethers. It was found that the NO2, F, and I groups increased the thioethers electron affinity by the greatest amount. Future work will involve the addition of these groups to the thioethers followed by tribological testing to assess their VPL properties.

  6. A laboratory evaluation of tagging-related mortality and tag loss in juvenile humpback chub

    USGS Publications Warehouse

    Ward, David L.; Persons, William R.; Young, Kirk; Stone, Dennis M.; Van Haverbeke, Randy; Knight, William R.

    2015-01-01

    We quantified tag retention, survival, and growth in juvenile, captive-reared Humpback Chub Gila cypha marked with three different tag types: (1) Biomark 12.5-mm, 134.2-kHz, full duplex PIT tags injected into the body cavity with a 12-gauge needle; (2) Biomark 8.4-mm, 134.2-kHz, full duplex PIT tags injected with a 16-gauge needle; and (3) Northwest Marine Technology visible implant elastomer (VIE) tags injected under the skin with a 29-gauge needle. Estimates of tag loss, tagging-induced mortality, and growth were evaluated for 60 d with each tag type for four different size-groups of fish: 40–49 mm, 50–59 mm, 60–69 mm, and 70–79 mm TL. Total length was a significant predictor of the probability of PIT tag retention and mortality for both 8-mm and 12-mm PIT tags, and the smallest fish had the highest rates of tag loss (12.5–30.0%) and mortality (7.5–20.0%). Humpback Chub of sizes 40–49 mm TL and tagged with VIE tags had no mortality but did have a 17.5% tag loss. Growth rates of all tagged fish were similar to controls. Our data indicate Humpback Chub can be effectively tagged using either 8-mm or 12-mm PIT tags with little tag loss or mortality at sizes as low as 65 mm TL.

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

    PubMed

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

    2012-08-01

    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. PMID:22706764

  8. [Fusion tags technology and their applications].

    PubMed

    Li, Yong-Jin; Chen, Yuan-Yuan; Bi, Li-Jun

    2006-07-01

    Fusion tags are originally developed to facilitate the purification of recombinant protein from crude extracts. In recent years, the discovery of different tags and the development of fusion strategy make the function of fusion tags diversified. However, there was no a cure-all fusion tag for different applications. We here give an overview of fusion tag technology and the different applications of fusion tags, including the purification, detection and oriented immobilization of recombinant protein, the visualization of bioevent in vivo, the enhancement of the yield of protein, the improvement of the solubility and stability of the expressed protein. PMID:16894881

  9. From the Cover: Imaging of receptor trafficking by using -bungarotoxin-binding-site-tagged receptors

    NASA Astrophysics Data System (ADS)

    Sekine-Aizawa, Yoko; Huganir, Richard L.

    2004-12-01

    -Amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors mediate excitatory synaptic transmission and are dynamically regulated during synaptic plasticity in the CNS. The membrane trafficking of AMPA receptors to synapses is critical for the regulation of the efficacy of excitatory synaptic transmission. Direct imaging of AMPA receptors in various cell compartments is important to dissecting the regulation of distinct steps in receptor membrane trafficking. In this study, we have developed an approach for the imaging of receptor trafficking with subunits tagged with a 13-aa -bungarotoxin (BTX)-binding site (BBS). The small polypeptide neurotoxin BTX has been used for decades to study the nicotinic acetylcholine receptor. Similar high-affinity ligands are rarely available for most receptors. Engineering the BBS tag into receptor subunits allowed the high-affinity binding of fluorescent, radioactive, and biotinylated BTX to the tagged receptor subunits. By using this approach, the total receptor expression, surface expression, internalization, and insertion of receptors into the plasma membrane could be visualized and quantified in fixed or live cells including cultured neurons. The BBS tag is a flexible approach for labeling membrane proteins and studying their dynamic trafficking. GFP | glutamate receptor | live imaging | synapses | tag

  10. Tandem affinity purification in Drosophila: the advantages of the GS-TAP system.

    PubMed

    Kyriakakis, Phillip; Tipping, Marla; Abed, Louka; Veraksa, Alexey

    2008-01-01

    Tandem affinity purification (TAP) has been widely used for the analysis of protein complexes. We investigated the parameters of the recently developed TAP method (GS-TAP) and its application in Drosophila. This new tag combination includes two Protein G modules and a streptavidin binding peptide (SBP), separated by one or two TEV protease cleavage sites. We made pMK33-based GS-TAP vectors to allow for generation of stable cell lines using hygromycin selection and inducible expression from a metallothionein promoter, as well as pUAST-based vectors that can be used for inducible expression in flies. Rescue experiments in flies demonstrated that the GS-TAP tag preserves the function of the tagged protein. We have done parallel purifications of proteins tagged with the new GS-TAP tag or with the conventional TAP tag (containing the Protein A and calmodulin binding peptide domains) at the amino terminus, using both cultured cells and embryos. A major difference between the two tags was in the levels of contaminating proteins, which were significantly lower in the GS-TAP purifications. The GS-TAP procedure also resulted in higher yield of the bait protein. Overall, GS-TAP is an improved method of protein complex purification because it provides a superior signal-to-noise ratio of the bait protein relative to contaminants in purified material. PMID:18719405

  11. Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins.

    PubMed

    Béhar, Ghislaine; Renodon-Cornière, Axelle; Mouratou, Barbara; Pecorari, Frédéric

    2016-04-01

    Affinity chromatography is a convenient way of purifying proteins, as a high degree of purity can be reached in one step. The use of tags has greatly contributed to the popularity of this technique. However, the addition of tags may not be desirable or possible for the production of biopharmaceuticals. There is thus a need for tailored artificial affinity ligands. We have developed the use of archaeal extremophilic proteins as scaffolds to generate affinity proteins (Affitins). Here, we explored the potential of Affitins as ligand to design affinity columns. Affitins specific for human immunoglobulin G (hIgG), bacterial PulD protein, and chicken egg lysozyme were immobilized on an agarose matrix. The columns obtained were functional and highly selective for their cognate target, even in the presence of exogenous proteins as found in cell culture media, ascites and bacterial lysates, which result in a high degree of purity (∼95%) and recovery (∼100%) in a single step. Anti-hIgG Affitin columns withstand repetitive cycles of purification and cleaning-in-place treatments with 0.25M NaOH as well as Protein A does. High levels of Affitin productions in Escherichia coli makes it possible to produce these affinity columns at low cost. Our results validate Affitins as a new class of tailored ligands for the affinity chromatography purification of potentially any proteins of interest including biopharmaceuticals. PMID:26952369

  12. Removal of the tag from His-tagged ILYd4, a human CD59 inhibitor, significantly improves its physical properties and its activity.

    PubMed

    Wu, Lin; Su, Sanbao; Liu, Fengming; Xu, Tao; Wang, Xiaoxiao; Huang, Yan; Sun, Xinlu; Ge, Xiaowen; Chen, Ting; Liu, Huixia; Wang, Chun; Chorev, Michael; Xu, Ting; Qin, Xuebin

    2012-01-01

    Complement dependent cytotoxicity (CDC) significantly contributes to Rituximab (RTX) and Ofatumumab (OFA) efficacies in the treatment of B-cell non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Human CD59 (hCD59) is a key complement regulatory protein that restricts the formation of the membrane attack complex and thereby inhibits CDC. hCD59 is an important determinant of the sensitivity of NHL and CLL to RTX and OFA treatment. Recently, we developed a specific and potent hCD59 inhibitor, His-tagged ILYd4, which consists of 30 amino acid sequences extending from the N-terminus of ILYd4. Our previously published results indicate that His-tagged ILYd4 can be used as a lead candidate to further develop a potential therapeutic adjuvant for RTX and OFA treatment of RTX-resistant NHL and CLL. However, these studies were conducted using ILYd4 tagged on the N-terminus with 30 additional amino acids (AA) containing 6 X His used for immobilized metal affinity chromatograph. As a further step towards the development of ILYd4-based therapeutics, we investigated the impact of the removal of this extraneous sequence on the anti-hCD59 activity. In this paper, we report the generation and characterization of tag-free ILYd4. We demonstrate that tag-free ILYd4 has over threefold higher anti-hCD59 activities than the His-tagged ILYd4. The enhanced RTX-mediated CDC effect on B-cell malignant cells comes from tag-free ILYd4's improved functionality and physical properties including better solubility, reduced tendency to aggregation, and greater thermal stability. Therefore, tag-free ILYd4 is a better candidate for the further development for the clinical application. PMID:22642361

  13. What Do Tag Games Teach?

    ERIC Educational Resources Information Center

    Belka, David

    2006-01-01

    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

  14. What Do Tag Games Teach?

    ERIC Educational Resources Information Center

    Belka, David

    2006-01-01

    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…

  15. Virtual colon tagging for electronic cleansing in dual-energy fecal-tagging CT colonography.

    PubMed

    Cai, Wenli; Kim, Se Hyung; Lee, June-Goo; Yoshida, Hiroyuki

    2012-01-01

    Partial volume effect (PVE) and tagging inhomogeneity are two major causes of artifacts in electronic cleansing (EC) for fecal-tagging CT colonography (CTC). Our purpose was to develop a novel method called "virtual tagging" for electronic cleansing in dual-energy fecal-tagging CTC. A three-material decomposition scheme was first applied in dual-energy CTC to decompose each voxel into a mixture of air, soft tissue, and iodine-tagged fecal material. The entire colonic lumen was then marked by virtually tagging the mixture portion of luminal air at each voxel. As a result, colon lumen including air and tagged materials was segmented and subtracted by their high values in virtually tagged images. Our virtual tagging scheme provides a cleansed colon that is free from artifacts caused by the PVE at air-tagging mixture and inhomogeneous tagging. PMID:23366740

  16. An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells.

    PubMed

    Bürckstümmer, Tilmann; Bennett, Keiryn L; Preradovic, Adrijana; Schütze, Gregor; Hantschel, Oliver; Superti-Furga, Giulio; Bauch, Angela

    2006-12-01

    Tandem affinity purification (TAP) is a generic two-step affinity purification protocol that enables the isolation of protein complexes under close-to-physiological conditions for subsequent analysis by mass spectrometry. Although TAP was instrumental in elucidating the yeast cellular machinery, in mammalian cells the method suffers from a low overall yield. We designed several dual-affinity tags optimized for use in mammalian cells and compared the efficiency of each tag to the conventional TAP tag. A tag based on protein G and the streptavidin-binding peptide (GS-TAP) resulted in a tenfold increase in protein-complex yield and improved the specificity of the procedure. This allows purification of protein complexes that were hitherto not amenable to TAP and use of less starting material, leading to higher success rates and enabling systematic interaction proteomics projects. Using the well-characterized Ku70-Ku80 protein complex as an example, we identified both core elements as well as new candidate effectors. PMID:17060908

  17. Isolation and purification of blood group antigens using immuno-affinity chromatography on short monolithic columns.

    PubMed

    Mnster, Andrea; Hiller, Oliver; Grger, Daniela; Blasczyk, Rainer; Kasper, Cornelia

    2011-02-01

    Monolithic columns have gained increasing attention as stationary phases for the separation of biomolecules and biopharmaceuticals. In the present work the performance of monolithic convective interaction media (CIM()) chromatography for the purification of blood group antigens was established. The proteins employed in this study are derived from blood group antigens Knops, JMH and Scianna, equipped both with a His-tag and with a V5-tag by which they can be purified. In a first step a monoclonal antibody directed against the V5-tag was immobilized on a CIM() Disk with epoxy chemistry. After this, the immobilized CIM() Disk was used in immuno-affinity chromatography to purify the three blood group antigens from cell culture supernatant. Up-scaling of the applied technology was carried out using CIM() Tubes. In comparison to conventional affinity chromatography, blood group antigens were also purified via His-tag using a HiTrap() metal-affinity column. The two purifications have been compared regarding purity, yield and purification speed. Using the monolithic support, it was possible to isolate the blood group antigens with a higher flow rate than using the conventional bed-packed column. PMID:21194702

  18. Tag removal in cardiac tagged MRI images using coupled dictionary learning.

    PubMed

    Makram, Abram W; Rushdi, Muhammad A; Khalifa, Ayman M; El-Wakad, Mohamed T

    2015-08-01

    Tagged Magnetic Resonance Imaging (tMRI) is considered to be the gold standard for quantitative assessment of the cardiac local functions. However, the tagging patterns and low myocardium-to-blood-pool contrast of tagged images bring great challenges to cardiac image processing and analysis tasks such as myocardium segmentation and tracking. Hence, there has been growing interest in techniques for removing tagging lines. In this work, a method for removing tagging patterns in tagged MR images using a coupled dictionary learning (CDL) model is proposed. In this model, identical sparse representations are assumed for image patches in the tagged MRI and corresponding cine MRI image spaces. First, we learn a dictionary for the tagged MRI image space. Then, we compute a dictionary for the cine MRI image space so that corresponding tagged and cine patches have the same sparse codes in terms of their respective dictionaries. Finally, in order to produce the de-tagged (cine version) of a test tagged image, the sparse codes of the tagged patches and the trained cine dictionary are used together to construct the de-tagged patches. We have tested this tag removal method on a dataset of tagged cardiac MR images. Our experimental results compared favorably with a recently proposed tag removal method that removes tags in the frequency domain using an optimal band-stop filter of harmonic peaks. PMID:26738129

  19. 50 CFR 20.81 - Tagging requirement.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 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 migratory game birds unless such birds are tagged as required by 20.36....

  20. 50 CFR 20.81 - Tagging requirement.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 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 migratory game birds unless such birds are tagged as required by 20.36....

  1. 50 CFR 20.81 - Tagging requirement.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 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 migratory game birds unless such birds are tagged as required by 20.36....

  2. 50 CFR 635.33 - Archival tags.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ...) Implantation report. Any person affixing or implanting an archival tag into a regulated species must obtain... catch, possess, retain, and land an Atlantic HMS in which an archival tag has been implanted or...

  3. Process intensification for the removal of poly-histidine fusion tags from recombinant proteins by an exopeptidase.

    PubMed

    Kuo, Wen-Hui K; Chase, Howard A

    2010-01-01

    This study describes the use of a hexa-histidine tagged exopeptidase for the cleavage of hexa-histidine tags from recombinant maltose binding protein (MBP) when both tagged species are bound to an immobilized metal affinity chromatography (IMAC) matrix. On-column exopeptidase cleavage only occurred when the cleavage buffer contained an imidazole concentration of 50 mM or higher. Two strategies were tested for the on-column tag cleavage by dipeptidylaminopeptidase (DAPase): (i) a post-load wash was performed after sample loading using cleavage buffers containing varying imidazole concentrations and (ii) a post-load wash was omitted following sample loading. In the presence of 50 mM imidazole, 46% of the originally adsorbed hexa-histidine tagged MBP was cleaved, released from the column, and recovered in a sample containing 100% native (i.e., completely detagged) MBP. This strategy renders the subsequent purification steps unnecessary as any tagged contaminants remained bound to the column. At higher imidazole concentrations, binding of both hexa-histidine tagged MBP and DAPase to the column was minimized, leading to characteristics of cleavage more closely resembling that of a batch cleavage. An on-column cleavage yield of 93% was achieved in the presence of 300 mM imidazole, albeit with contamination of the detagged protein with tag fragments and partially tagged MBP. The success of the on-column exopeptidase cleavage makes the integration of the poly-histidine tag removal protocol within the IMAC protein capture step possible. The many benefits of using commercially available exopeptidases, such as DAPase, for poly-histidine tag removal can now be combined with the on-column tag cleavage operation. PMID:19785040

  4. HaloTag technology for specific and covalent labeling of fusion proteins.

    PubMed

    Benink, Hlne A; Urh, Marjeta

    2015-01-01

    Appending proteins of interest to fluorescent protein tags such as GFP has revolutionized how proteins are studied in the cellular environment. Over the last few decades many varieties of fluorescent proteins have been generated, each bringing new capability to research. However, taking full advantage of standard fluorescent proteins with advanced and differential features requires significant effort on the part of the researcher. This approach necessitates that many genetic fusions be generated and confirmed to function properly in cells with the same protein of interest. To lessen this burden, a newer category of protein fusion tags termed "self-labeling protein tags" has been developed. This approach utilizes a single protein tag, the function of which can be altered by attaching various chemical moieties (fluorescent labels, affinity handles, etc.). In this way a single genetically encoded protein fusion can easily be given functional diversity and adaptability as supplied by synthetic chemistry. Here we present protein labeling methods using HaloTag technology; comprised of HaloTag protein and the collection of small molecules designed to bind it specifically and provide it with varied functionalities. For imaging purposes these small molecules, termed HaloTag ligands, contain distinct fluorophores. Due to covalent and rapid binding between HaloTag protein and its ligands, labeling is permanent and efficient. Many of these ligands have been optimized for permeability across cellular membranes allowing for live cell labeling and imaging analysis. Nonpermeable ligands have also been developed for specific labeling of surface proteins. Overall, HaloTag is a versatile technology that empowers the end user to label a protein of interest with the choice of different fluorophores while alleviating the need for generation of multiple genetic fusions. PMID:25560071

  5. Implanting Telemetry Tag in Pallid Sturgeon

    USGS Multimedia Gallery

    Acoustic telemetry tags have to be fairly small to fit inside a five to fifteen pound pallid sturgeon.  The average tag is about the size of your ring finger (60-90 mm long and 16 mm diameter).  That means the battery that powers the acoustic tag is small, too.  A small battery means ...

  6. Method for designing gas tag compositions

    DOEpatents

    Gross, Kenny C. (1433 Carriage La., Bolingbrook, IL 60440)

    1995-01-01

    For use in the manufacture of gas tags such as employed in a nuclear reactor gas tagging failure detection system, a method for designing gas tagging compositions utilizes an analytical approach wherein the final composition of a first canister of tag gas as measured by a mass spectrometer is designated as node #1. Lattice locations of tag nodes in multi-dimensional space are then used in calculating the compositions of a node #2 and each subsequent node so as to maximize the distance of each node from any combination of tag components which might be indistinguishable from another tag composition in a reactor fuel assembly. Alternatively, the measured compositions of tag gas numbers 1 and 2 may be used to fix the locations of nodes 1 and 2, with the locations of nodes 3-N then calculated for optimum tag gas composition. A single sphere defining the lattice locations of the tag nodes may be used to define approximately 20 tag nodes, while concentric spheres can extend the number of tag nodes to several hundred.

  7. Method for designing gas tag compositions

    DOEpatents

    Gross, K.C.

    1995-04-11

    For use in the manufacture of gas tags such as employed in a nuclear reactor gas tagging failure detection system, a method for designing gas tagging compositions utilizes an analytical approach wherein the final composition of a first canister of tag gas as measured by a mass spectrometer is designated as node No. 1. Lattice locations of tag nodes in multi-dimensional space are then used in calculating the compositions of a node No. 2 and each subsequent node so as to maximize the distance of each node from any combination of tag components which might be indistinguishable from another tag composition in a reactor fuel assembly. Alternatively, the measured compositions of tag gas numbers 1 and 2 may be used to fix the locations of nodes 1 and 2, with the locations of nodes 3-N then calculated for optimum tag gas composition. A single sphere defining the lattice locations of the tag nodes may be used to define approximately 20 tag nodes, while concentric spheres can extend the number of tag nodes to several hundred. 5 figures.

  8. Identification of protein interacting partners using tandem affinity purification.

    PubMed

    Bailey, Dalan; Urena, Luis; Thorne, Lucy; Goodfellow, Ian

    2012-01-01

    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 identification(1). 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 yeast(2,3) but more recently has been adapted to use in mammalian cells(4-8). As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E(9,10).The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation(10). 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 sequence(8). 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. PMID:22395237

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

    PubMed

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

    2011-09-20

    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

  10. Infrared tag and track technique

    DOEpatents

    Partin, Judy K.; Stone, Mark L.; Slater, John; Davidson, James R.

    2007-12-04

    A method of covertly tagging an object for later tracking includes providing a material capable of at least one of being applied to the object and being included in the object, which material includes deuterium; and performing at least one of applying the material to the object and including the material in the object in a manner in which in the appearance of the object is not changed, to the naked eye.

  11. Electronic Tag and Position Sensor

    SciTech Connect

    Not Available

    1992-01-20

    The intent of this study phase program was to adequately define the Electronic Tag and Position Sensor chip so as to be able to price and schedule the full design and development culminating in a silicon IC. Therefore, even though Hughes Aircraft Company feels that the approach submitted in this document is what should be developed, it is still considered preliminary and could change as the full design is developed.

  12. The ABRF Edman Sequencing Research Group 2008 Study: Investigation into Homopolymeric Amino Acid N-Terminal Sequence Tags and Their Effects on Automated Edman Degradation

    PubMed Central

    Thoma, R. S.; Smith, J. S.; Sandoval, W.; Leone, J. W.; Hunziker, P.; Hampton, B.; Linse, K. D.; Denslow, N. D.

    2009-01-01

    The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and executes interlaboratory studies investigating the use of automated Edman degradation for protein and peptide analysis. In 2008, the ESRG enlisted the help of core sequencing facilities to investigate the effects of a repeating amino acid tag at the N-terminus of a protein. Commonly, to facilitate protein purification, an affinity tag containing a polyhistidine sequence is conjugated to the N-terminus of the protein. After expression, polyhistidine-tagged protein is readily purified via chelation with an immobilized metal affinity resin. The addition of the polyhistidine tag presents unique challenges for the determination of protein identity using Edman degradation chemistry. Participating laboratories were asked to sequence one protein engineered in three configurations: with an N-terminal polyhistidine tag; with an N-terminal polyalanine tag; or with no tag. Study participants were asked to return a data file containing the uncorrected amino acid picomole yields for the first 17 cycles. Initial and repetitive yield (R.Y.) information and the amount of lag were evaluated. Information about instrumentation and sample treatment was also collected as part of the study. For this study, the majority of participating laboratories successfully called the amino acid sequence for 17 cycles for all three test proteins. In general, laboratories found it more difficult to call the sequence containing the polyhistidine tag. Lag was observed earlier and more consistently with the polyhistidine-tagged protein than the polyalanine-tagged protein. Histidine yields were significantly less than the alanine yields in the tag portion of each analysis. The polyhistidine and polyalanine protein-R.Y. calculations were found to be equivalent. These calculations showed that the nontagged portion from each protein was equivalent. The terminal histidines from the tagged portion of the protein were demonstrated to be responsible for the high lag during N-terminal sequence analysis. PMID:19721823

  13. Phase modulation in RF tag

    DOEpatents

    Carrender, Curtis Lee; Gilbert, Ronald W.

    2007-02-20

    A radio frequency (RF) communication system employs phase-modulated backscatter signals for RF communication from an RF tag to an interrogator. The interrogator transmits a continuous wave interrogation signal to the RF tag, which based on an information code stored in a memory, phase-modulates the interrogation signal to produce a backscatter response signal that is transmitted back to the interrogator. A phase modulator structure in the RF tag may include a switch coupled between an antenna and a quarter-wavelength stub; and a driver coupled between the memory and a control terminal of the switch. The driver is structured to produce a modulating signal corresponding to the information code, the modulating signal alternately opening and closing the switch to respectively decrease and increase the transmission path taken by the interrogation signal and thereby modulate the phase of the response signal. Alternatively, the phase modulator may include a diode coupled between the antenna and driver. The modulating signal from the driver modulates the capacitance of the diode, which modulates the phase of the response signal reflected by the diode and antenna.

  14. A genome-scale resource for in vivo tag-based protein function exploration in C. elegans.

    PubMed

    Sarov, Mihail; Murray, John I; Schanze, Kristin; Pozniakovski, Andrei; Niu, Wei; Angermann, Karolin; Hasse, Susanne; Rupprecht, Michaela; Vinis, Elisabeth; Tinney, Matthew; Preston, Elicia; Zinke, Andrea; Enst, Susanne; Teichgraber, Tina; Janette, Judith; Reis, Kadri; Janosch, Stephan; Schloissnig, Siegfried; Ejsmont, Radoslaw K; Slightam, Cindie; Xu, Xiao; Kim, Stuart K; Reinke, Valerie; Stewart, A Francis; Snyder, Michael; Waterston, Robert H; Hyman, Anthony A

    2012-08-17

    Understanding the in vivo dynamics of protein localization and their physical interactions is important for many problems in biology. To enable systematic protein function interrogation in a multicellular context, we built a genome-scale transgenic platform for in vivo expression of fluorescent- and affinity-tagged proteins in Caenorhabditis elegans under endogenous cis regulatory control. The platform combines computer-assisted transgene design, massively parallel DNA engineering, and next-generation sequencing to generate a resource of 14,637 genomic DNA transgenes, which covers 73% of the proteome. The multipurpose tag used allows any protein of interest to be localized in vivo or affinity purified using standard tag-based assays. We illustrate the utility of the resource by systematic chromatin immunopurification and automated 4D imaging, which produced detailed DNA binding and cell/tissue distribution maps for key transcription factor proteins. PMID:22901814

  15. Sensor-based material tagging system

    SciTech Connect

    Vercellotti, L.C.; Cox, R.W.; Ravas, R.J.; Schlotterer, J.C. )

    1991-01-01

    This paper reports that 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 or beacon mode. Radiation measurement was also considered, but the background from nearby containers was found to be excessive.

  16. His-tag impact on structure.

    PubMed

    Carson, Mike; Johnson, David H; McDonald, Heather; Brouillette, Christie; Delucas, Lawrence J

    2007-03-01

    Crystallographers are increasingly determining structures of protein constructs that include His tags. Many have taken for granted that these tags have little effect on the native structure. This paper surveys and compares crystal structures with and without His tags. It is observed that actual refined tag residues fitted into density occur in less that 10% of the tagged sequences. However, higher resolution crystals are observed when this occurs. It is shown that these purification tags generally have no significant effect on the structure of the native protein. Resolution and R factors are not affected, but the overall B factors are slightly higher. Additional annotation in the PDB format to make tag definition explicit is suggested. PMID:17327666

  17. Frequency-domain simulation of MR tagging.

    PubMed

    Crum, W R; Berry, E; Ridgway, J P; Sivananthan, U M; Tan, L B; Smith, M A

    1998-01-01

    Simulation of MR images is a useful tool for offline sequence development and as an aid to understanding image formation. One particular application of simulation is MR tagging, which is used for tracking myocardial motion. Simple spatial-domain methods cannot adequately represent effects common in these images, such as motion artifact and signal wrap. An existing frequency-domain model is shown to be inappropriate for tagged images, and an extension based on the Bloch equations and Fourier shift theorem is described to correct this. Software incorporating the new model is used to generate ideal tag intensity profiles and to accurately simulate tagged images. The shifted k-space patterns associated with tagged images, and their dependence on the order of the binomial tagging sequence, are explained. An application of the Fourier shift theorem is suggested that allows more rapid simulation of static tagged images. PMID:9786140

  18. Molecular Cloning and Characterization of a (Lys)6-Tagged Sulfide-Reactive Hemoglobin I from Lucina pectinata.

    PubMed

    Díaz-Ayala, Ramonita; Moya-Rodríguez, Andrés; Pietri, Ruth; Cadilla, Carmen L; López-Garriga, Juan

    2015-12-01

    A poly-Lys tag was fused to the Lucina pectinata hemoglobin I (HbI) coding sequence and purified using an efficient and fast process. HbI is a hemeprotein that binds hydrogen sulfide (H2S) with high affinity and it has been used to understand physiologically relevant reactions of this signaling molecule. The (Lys)6-tagged rHbI construct was expressed in E. coli and purified by immobilization on a cation exchange matrix, followed by size-exclusion chromatography. The identity, structure, and function of the (Lys)6-tagged rHbI were assessed by mass spectrometry, small and wide X-ray scattering, optical spectroscopy, and kinetic analysis. The scattering and spectroscopic results showed that the (Lys)6-tagged rHbI is structurally and functionally analogous to the native protein as well as to the (His)6-tagged rHbI. Kinetics studies with H2S indicated that the association (k on) and dissociation (k off) rate constants were 1.4 × 10(5)/M/s and 0.1 × 10(-3)/s, respectively. This results confirmed that the (Lys)6-tagged rHbI binds H2S with the same high affinity as its homologue. PMID:26482241

  19. pAUL: A Gateway-Based Vector System for Adaptive Expression and Flexible Tagging of Proteins in Arabidopsis

    PubMed Central

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

    2013-01-01

    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

  20. High throughput generation of tagged stable cell lines for proteomic analysis

    PubMed Central

    Torres, Jorge Z.; Miller, Julie J.; Jackson, Peter K.

    2014-01-01

    We present an optimized system for rapid generation of Localization and Affinity Purification (LAP)-tagged mammalian stable cell lines that facilitates complex purification and interacting protein identification. The improved components of this method, including the flexibility of inducible expression, circumvent issues associated with toxicity, clonal selection, sample yields and time to data acquisition. We have applied this method to the study of cell cycle regulators and novel microtubule-associated proteins. PMID:19405035

  1. High-throughput generation of tagged stable cell lines for proteomic analysis.

    PubMed

    Torres, Jorge Z; Miller, Julie J; Jackson, Peter K

    2009-05-01

    We present an optimized system for rapid generation of localization and affinity purification-tagged mammalian stable cell lines that facilitates complex purification and interacting protein identification. The improved components of this method, including the flexibility of inducible expression, circumvent issues associated with toxicity, clonal selection, sample yields and time to data acquisition. We have applied this method to the study of cell-cycle regulators and novel microtubule-associated proteins. PMID:19405035

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

    ERIC Educational Resources Information Center

    Bailey, Cheryl P.

    2009-01-01

    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

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

    ERIC Educational Resources Information Center

    Bailey, Cheryl P.

    2009-01-01

    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…

  4. Tag retention, growth, and survival of red swamp crayfish marked with a visible implant tag

    USGS Publications Warehouse

    Isely, J.J.; Stockett, P.E.

    2001-01-01

    Eighty juvenile (means: 42.4 mm total length, 1.6 g) red swamp crayfish Procambarus clarkii were implanted with sequentially numbered visible implant tags and held in the laboratory. Tags were injected transversely into the musculature just beneath the exoskeleton of the third abdominal segment from the cephalothorax; tags were visible upon inspection. An additional 20 crayfish were left untagged and served as controls. After 150 d, tag retention was 80% and all tags were readable. No tagged crayfish died during the study, and no differences in total length or weight were detected between tagged and control crayfish. All individuals molted at least three times during the 150-d study, and some individuals molted up to six times, suggesting that most tags would be permanently retained. The readability in the field without specialized equipment makes the visible implant tag ideal for studies of crayfish ecology, management, and culture.

  5. The structure of the SBP-Tagstreptavidin complex reveals a novel helical scaffold bridging binding pockets on separate subunits

    SciTech Connect

    Barrette-Ng, Isabelle H.; Wu, Sau-Ching; Tjia, Wai-Mui; Wong, Sui-Lam; Ng, Kenneth K. S.

    2013-05-01

    The structure of the SBP-Tagstreptavidin complex reveals a novel mode of peptide recognition in which a single peptide binds simultaneously to biotin-binding pockets from adjacent subunits of streptavidin. The molecular details of peptide recognition suggest how the SBP-Tag can be further modified to become an even more useful tag for a wider range of biotechnological applications. The 38-residue SBP-Tag binds to streptavidin more tightly (K{sub d} ? 2.54.9 nM) than most if not all other known peptide sequences. Crystallographic analysis at 1.75 resolution shows that the SBP-Tag binds to streptavidin in an unprecedented manner by simultaneously interacting with biotin-binding pockets from two separate subunits. An N-terminal HVV peptide sequence (residues 1214) and a C-terminal HPQ sequence (residues 3133) form the bulk of the direct interactions between the SBP-Tag and the two biotin-binding pockets. Surprisingly, most of the peptide spanning these two sites (residues 1728) adopts a regular ?-helical structure that projects three leucine side chains into a groove formed at the interface between two streptavidin protomers. The crystal structure shows that residues 110 and 3538 of the original SBP-Tag identified through in vitro selection and deletion analysis do not appear to contact streptavidin and thus may not be important for binding. A 25-residue peptide comprising residues 1134 (SBP-Tag2) was synthesized and shown using surface plasmon resonance to bind streptavidin with very similar affinity and kinetics when compared with the SBP-Tag. The SBP-Tag2 was also added to the C-terminus of ?-lactamase and was shown to be just as effective as the full-length SBP-Tag in affinity purification. These results validate the molecular structure of the SBP-Tagstreptavidin complex and establish a minimal bivalent streptavidin-binding tag from which further rational design and optimization can proceed.

  6. Radio tag retention and tag-related mortality among adult sockeye salmon

    USGS Publications Warehouse

    Ramstad, K.M.; Woody, C.A.

    2003-01-01

    Tag retention and tag-related mortality are concerns for any tagging study but are rarely estimated. We assessed retention and mortality rates for esophageal radio tag implants in adult sockeye salmon Oncorhynchus nerka. Migrating sockeye salmon captured at the outlet of Lake Clark, Alaska, were implanted with one of four different radio tags (14.5 ?? 43 mm [diameter ?? length], 14.5 ?? 49 mm, 16 ?? 46 mm, and 19 ?? 51 mm). Fish were observed for 15 to 35 d after tagging to determine retention and mortality rates. The overall tag retention rate was high (0.98; 95% confidence interval [CI], 0.92-1.00; minimum, 33 d), with one loss of a 19-mm ?? 51-mm tag. Mortality of tagged sockeye salmon (0.02; 95% CI, 0-0.08) was similar to that of untagged controls (0.03 [0-0.15]). Sockeye salmon with body lengths (mid-eye to tail fork) of 585-649 mm retained tags as large as 19 ?? 51 mm and those with body lengths of 499-628 mm retained tags as small as 14.5 ?? 43 mm for a minimum of 33 d with no increase in mortality. The tags used in this study represent a suite of radio tags that vary in size, operational life, and cost but that are effective in tracking adult anadromous salmon with little tag loss or increase in fish mortality.

  7. Dual tagging as an approach to isolate endogenous chromatin remodeling complexes from Saccharomyces cerevisiae.

    PubMed

    Lin, Tzong-Yuan; Voronovsky, Andriy; Raabe, Monika; Urlaub, Henning; Sander, Bjoern; Golas, Monika M

    2015-03-01

    Affinity isolation has been an essential technique for molecular studies of cellular assemblies, such as the switch/sucrose non-fermentable (SWI/SNF) family of ATP-dependent chromatin remodeling complexes. However, even biochemically pure isolates can contain heterogeneous mixtures of complexes and their components. In particular, purification strategies that rely on affinity tags fused to only one component of a complex may be susceptible to this phenomenon. This study demonstrates that fusing purification tags to two different proteins enables the isolation of intact complexes of remodels the structure of chromatin (RSC). A Protein A tag was fused to one of the RSC proteins and a Twin-Strep tag to another protein of the complex. By mass spectrometry, we demonstrate the enrichment of the RSC complexes. The complexes had an apparent Svedberg value of about 20S, as shown by glycerol gradient ultracentrifugation. Additionally, purified complexes were demonstrated to be functional. Electron microscopy and single-particle analyses revealed a conformational rearrangement of RSC upon interaction with acetylated histone H3 peptides. This purification method is useful to purify functionally active, structurally well-defined macromolecular assemblies. PMID:25486077

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

    PubMed Central

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

    2009-01-01

    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 of plasmids to express sRNAs with any of three widely used aptamer sequences (MS2, boxB, eIF4A), and systematically tested how the aptamer tagging impacted on intracellular accumulation and target regulation of the Salmonella GcvB, InvR or RybB sRNAs. In addition, we successfully tagged the chromosomal rybB gene with MS2 to observe that RybB-MS2 is fully functional as an envelope stress-induced repressor of ompN mRNA following induction of sigmaE. We further demonstrate that the common sRNA-binding protein, Hfq, co-purifies with MS2-tagged sRNAs of Salmonella. The presented affinity purification strategy may facilitate the isolation of in vivo assembled sRNA–protein complexes in a wide range of bacteria. PMID:19726584

  9. BONCAT: metabolic labeling, click chemistry, and affinity purification of newly synthesized proteomes.

    PubMed

    Landgraf, Peter; Antileo, Elmer R; Schuman, Erin M; Dieterich, Daniela C

    2015-01-01

    Metabolic labeling of proteins using classical radioisotope-labeled amino acids has enabled the analysis and function of protein synthesis for many biological processes but cannot be combined with modern high-throughput mass spectrometry analysis. This chapter describes the unbiased identification of a whole de novo synthesized proteome of cultured cells or of a translationally active subcellular fraction of the mammalian brain. This technique relies on the introduction of a small bioorthogonal reactive group by metabolic labeling accomplished by replacing the amino acid methionine by the azide-bearing methionine surrogate azidohomoalanine (AHA) or the amino acid homopropargylglycine (HPG). Subsequently an alkyne- or azide-bearing affinity tag is covalently attached to the group by "click chemistry"-a copper(I)-catalyzed [3+2] azide-alkyne cycloaddition. Affinity tag-labeled proteins can be analyzed in candidate-based approaches by conventional biochemical methods or with high-throughput mass spectrometry. PMID:25560077

  10. Directional Radio-Frequency Identification Tag Reader

    NASA Technical Reports Server (NTRS)

    Medelius, Pedro J.; Taylor, John D.; Henderson, John J.

    2004-01-01

    A directional radio-frequency identification (RFID) tag reader has been designed to facilitate finding a specific object among many objects in a crowded room. The device could be an adjunct to an electronic inventory system that tracks RFID-tagged objects as they move through reader-equipped doorways. Whereas commercial RFID-tag readers do not measure directions to tagged objects, the device is equipped with a phased-array antenna and a received signal-strength indicator (RSSI) circuit for measuring direction. At the beginning of operation, it is set to address only the RFID tag of interest. It then continuously transmits a signal to interrogate that tag while varying the radiation pattern of the antenna. It identifies the direction to the tag as the radiation pattern direction of peak strength of the signal returned by the tag. An approximate distance to the tag is calculated from the peak signal strength. The direction and distance can be displayed on a screen. A prototype containing a Yagi antenna was found to be capable of detecting a 915.5-MHz tag at a distance of approximately equal to 15 ft (approximately equal to 4.6 m).

  11. Tagged-weak {pi} method

    SciTech Connect

    Margaryan, A.; Hashimoto, O.; Kakoyan, V.; Knyazyan, S.; Tang, L.

    2011-02-15

    A new 'tagged-weak {pi} method' is proposed for determination of electromagnetic transition probabilities B(E2) and B(M1) of the hypernuclear states with lifetimes of {approx}10{sup -10} s. With this method, we are planning to measure B(E2) and B(M1) for light hypernuclei at JLab. The results of Monte Carlo simulations for the case of E2(5/2{sup +}, 3/2{sup +} {yields} 1/2{sup +}) transitions in {sub {Lambda}}{sup 7}He hypernuclei are presented.

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

    PubMed Central

    Strohmaier, Markus; Krner, Christian; Kern, Roman

    2012-01-01

    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

  13. Affine toric SL(2)-embeddings

    NASA Astrophysics Data System (ADS)

    Gaifullin, S. A.

    2008-04-01

    In the theory of affine SL(2)-embeddings, which was constructed in 1973 by Popov, a locally transitive action of the group SL(2) on a normal affine three-dimensional variety X is determined by a pair (p/q,r), where 0 is a rational number written as an irreducible fraction and called the height of the action, while r is a positive integer that is the order of the stabilizer of a generic point. In the present paper it is shown that the variety X is toric, that is, it admits a locally transitive action of an algebraic torus if and only if the number r is divisible by q-p. For that, the following criterion for an affine G/H-embedding to be toric is proved. Let X be a normal affine variety, G a simply connected semisimple group acting regularly on X, and H\\subset G a closed subgroup such that the character group \\mathfrak{X}(H) of the group H is finite. If an open equivariant embedding G/H\\hookrightarrow X is defined, then X is toric if and only if there exist a quasitorus \\widehat T and a (G\\times\\widehat T)-module V such that X\\stackrel G\\cong V/\\!/\\widehat T. In the substantiation of this result a key role is played by Cox's construction in toric geometry. Bibliography: 12 titles.

  14. Affine Contractions on the Plane

    ERIC Educational Resources Information Center

    Celik, D.; Ozdemir, Y.; Ureyen, M.

    2007-01-01

    Contractions play a considerable role in the theory of fractals. However, it is not easy to find contractions which are not similitudes. In this study, it is shown by counter examples that an affine transformation of the plane carrying a given triangle onto another triangle may not be a contraction even if it contracts edges, heights or medians.

  15. Affine Contractions on the Plane

    ERIC Educational Resources Information Center

    Celik, D.; Ozdemir, Y.; Ureyen, M.

    2007-01-01

    Contractions play a considerable role in the theory of fractals. However, it is not easy to find contractions which are not similitudes. In this study, it is shown by counter examples that an affine transformation of the plane carrying a given triangle onto another triangle may not be a contraction even if it contracts edges, heights or medians.…

  16. Removal of poly-histidine fusion tags from recombinant proteins purified by expanded bed adsorption.

    PubMed

    Abdullah, N; Chase, H A

    2005-11-20

    Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His(6) by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His(6) to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions. PMID:16080185

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

    SciTech Connect

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

    2007-07-01

    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.

  18. Purification of his-tagged proteins using fractogel-cobalt.

    PubMed

    Tom, Roseanne; Bisson, Louis; Durocher, Yves

    2008-01-01

    INTRODUCTIONFast and efficient production of recombinant proteins (r-proteins) remains a major challenge for the academic and biopharmaceutical communities. Such proteins often need to be as pure as possible before any characterization study can begin. Although many types of protein tag are available, histidine is the most popular. Although small-scale immobilized metal-affinity column (IMAC) purification of such proteins (e.g., <500 mL of culture medium) can easily be achieved using gravity chromatography columns, larger volumes can be processed with the aid of automated chromatography systems. This protocol describes an IMAC purification technique for secreted proteins using a cobalt-loaded resin. Preliminary small-scale trials using this technique can be used to determine the production scale that will be needed to provide enough pure material for a given study. PMID:21356796

  19. Sensor-based material tagging system

    SciTech Connect

    Vercellotti, L.C.; Cox, R.W.; Ravas, R.J.; Schlotterer, J.C.

    1991-12-31

    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.

  20. Sensor-based material tagging system

    SciTech Connect

    Vercellotti, L.C.; Cox, R.W.; Ravas, R.J.; Schlotterer, J.C. . Science and Technology Center)

    1991-01-01

    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.

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

    NASA Astrophysics Data System (ADS)

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

    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.

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

    PubMed

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

    2011-07-01

    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

  3. Production of a His-tagged canecystatin in transgenic sugarcane.

    PubMed

    Henrique-Silva, Flavio; Soares-Costa, Andrea

    2012-01-01

    Transgenic plants have been widely used as expression systems of recombinant proteins in recent years because it can be an efficient alternative for the large-scale production of proteins. This is an area with great potential but is still not much explored. Indeed, this system can bring a breakthrough in the expression of any protein. The model used here as a protein factory was sugarcane, a crop of great global importance. This chapter describes the system that has been adopted in the routine production of transgenic sugarcane coupled with protein purification protocol. In this chapter, we describe production of transgenic sugarcane expressing a His-tagged cystatin under the control of the maize ubiquitin promoter. A transformed sugarcane plant presented high levels of protein expression and was selected for the purification of this protein through affinity chromatography in a nickel column. These studies demonstrate that sugarcane can be a viable expression system for recombinant protein production and that the His-tag purification strategy used to isolate the purified protein was effective. PMID:22351027

  4. To tag or not to tag: animal welfare, conservation and stakeholder considerations in fish tracking studies that use electronic tags

    SciTech Connect

    Cooke, Steven J.; Nguyen, Vivian M.; Murchie, Karen J.; Thiem, Jason D.; Donaldson, Michael R.; Hinch, Scott G.; Brown, Richard S.; Fisk, Aaron

    2013-11-01

    The advent and widespread adoption of electronic tags (including biotelemetry and biologging devices) for tracking animals has provided unprecedented information on the biology, management, and conservation of fish in the world’s oceans and inland waters. However, use of these tools is not without controversy. Even when scientific and management objectives may best be achieved using electronic tags, it is increasingly important to further consider other factors such as the welfare of tagged animals (i.e., the role of training and science-based surgical guidelines, anesthetic use, inability to maintain sterile conditions in field environments), the ethics of tagging threatened species vs. using surrogates, stakeholder perspectives on tagging (including aboriginals), as well as use of data emanating from such studies (e.g., by fishers to facilitate exploitation). Failure to do so will have the potential to create conflict and undermine scientific, management and public confidence in the use of this powerful tool. Indeed, there are already a number of examples of where tracking studies using electronic tags have been halted based on concerns raised by researchers, authorities, or stakeholders. Here we present a candid evaluation of several factors that should be considered when determining when to tag or not to tag fish with electronic devices. It is not our objective to judge the merit of previous studies. Rather, we hope to stimulate debate and discussion regarding the use of electronic tags to study fish. Relatedly, there is a need for more research to address these questions (e.g., what level of cleanliness is needed when conducting surgeries, what type of training should be required for fish surgery) including human dimensions studies to understand perspectives of different actors including society as a whole with respect to tagging and tracking studies.

  5. Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

    PubMed

    Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

    2016-05-01

    Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. PMID:26805756

  6. Photodissociation of Charge Tagged Peptides

    NASA Astrophysics Data System (ADS)

    He, Yi; Parthasarathi, Ramakrishnan; Raghavachari, Krishnan; Reilly, James P.

    2012-07-01

    Tris(2,4,6-trimethoxyphenyl) phosphonium acetyl (TMPP-Ac) was previously introduced to improve the mass spectrometric sequence analysis of peptides by fixing a permanent charge at the N-termini. However, peptides containing arginine residues did not fragment efficiently after TMPP-Ac modification. In this work, we combine charge derivatization with photodissociation. The fragmentation of TMPP-derivatized peptides is greatly improved and a series of N-terminal fragments is generated with complete sequence information. Arginine has a special effect on the fragmentation of the TMPP tagged peptides when it is the N-terminal peptide residue. Theoretical and experimental results suggest that this is due to hydrogen transfer from the charged N-terminus to the hydrogen-deficient peptide sequence.

  7. Engineering the ATLAS TAG Browser

    NASA Astrophysics Data System (ADS)

    Zhang, Qizhi; ATLAS Collaboration

    2011-12-01

    ELSSI is a web-based event metadata (TAG) browser and event-level selection service for ATLAS. In this paper, we describe some of the challenges encountered in the process of developing ELSSI, and the software engineering strategies adopted to address those challenges. Approaches to management of access to data, browsing, data rendering, query building, query validation, execution, connection management, and communication with auxiliary services are discussed. We also describe strategies for dealing with data that may vary over time, such as run-dependent trigger decision decoding. Along with examples, we illustrate how programming techniques in multiple languages (PHP, JAVASCRIPT, XML, AJAX, and PL/SQL) have been blended to achieve the required results. Finally, we evaluate features of the ELSSI service in terms of functionality, scalability, and performance.

  8. Protein purification by affinity precipitation.

    PubMed

    Hilbrig, Frank; Freitag, Ruth

    2003-06-25

    Developing the most efficient strategy for the purification of a (recombinant) protein especially at large scale remains a challenge. A typical problem of the downstream process of mammalian cell products is, for instance, the early capture of the highly diluted product from the complex process stream. Affinity precipitation has been suggested in this context. The technique is known for over 20 years, but has recently received more attention due to the development of new materials for its implementation, but also because it seems ideally suited to specific product capture at large scale. The present review gives a comprehensive overview over this technique. Besides an introduction to the basic principle and a brief summary of the historical development, the main focus is on the current state-of-art of the technique, the available materials, important recent applications, as well as process design strategies and operating procedures. Special consideration is given to affinity precipitation for product recovery at large scale. PMID:12767322

  9. Transposon tagging in diploid strawberry.

    PubMed

    Veilleux, Richard E; Mills, Kerri P; Baxter, Aaron J; Upham, Kendall T; Ferguson, Tammy J; Holt, Sarah Hudson; Lu, Nan; Ruiz-Rojas, Juan J; Pantazis, Christopher J; Davis, Cherish M; Lindsay, Robert C; Powell, Frankie L; Dan, Yinghui; Dickerman, Allan W; Oosumi, Teruko; Shulaev, Vladimir

    2012-10-01

    Fragaria vesca was transformed with a transposon tagging construct harbouring amino terminally deleted maize transposase and EGFP (Ac element), NPTII, CaMV 35S promoter (P35S) driving transposase and mannopine synthase promoter (Pmas) driving EGFP (Ds element). Of 180 primary transgenics, 48 were potential launch pads, 72 were multiple insertions or chimaeras, and 60 exhibited somatic transposition. T? progeny of 32 putative launch pads were screened by multiplex PCR for transposition. Evidence of germ-line transposition occurred in 13 putative launch pads; however, the transposition frequency was too low in three for efficient recovery of transposants. The transposition frequency in the remaining launch pads ranged from 16% to 40%. After self-pollination of the T? launch pads, putative transposants in the T? generation were identified by multiplex PCR. Sequencing of hiTAIL-PCR products derived from nested primers within the Ds end sequences (either P35S at the left border or the inverted repeat at the right border) of T? plants revealed transposition of the Ds element to distant sites in the strawberry genome. From more than 2400 T? plants screened, 103 unique transposants have been identified, among which 17 were somatic transpositions observed in the T? generation. Ds insertion sites were dispersed among various gene elements [exons (15%), introns (23%), promoters (30%), 3' UTRs (17%) as well as intergenically (15%)]. Three-primer (one on either side of the Ds insertion and one within the Ds T-DNA) PCR could be used to identify homozygous T? transposon-tagged plants. The mutant collection has been catalogued in an on-line database. PMID:22845757

  10. Method and apparatus for manufacturing gas tags

    DOEpatents

    Gross, Kenny C. (Bolingbrook, IL); Laug, Matthew T. (Idaho Falls, ID)

    1996-01-01

    For use in the manufacture of gas tags employed in a gas tagging failure detection system for a nuclear reactor, a plurality of commercial feed gases each having a respective noble gas isotopic composition are blended under computer control to provide various tag gas mixtures having selected isotopic ratios which are optimized for specified defined conditions such as cost. Using a new approach employing a discrete variable structure rather than the known continuous-variable optimization problem, the computer controlled gas tag manufacturing process employs an analytical formalism from condensed matter physics known as stochastic relaxation, which is a special case of simulated annealing, for input feed gas selection. For a tag blending process involving M tag isotopes with N distinct feed gas mixtures commercially available from an enriched gas supplier, the manufacturing process calculates the cost difference between multiple combinations and specifies gas mixtures which approach the optimum defined conditions. The manufacturing process is then used to control tag blending apparatus incorporating tag gas canisters connected by stainless-steel tubing with computer controlled valves, with the canisters automatically filled with metered quantities of the required feed gases.

  11. Method and apparatus for manufacturing gas tags

    DOEpatents

    Gross, K.C.; Laug, M.T.

    1996-12-17

    For use in the manufacture of gas tags employed in a gas tagging failure detection system for a nuclear reactor, a plurality of commercial feed gases each having a respective noble gas isotopic composition are blended under computer control to provide various tag gas mixtures having selected isotopic ratios which are optimized for specified defined conditions such as cost. Using a new approach employing a discrete variable structure rather than the known continuous-variable optimization problem, the computer controlled gas tag manufacturing process employs an analytical formalism from condensed matter physics known as stochastic relaxation, which is a special case of simulated annealing, for input feed gas selection. For a tag blending process involving M tag isotopes with N distinct feed gas mixtures commercially available from an enriched gas supplier, the manufacturing process calculates the cost difference between multiple combinations and specifies gas mixtures which approach the optimum defined conditions. The manufacturing process is then used to control tag blending apparatus incorporating tag gas canisters connected by stainless-steel tubing with computer controlled valves, with the canisters automatically filled with metered quantities of the required feed gases. 4 figs.

  12. A Radio Tag for Big Whales

    ERIC Educational Resources Information Center

    Watkins, William A.

    1978-01-01

    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)

  13. 9 CFR 2.54 - Lost tags.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Lost tags. 2.54 Section 2.54 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.54 Lost tags. Each dealer or exhibitor shall be...

  14. 9 CFR 2.54 - Lost tags.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Lost tags. 2.54 Section 2.54 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.54 Lost tags. Each dealer or exhibitor shall be...

  15. 9 CFR 2.54 - Lost tags.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Lost tags. 2.54 Section 2.54 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.54 Lost tags. Each dealer or exhibitor shall be...

  16. 9 CFR 2.54 - Lost tags.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Lost tags. 2.54 Section 2.54 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.54 Lost tags. Each dealer or exhibitor shall be...

  17. 9 CFR 2.54 - Lost tags.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Lost tags. 2.54 Section 2.54 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.54 Lost tags. Each dealer or exhibitor shall be...

  18. TAG (Teaching Active Games) for the Holidays

    ERIC Educational Resources Information Center

    Erwin, Heather E.; Bachtel, Amy

    2007-01-01

    Holidays present the perfect opportunity for physical educators to utilize creative TAG (Teaching Active Games) games to offer maximum physical activity opportunities for their students. The TAG ideas in this article offer physical education teachers quick, instant activities that involve very little equipment, time management, or instruction. At

  19. 50 CFR 20.81 - Tagging requirement.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 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 migratory game birds unless such birds are tagged as required by § 20.36....

  20. 50 CFR 20.81 - Tagging requirement.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 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 migratory game birds unless such birds are tagged as required by § 20.36....

  1. Harnessing Collective Knowledge Inherent in Tag Clouds

    ERIC Educational Resources Information Center

    Cress, U.; Held, C.

    2013-01-01

    Tagging systems represent the conceptual knowledge of a community. We experimentally tested whether people harness this collective knowledge when navigating through the Web. As a within-factor we manipulated people's prior knowledge (no knowledge vs. prior knowledge that was congruent/incongruent to the collective knowledge inherent in the tags).

  2. Harnessing Collective Knowledge Inherent in Tag Clouds

    ERIC Educational Resources Information Center

    Cress, U.; Held, C.

    2013-01-01

    Tagging systems represent the conceptual knowledge of a community. We experimentally tested whether people harness this collective knowledge when navigating through the Web. As a within-factor we manipulated people's prior knowledge (no knowledge vs. prior knowledge that was congruent/incongruent to the collective knowledge inherent in the tags).…

  3. Fuel and control assembly tag gas

    SciTech Connect

    Not Available

    1986-01-01

    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.

  4. Notes on SAW Tag Interrogation Techniques

    NASA Technical Reports Server (NTRS)

    Barton, Richard J.

    2010-01-01

    We consider the problem of interrogating a single SAW RFID tag with a known ID and known range in the presence of multiple interfering tags under the following assumptions: (1) The RF propagation environment is well approximated as a simple delay channel with geometric power-decay constant alpha >/= 2. (2) The interfering tag IDs are unknown but well approximated as independent, identically distributed random samples from a probability distribution of tag ID waveforms with known second-order properties, and the tag of interest is drawn independently from the same distribution. (3) The ranges of the interfering tags are unknown but well approximated as independent, identically distributed realizations of a random variable rho with a known probability distribution f(sub rho) , and the tag ranges are independent of the tag ID waveforms. In particular, we model the tag waveforms as random impulse responses from a wide-sense-stationary, uncorrelated-scattering (WSSUS) fading channel with known bandwidth and scattering function. A brief discussion of the properties of such channels and the notation used to describe them in this document is given in the Appendix. Under these assumptions, we derive the expression for the output signal-to-noise ratio (SNR) for an arbitrary combination of transmitted interrogation signal and linear receiver filter. Based on this expression, we derive the optimal interrogator configuration (i.e., transmitted signal/receiver filter combination) in the two extreme noise/interference regimes, i.e., noise-limited and interference-limited, under the additional assumption that the coherence bandwidth of the tags is much smaller than the total tag bandwidth. Finally, we evaluate the performance of both optimal interrogators over a broad range of operating scenarios using both numerical simulation based on the assumed model and Monte Carlo simulation based on a small sample of measured tag waveforms. The performance evaluation results not only provide guidelines for proper interrogator design, but also provide some insight on the validity of the assumed signal model. It should be noted that the assumption that the impulse response of the tag of interest is known precisely implies that the temperature and range of the tag are also known precisely, which is generally not the case in practice. However, analyzing interrogator performance under this simplifying assumption is much more straightforward and still provides a great deal of insight into the nature of the problem.

  5. Use of the Nanofitin Alternative Scaffold as a GFP-Ready Fusion Tag

    PubMed Central

    Huet, Simon; Gorre, Harmony; Perrocheau, Anaëlle; Picot, Justine; Cinier, Mathieu

    2015-01-01

    With the continuous diversification of recombinant DNA technologies, the possibilities for new tailor-made protein engineering have extended on an on-going basis. Among these strategies, the use of the green fluorescent protein (GFP) as a fusion domain has been widely adopted for cellular imaging and protein localization. Following the lead of the direct head-to-tail fusion of GFP, we proposed to provide additional features to recombinant proteins by genetic fusion of artificially derived binders. Thus, we reported a GFP-ready fusion tag consisting of a small and robust fusion-friendly anti-GFP Nanofitin binding domain as a proof-of-concept. While limiting steric effects on the carrier, the GFP-ready tag allows the capture of GFP or its blue (BFP), cyan (CFP) and yellow (YFP) alternatives. Here, we described the generation of the GFP-ready tag from the selection of a Nanofitin variant binding to the GFP and its spectral variants with a nanomolar affinity, while displaying a remarkable folding stability, as demonstrated by its full resistance upon thermal sterilization process or the full chemical synthesis of Nanofitins. To illustrate the potential of the Nanofitin-based tag as a fusion partner, we compared the expression level in Escherichia coli and activity profile of recombinant human tumor necrosis factor alpha (TNFα) constructs, fused to a SUMO or GFP-ready tag. Very similar expression levels were found with the two fusion technologies. Both domains of the GFP-ready tagged TNFα were proved fully active in ELISA and interferometry binding assays, allowing the simultaneous capture by an anti-TNFα antibody and binding to the GFP, and its spectral mutants. The GFP-ready tag was also shown inert in a L929 cell based assay, demonstrating the potent TNFα mediated apoptosis induction by the GFP-ready tagged TNFα. Eventually, we proposed the GFP-ready tag as a versatile capture and labeling system in addition to expected applications of anti-GFP Nanofitins (as illustrated with previously described state-of-the-art anti-GFP binders applied to living cells and in vitro applications). Through a single fusion domain, the GFP-ready tagged proteins benefit from subsequent customization within a wide range of fluorescence spectra upon indirect binding of a chosen GFP variant. PMID:26539718

  6. Intrinsic-surface-tag image authentication

    SciTech Connect

    Palm, R.G.; DeVolpi, A.

    1991-12-01

    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.

  7. Biofabrication of ZnS:Mn luminescent nanocrystals using histidine, hexahistidine, and His-tagged proteins: a comparison study

    PubMed Central

    Zhou, Weibin; Baneyx, Franois

    2013-01-01

    The ubiquitous hexahistidine purification tag has been used to conjugate proteins to the shell of CdSe:ZnS quantum dots (QDs) due to its affinity for surface-exposed Zn2+ ions but little attention has been paid to the potential of His-tagged proteins for mineralizing luminescent ZnS nanocrystals. Here, we compare the ability of free histidine, a His tag peptide, His-tagged thioredoxin (TrxA, a monomeric protein), and N- and C-terminally His-tagged versions of Hsp31 (a homodimeric protein) to support the synthesis of Mn-doped ZnS nanocrystals from aqueous precursors under mild conditions of pH (8.2) and temperature (37C). We find that: (1) it is possible to produce poor quality QDs when histidine is used at high (8 mM) concentration; (2) an increase in local histidine concentration through repetition of the amino acid as a His tag decreases the amount of needed reagent ?10-fold and improves optical properties; (3) fusion of the same His tag to TrxA allows for ZnS:Mn QDs mineralization at micromolar concentrations; and (4) doubling the local hexahistidine concentration by exploiting Hsp31 dimerization further improves nanocrystal luminescence with the brightest particles obtained when His tags are spatially co-localized at the Hsp31 N-termini. Although hexahistidine tracts are not as efficient as combinatorially selected ZnS binding peptides at QD synthesis, it should be possible to use the large number of available His-tagged proteins and the synthesis approach described herein to produce luminescent nanoparticles whose protein shell carries a broad range of functions. PMID:25013361

  8. Production of N-acetyl-D-neuraminic acid using two sequential enzymes overexpressed as double-tagged fusion proteins

    PubMed Central

    Wang, Tzu-Hsien; Chen, Ying-Yin; Pan, Hsin-Hung; Wang, Feng-Pao; Cheng, Chung-Hsien; Lee, Wen-Chien

    2009-01-01

    Background Two sequential enzymes in the production of sialic acids, N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), were overexpressed as double-tagged gene fusions. Both were tagged with glutathione S-transferase (GST) at the N-terminus, but at the C-terminus, one was tagged with five contiguous aspartate residues (5D), and the other with five contiguous arginine residues (5R). Results Both fusion proteins were overexpressed in Escherichia coli and retained enzymatic activity. The fusions were designed so their surfaces were charged under enzyme reaction conditions, which allowed isolation and immobilization in a single step, through a simple capture with either an anionic or a cationic exchanger (Sepharose Q or Sepharose SP) that electrostatically bound the 5D or 5R tag. The introduction of double tags only marginally altered the affinity of the enzymes for their substrates, and the double-tagged proteins were enzymatically active in both soluble and immobilized forms. Combined use of the fusion proteins led to the production of N-acetyl-D-neuraminic acid (Neu5Ac) from N-acetyl-D-glucosamine (GlcNAc). Conclusion Double-tagged gene fusions were overexpressed to yield two enzymes that perform sequential steps in sialic acid synthesis. The proteins were easily immobilized via ionic tags onto ionic exchange resins and could thus be purified by direct capture from crude protein extracts. The immobilized, double-tagged proteins were effective for one-pot enzymatic production of sialic acid. PMID:19586552

  9. Tags and seals for arms control verification

    SciTech Connect

    DeVolpi, A.

    1990-09-18

    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.

  10. Enhanced UHF RFID tags for drug tracing.

    PubMed

    Catarinucci, Luca; Colella, Riccardo; De Blasi, Mario; Patrono, Luigi; Tarricone, Luciano

    2012-12-01

    Radio Frequency Identification (RFID) technology is playing a crucial role for item-level tracing systems in healthcare scenarios. The pharmaceutical supply chain is a fascinating application context, where RFID can guarantee transparency in the drug flow, supporting both suppliers and consumers against the growing counterfeiting problem. In such a context, the choice of the most adequate RFID tag, in terms of shape, frequency, size and reading range, is crucial. The potential presence of items containing materials hostile to the electromagnetic propagation exasperates the problem. In addition, the peculiarities of the different RFID-based checkpoints make even more stringent the requirements for the tag. In this work, the performance of several commercial UHF RFID tags in each step of the pharmaceutical supply chain has been evaluated, confirming the expected criticality. On such basis, a guideline for the electromagnetic design of new high-performance tags capable to overcome such criticalities has been defined. Finally, driven by such guidelines, a new enhanced tag has been designed, realized and tested. Due to patent pending issues, the antenna shape is not shown. Nevertheless, the optimal obtained results do not lose their validity. Indeed, on the one hand they demonstrate that high performance item level tracing systems can actually be implemented also in critical operating conditions. On the other hand, they encourage the tag designer to follow the identified guidelines so to realize enhanced UHF tags. PMID:22048779

  11. Fabrication process for a flexible tag microlab

    NASA Astrophysics Data System (ADS)

    Abad, E.; Mazzolai, B.; Juarros, A.; Gmez, D.; Mondini, A.; Sayhan, I.; Krenkow, A.; Becker, Th.

    2007-05-01

    The aim of this paper is to present an integrated process flow for a smart tag with integrated sensors and RFID communication, a Flexible Tag Microlab (FTM). The heart of the designed container tracing system is an RFID system (Reader + Tag) with gas sensing capabilities on board. In the former prototypes, the chemical sensors were integrated on the reader, whereas the tags where addressed like conventional RFID-tags containing also physical (temperature, humidity and light) sensors. However, this paper will show how the gas sensing reader functionalities are being transferred to the tag, reaching a flexible tag microlab, which represents a real innovation in the field of flexible labels. Key issues for the realisation of the FTM, such us flexible substrates and gas sensor integration technologies will be presented. The process flow employed for the two metal levels interconnect fabrication will be described in detail. The material used is the DuPont TM Pyralux (R) AP 8525R double-sided copper-clad laminate, formed by a Kapton foil with a copper layer on each side. The vias and windows openings are performed by femtosecond laser ablation. The copper interconnections are realized by photolithography and wet chemical etching. The MOX sensors hotplates specially developed to fulfil the FTM constrains in terms of low power consumption has been used to prove two integration technologies into the flexible substrates: Chip on Flex (COF) wire bonding and Anisotropic Conductive Adhesive (ACA) flip chip bonding. Both technologies will be compared and benchmarked for future product developments.

  12. Transcriptome annotation using tandem SAGE tags

    PubMed Central

    Rivals, Eric; Boureux, Anthony; Lejeune, Mireille; Ottones, Florence; Pecharromàn Pérez, Oscar; Tarhio, Jorma; Pierrat, Fabien; Ruffle, Florence; Commes, Thérèse; Marti, Jacques

    2007-01-01

    Analysis of several million expressed gene signatures (tags) revealed an increasing number of different sequences, largely exceeding that of annotated genes in mammalian genomes. Serial analysis of gene expression (SAGE) can reveal new Poly(A) RNAs transcribed from previously unrecognized chromosomal regions. However, conventional SAGE tags are too short to identify unambiguously unique sites in large genomes. Here, we design a novel strategy with tags anchored on two different restrictions sites of cDNAs. New transcripts are then tentatively defined by the two SAGE tags in tandem and by the spanning sequence read on the genome between these tagged sites. Having developed a new algorithm to locate these tag-delimited genomic sequences (TDGS), we first validated its capacity to recognize known genes and its ability to reveal new transcripts with two SAGE libraries built in parallel from a single RNA sample. Our algorithm proves fast enough to experiment this strategy at a large scale. We then collected and processed the complete sets of human SAGE tags to predict yet unknown transcripts. A cross-validation with tiling arrays data shows that 47% of these TDGS overlap transcriptional active regions. Our method provides a new and complementary approach for complex transcriptome annotation. PMID:17709346

  13. Communication methods, systems, apparatus, and devices involving RF tag registration

    DOEpatents

    Burghard, Brion J.; Skorpik, James R.

    2008-04-22

    One technique of the present invention includes a number of Radio Frequency (RF) tags that each have a different identifier. Information is broadcast to the tags from an RF tag interrogator. This information corresponds to a maximum quantity of tag response time slots that are available. This maximum quantity may be less than the total number of tags. The tags each select one of the time slots as a function of the information and a random number provided by each respective tag. The different identifiers are transmitted to the interrogator from at least a subset of the RF tags.

  14. [Determination of L-dopa and dopamine in rat brain microdialysate by ultra high performance liquid chromatography-tandem mass spectrometry using stable isotope-coded derivatization coupled with dispersive liquid-liquid microextraction].

    PubMed

    Qi, Weimei; Zhao, Xian-en; Qi, Yong; Sun, Zhiwei; Chen, Guang; You, Jinmao; Suo, Yourui

    2015-09-01

    The sensitive detection method of levodopa (L-DOPA) and dopamine (DA) in rat brain microdialysate of Parkinson's disease (PD) is an essential tool for the clinical study and attenuated synergistic drug screening for L-DOPA from traditional Chinese medicines. Using d0/d3-10-methyl-acridone-2-sulfonyl chloride (d0/d3-MASC) as stable isotope derivatization reagent, a novel ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for L-DOPA and DA by stable isotope- coded derivatization coupled with ultrasonic-assisted dispersive liquid-liquid microextraction (UA-DLLME). d3-MASC (light) and d3-MASC (heavy) were used as derivatization reagents for microdialysate samples and standards, respectively. Mixtures of the two solutions were prepared by UA-DLLME for UHPLC-MS/MS analysis with multiple reaction monitoring (MRM) mode. With d3-MASC heavy derivatives as internal standards for corresponding light derivatives from samples, the stable isotope internal standard quantification for L-DOPA and DA was carried out. The stable derivatives were obtained in aqueous acetonitrile (pH 10.8 sodium carbonate-sodium bicarbonate buffer) at 37 C for 3.0 min, and then were separated within 2.0 min using gradient elution. Linear range was 0.20-1500.0 nmol/L (R > 0.994). LODs were 0.005 and 0.009 nmol/L for DA and L-DOPA (S/N = 3), respectively. This method was validated, and it showed obvious advantages in comparing with the reported methods in terms of sensitivity, analysis speed and anti-matrix interference. This method has been successfully applied to the study of effect of Shouwu Fang on L-DOPA and DA concentration fluctuations in PD rat brain microdialysate. PMID:26753287

  15. Tag retention, growth, and survival of red swamp crayfish Procambarus clarkii marked with coded wire tags

    USGS Publications Warehouse

    Isely, J.J.; Eversole, A.G.

    1998-01-01

    Juvenile red swamp crayfish (or crawfish), Procambarus clarkii (20-41 mm in total length) were collected from a crayfish culture pond by dipnetting and tagged with sequentially numbered, standard length, binary-coded wire tags. Four replicates of 50 crayfish were impaled perpendicular to the long axis of the abdomen with a fixed needle. Tags were injected transversely into the ventral surface of the first or second abdominal segment and were imbedded in the musculature just beneath the abdominal sternum. Tags were visible upon inspection. Additionally, two replicates of 50 crayfish were not tagged and were used as controls. Growth, survival, and tag retention were evaluated after 7 d in individual containers, after 100 d in aquaria, and after 200 d in field cages. Tag retention during each sample period was 100%, and average mortality of tagged crayfish within 7 d of tagging was 1%. Mortality during the remainder of the study was high (75-91%) but was similar between treatment and control samples. Most of the deaths were probably due to cannibalism. Average total length increased threefold during the course of the study, and crayfish reached maturity. Because crayfish were mature by the end of the study, we concluded that the coded wire tag was retained through the life history of the crayfish.

  16. Expression and affinity purification of recombinant proteins from plants

    NASA Technical Reports Server (NTRS)

    Desai, Urvee A.; Sur, Gargi; Daunert, Sylvia; Babbitt, Ruth; Li, Qingshun

    2002-01-01

    With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system. Copyright 2002 Elsevier Science (USA).

  17. Time-Tag Generation Script

    NASA Technical Reports Server (NTRS)

    Jackson, Dan E.

    2010-01-01

    Time-Tag Generation Script (TTaGS) is an application program, written in the AWK scripting language, for generating commands for aiming one Ku-band antenna and two S-band antennas for communicating with spacecraft. TTaGS saves between 2 and 4 person-hours per every 24 hours by automating the repetitious process of building between 150 and 180 antenna-control commands. TTaGS reads a text database of communication satellite schedules and a text database of satellite rise and set times and cross-references items in the two databases. It then compares the scheduled start and stop with the geometric rise and set to compute the times to execute antenna control commands. While so doing, TTaGS determines whether to generate commands for guidance, navigation, and control computers to tell them which satellites to track. To help prevent Ku-band irradiation of the Earth, TTaGS accepts input from the user about horizon tolerance and accordingly restricts activation and effects deactivation of the transmitter. TTaGS can be modified easily to enable tracking of additional satellites and for such other tasks as reading Sun-rise/set tables to generate commands to point the solar photovoltaic arrays of the International Space Station at the Sun.

  18. Tagging Water Sources in Atmospheric Models

    NASA Technical Reports Server (NTRS)

    Bosilovich, M.

    2003-01-01

    Tagging of water sources in atmospheric models allows for quantitative diagnostics of how water is transported from its source region to its sink region. In this presentation, we review how this methodology is applied to global atmospheric models. We will present several applications of the methodology. In one example, the regional sources of water for the North American Monsoon system are evaluated by tagging the surface evaporation. In another example, the tagged water is used to quantify the global water cycling rate and residence time. We will also discuss the need for more research and the importance of these diagnostics in water cycle studies.

  19. The affine quantum gravity programme

    NASA Astrophysics Data System (ADS)

    Klauder, John R.

    2002-02-01

    The central principle of affine quantum gravity is securing and maintaining the strict positivity of the matrix hat g ab(x)} composed of the spatial components of the local metric operator. On spectral grounds, canonical commutation relations are incompatible with this principle, and they must be replaced by noncanonical, affine commutation relations. Due to the partial second-class nature of the quantum gravitational constraints, it is advantageous to use the recently developed projection operator method, which treats all quantum constraints on an equal footing. Using this method, enforcement of regularized versions of the gravitational operator constraints is formulated quite naturally by means of a novel and relatively well-defined functional integral involving only the same set of variables that appears in the usual classical formulation. It is anticipated that skills and insight to study this formulation can be developed by studying special, reduced-variable models that still retain some basic characteristics of gravity, specifically a partial second-class constraint operator structure. Although perturbatively nonrenormalizable, gravity may possibly be understood nonperturbatively from a hard-core perspective that has proved valuable for specialized models. Finally, developing a procedure to pass to the genuine physical Hilbert space involves several interconnected steps that require careful coordination.

  20. Indian Craniometric Variability and Affinities

    PubMed Central

    Raghavan, Pathmanathan; Bulbeck, David; Pathmanathan, Gayathiri; Rathee, Suresh Kanta

    2013-01-01

    Recently published craniometric and genetic studies indicate a predominantly indigenous ancestry of Indian populations. We address this issue with a fuller coverage of Indian craniometrics than any done before. We analyse metrical variability within Indian series, Indians' sexual dimorphism, differences between northern and southern Indians, index-based differences of Indian males from other series, and Indians' multivariate affinities. The relationship between a variable's magnitude and its variability is log-linear. This relationship is strengthened by excluding cranial fractions and series with a sample size less than 30. Male crania are typically larger than female crania, but there are also shape differences. Northern Indians differ from southern Indians in various features including narrower orbits and less pronounced medial protrusion of the orbits. Indians resemble Veddas in having small crania and similar cranial shape. Indians' wider geographic affinities lie with “Caucasoid” populations to the northwest, particularly affecting northern Indians. The latter finding is confirmed from shape-based Mahalanobis-D distances calculated for the best sampled male and female series. Demonstration of a distinctive South Asian craniometric profile and the intermediate status of northern Indians between southern Indians and populations northwest of India confirm the predominantly indigenous ancestry of northern and especially southern Indians. PMID:24455409

  1. Multi-exemplar affinity propagation.

    PubMed

    Wang, Chang-Dong; Lai, Jian-Huang; Suen, Ching Y; Zhu, Jun-Yong

    2013-09-01

    The affinity propagation (AP) clustering algorithm has received much attention in the past few years. AP is appealing because it is efficient, insensitive to initialization, and it produces clusters at a lower error rate than other exemplar-based methods. However, its single-exemplar model becomes inadequate when applied to model multisubclasses in some situations such as scene analysis and character recognition. To remedy this deficiency, we have extended the single-exemplar model to a multi-exemplar one to create a new multi-exemplar affinity propagation (MEAP) algorithm. This new model automatically determines the number of exemplars in each cluster associated with a super exemplar to approximate the subclasses in the category. Solving the model is NP-hard and we tackle it with the max-sum belief propagation to produce neighborhood maximum clusters, with no need to specify beforehand the number of clusters, multi-exemplars, and superexemplars. Also, utilizing the sparsity in the data, we are able to reduce substantially the computational time and storage. Experimental studies have shown MEAP's significant improvements over other algorithms on unsupervised image categorization and the clustering of handwritten digits. PMID:23868781

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

    SciTech Connect

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

    2008-02-01

    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.

  3. Evacuation of Passive Integrated Transponder (PIT) Tags from Northern Pikeminnow Consuming Tagged Juvenile Chinook Salmon

    USGS Publications Warehouse

    Petersen, J.H.; Barfoot, C.A.

    2003-01-01

    Prey fish implanted with passive integrated transponder (PIT) tags can be used in predation studies if the timing of tag evacuation from the predators is understood. Laboratory experiments were conducted to determine how PIT tags in juvenile Chinook salmon Oncorhynchus tshawytscha that were consumed by northern pikeminnow Ptychocheilus oregonensis were evacuated in relation to various parameters. The rate of evacuation was directly related to temperature, while predator size and the number of prey consumed had less effect on the timing of tag evacuation. A power model was fitted to predict the proportion of tags expected to be evacuated at different intervals after ingestion. These results could be used in planning field or laboratory predation experiments with PIT-tagged prey fish.

  4. Mass-producible micro-holographic tags

    SciTech Connect

    Sweatt, W.C.; Ray-Chaudhuri, A.K.; Kravitz, S.H.; Warren, M.E.; Stulen, R.H.; Tichenor, D.A.; Krenz, K.D. |; Descour, M.R.; Underwood, J.H.

    1996-06-01

    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.

  5. 50 CFR 635.33 - Archival tags.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... catch, possess, retain, and land an Atlantic HMS in which an archival tag has been implanted or affixed... consistent with the fishing gear and activity which resulted in the catch. In the event such fishing gear...

  6. 50 CFR 635.33 - Archival tags.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... catch, possess, retain, and land an Atlantic HMS in which an archival tag has been implanted or affixed... consistent with the fishing gear and activity which resulted in the catch. In the event such fishing gear...

  7. Magnetic vector field tag and seal

    DOEpatents

    Johnston, Roger G.; Garcia, Anthony R.

    2004-08-31

    One or more magnets are placed in a container (preferably on objects inside the container) and the magnetic field strength and vector direction are measured with a magnetometer from at least one location near the container to provide the container with a magnetic vector field tag and seal. The location(s) of the magnetometer relative to the container are also noted. If the position of any magnet inside the container changes, then the measured vector fields at the these locations also change, indicating that the tag has been removed, the seal has broken, and therefore that the container and objects inside may have been tampered with. A hollow wheel with magnets inside may also provide a similar magnetic vector field tag and seal. As the wheel turns, the magnets tumble randomly inside, removing the tag and breaking the seal.

  8. Intrinsic-surface-tag image authentication

    SciTech Connect

    Palm, R.G.; DeVolpi, A.

    1991-12-01

    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.

  9. User Interface Program for secure electronic tags

    SciTech Connect

    Cai, Y.; Koehl, E.R.; Carlson, R.D.; Raptis, A.C.

    1995-05-01

    This report summarizes and documents the efforts of Argonne National Laboratory (ANL) in developing a secure tag communication user interface program comprising a tag monitor and a communication tool. This program can perform the same functions as the software that was developed at the Lawrence Livermore National Laboratory (LLNL), but it is enhanced with a user-friendly screen. It represents the first step in updating the TRANSCOM Tracking System (TRANSCOM) by incorporating a tag communication screen menu into the main menu of the TRANSCOM user program. A working version of TRANSCOM, enhanced with ANL secure-tag graphics, will strongly support the Department of Energy Warhead Dismantlement/Special Nuclear Materials Control initiatives. It will allow commercial satellite tracking of the movements and operational activities of treaty-limited items and transportation vehicles throughout Europe and the former USSR, as well as the continental US.

  10. Rapid analysis of protein expression and solubility with the SpyTag-SpyCatcher system.

    PubMed

    Dovala, Dustin; Sawyer, William S; Rath, Christopher M; Metzger, Louis E

    2016-01-01

    Successful isolation of well-folded and active protein often first requires the creation of many constructs. These are needed to assess the effects of truncations, insertions, mutations, and the presence and position of different affinity tags. Determining which constructs yield the highest expression and solubility requires the investigator to express and partially purify each construct, and, in the case of low-expressing proteins, to follow the protein using time-consuming Western blots. Even then, many proteins form soluble aggregates, which may only be apparent after more extensive purification via size exclusion chromatography. In this work, we have utilized a covalent bond-forming tag/domain pair, known as SpyTag/SpyCatcher, to rapidly and specifically attach a fluorescent label to proteins of interest in cellular lysates. Once labeled, tagged proteins can easily be followed via SDS-PAGE and fluorescence size exclusion chromatography (F-SEC) to assess expression levels, solubility, and monodispersity without the need for purification. These techniques enable rapid and facile analysis of proteins, which may greatly facilitate optimization of protein expression constructs. PMID:26405011

  11. The unusual metal ion binding ability of histidyl tags and their mutated derivatives.

    PubMed

    Brasili, Davide; Watly, Joanna; Simonovsky, Eyal; Guerrini, Remo; Barbosa, Nuno A; Wieczorek, Robert; Remelli, Maurizio; Kozlowski, Henryk; Miller, Yifat

    2016-04-01

    Polyhistidine-tags are often used for the affinity purification of polyhistidine-tagged recombinant proteins. These sequences are also found in nature and are often highly conserved across different species. However, their exact role in the biological systems is not clear. The purpose of this work is to shed light on the behavior of poly-His sequences in their interactions with metal ions. This work illustrates the first study of novel poly-(His-Ala) peptides that bind Cu(ii) applying both experimental techniques and extensive computational tools. The studied novel peptides are analogues of the short protected fragment of the pHpG (EDDH9GVG10) peptide, which was found in the venom of Atheris squamigera. Our study presents the properties of metal ion binding-histidine tag complexes and their mutated derivatives. The Cu(ii) binding ability in pHG (Ac-EDDH9G-NH2) is more efficient than in the mutated derivatives, although the number of imidazoles that bind to Cu(ii) ions are similar. Finally, the formation of an α-helical structure is observed in pHG and in one of the mutated derivatives, indicating the importance of the sequence in the poly-(His-Ala) tags. PMID:26923149

  12. Deciphering the Cellular Targets of Bioactive Compounds Using a Chloroalkane Capture Tag.

    PubMed

    Ohana, Rachel Friedman; Kirkland, Thomas A; Woodroofe, Carolyn C; Levin, Sergiy; Uyeda, H Tetsuo; Otto, Paul; Hurst, Robin; Robers, Matthew B; Zimmerman, Kris; Encell, Lance P; Wood, Keith V

    2015-10-16

    Phenotypic screening of compound libraries is a significant trend in drug discovery, yet success can be hindered by difficulties in identifying the underlying cellular targets. Current approaches rely on tethering bioactive compounds to a capture tag or surface to allow selective enrichment of interacting proteins for subsequent identification by mass spectrometry. Such methods are often constrained by ineffective capture of low affinity and low abundance targets. In addition, these methods are often not compatible with living cells and therefore cannot be used to verify the pharmacological activity of the tethered compounds. We have developed a novel chloroalkane capture tag that minimally affects compound potency in cultured cells, allowing binding interactions with the targets to occur under conditions relevant to the desired cellular phenotype. Subsequent isolation of the interacting targets is achieved through rapid lysis and capture onto immobilized HaloTag protein. Exchanging the chloroalkane tag for a fluorophore, the putative targets identified by mass spectrometry can be verified for direct binding to the compound through resonance energy transfer. Using the interaction between histone deacetylases (HDACs) and the inhibitor, Vorinostat (SAHA), as a model system, we were able to identify and verify all the known HDAC targets of SAHA as well as two previously undescribed targets, ADO and CPPED1. The discovery of ADO as a target may provide mechanistic insight into a reported connection between SAHA and Huntington's disease. PMID:26162280

  13. Multifunctional protein labeling via enzymatic N-terminal tagging and elaboration by click chemistry.

    PubMed

    Heal, William P; Wright, Megan H; Thinon, Emmanuelle; Tate, Edward W

    2012-01-01

    A protocol for selective and site-specific enzymatic labeling of proteins is described. The method exploits the protein co-/post-translational modification known as myristoylation, the transfer of myristic acid (a 14-carbon saturated fatty acid) to an N-terminal glycine catalyzed by the enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT). Escherichia coli, having no endogenous NMT, is used for the coexpression of both the transferase and the target protein to be labeled, which participate in the in vivo N-terminal attachment of synthetically derived tagged analogs of myristic acid bearing a 'clickable' tag. This tag is a functional group that can undergo bio-orthogonal ligation via 'click' chemistry, for example, an azide, and can be used as a handle for further site-specific labeling in vitro. Here we provide protocols for in vivo N-terminal tagging of recombinant protein, and the synthesis and application of multifunctional reagents that enable protein labeling via click chemistry for affinity purification and detection by fluorescence. In addition to general N-terminal protein labeling, the protocol would be of particular use in providing evidence for native myristoylation of proteins of interest, proof of activity/selectivity of NMTs and cross-species reactivity of NMTs without resorting to the use of radioactive isotopes. PMID:22193303

  14. 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

    Tykvart, J.; Sacha, P.; Barinka, C.; Knedlik, T.; Starkova, J.; Lubkowski, J.; Konvalinka, J.

    2012-02-07

    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.

  15. Solid tags for identifying failed reactor components

    DOEpatents

    Bunch, Wilbur L. (Richland, WA); Schenter, Robert E. (Richland, WA)

    1987-01-01

    A solid tag material which generates stable detectable, identifiable, and measurable isotopic gases on exposure to a neutron flux to be placed in a nuclear reactor component, particularly a fuel element, in order to identify the reactor component in event of its failure. Several tag materials consisting of salts which generate a multiplicity of gaseous isotopes in predetermined ratios are used to identify different reactor components.

  16. b-tagging at D0

    SciTech Connect

    Hanagaki, K.; /Fermilab

    2005-07-01

    Many high p{sub T} physics analyses at the Tevatron contain a b-quark and hence a b-jet in the final states. We report on the b-jet identification methods in D0 and their performance. For 0.5% of light jet tagging rate, 40 or 45% of b-jet tagging efficiency is achieved for jets with 35 < E{sub T} < 55 GeV and |{eta}| < 1.2.

  17. Survival and tag loss in Moapa White River springfish implanted with passive integrated transponder tags

    USGS Publications Warehouse

    Dixon, Christopher J.; Mesa, Matthew G.

    2011-01-01

    We monitored survival and tag loss among Moapa White River springfish Crenichthys baileyi moapae that were surgically implanted with passive integrated transponder (PIT; 9 2 mm) tags. The fish used in the study ranged from 40 to 67 mm in total length and from 1.0 to 6.5 g in mass; the PIT tag: body weight ratios were 1.06.1%. Fish were held for 41 d in live cages within a small, warm desert stream. Survival did not differ between untagged control fish (94.5%) and tagged fish (95.6%). Survival did not appear to be influenced by fish size or PIT tag: body weight ratio, but the small number of fish that died precluded a detailed analysis. Tag retention was 100% among the 86 fish that survived over the 41 d. Our results suggest that surgically implanting 9-mm PIT tags into Moapa White River springfish as small as 40 mm is an effective method for marking them because it has minimal impacts on survival and tag retention is high. More work is needed on the effects of PIT tagging on growth and other performance metrics of springfish and other small desert fishes.

  18. TagSmart: analysis and visualization for yeast mutant fitness data measured by tag microarrays

    PubMed Central

    Kim, Chulyun; Kim, Sangkyum; Dorer, Russell; Xie, Dan; Han, Jiawei; Zhong, Sheng

    2007-01-01

    Background A nearly complete collection of gene-deletion mutants (96% of annotated open reading frames) of the yeast Saccharomyces cerevisiae has been systematically constructed. Tag microarrays are widely used to measure the fitness of each mutant in a mutant mixture. The tag array experiments can have a complex experimental design, such as time course measurements and drug treatment with multiple dosages. Results TagSmart is a web application for analysis and visualization of Saccharomyces cerevisiae mutant fitness data measured by tag microarrays. It implements a robust statistical approach to assess the concentration differences among S. cerevisiae mutant strains. It also provides an interactive environment for data analysis and visualization. TagSmart has the following advantages over previously described analysis procedures: 1) it is user-friendly software rather than merely a description of analytical procedure; 2) It can handle complicated experimental designs, such as multiple time points and treatment with multiple dosages; 3) it has higher sensitivity and specificity; 4) It allows users to mask out "bad" tags in the analysis. Two biological tests were performed to illustrate the performance of TagSmart. First, we generated titration mixtures of mutant strains, in which the relative concentration of each strain was controlled. We used tag microarrays to measure the numbers of tag copies in each titration mixture. The data was analyzed with TagSmart and the result showed high precision and recall. Second, TagSmart was applied to a dataset in which heterozygous deletion strain mixture pools were treated with a new drug, Cincreasin. TagSmart identified 53 mutant strains as sensitive to Cincreasin treatment. We individually tested each identified mutant, and found 52 out of the 53 predicted mutants were indeed sensitive to Cincreasin. Conclusion TagSmart is provided "as is" to analyze tag array data produced by Affymetrix and Agilent arrays. TagSmart web application is assessable by Windows, Mac, and Linux users. It also has a downloadable version for execution on PCs running Windows. TagSmart is available for academic use at: PMID:17442117

  19. Survival and tag retention of Pacific lamprey larvae and macrophthalmia marked with coded wire tags

    USGS Publications Warehouse

    Meeuwig, M.H.; Puls, A.L.; Bayer, J.M.

    2007-01-01

    We examined the survival, tag retention, and growth of Pacific lamprey Lampetra tridentata larvae and macrophthalmia marked with standard-length decimal coded wire tags and exposed to two levels of handling stress. The survival of marked individuals did not differ from that of unmarked individuals at either life stage for the duration of the experiment (56 d). Tag retention was 100% for all treatment combinations except larvae that were handled frequently (93 ?? 3%). The majority of tag loss occurred within 28 d of marking, and no tag loss was observed between 42 and 56 d after marking. The individuals that lost tags were among the smallest marked, and a logistic regression model indicated a relationship between larva length and the probability of tag retention. Size of larvae (length and mass) and macrophthalmia (mass) decreased over the duration of the experiment; however, changes in size were systematic among treatment combinations, indicating that factors other than tagging or handling affected growth. These data indicate that coded wire tags may be useful for field-based studies of Pacific lamprey larvae and macrophthalmia.

  20. Onboard tagging for smart medical devices.

    PubMed

    Li, Kejia; Warren, Steve

    2011-01-01

    Most medical devices are 'dumb:' their role is to acquire, display, and forward data. They make few if any operational decisions based on those data. Onboard tagging is a means whereby a device can embed information about itself, its data, and the sensibility of those data into its data stream. This diagnostic add-on offers a move toward 'smart' devices that will have the ability to affect changes in operational modes based on onboard contextual decision making, such as decisions to avoid needless wireless transmission of corrupt data. This paper presents a description of three types of onboard tags that relate to device hardware (type I tag), signal statistics (type II tag), and signal viability for the intended application (type III tag). A custom wireless pulse oximeter is presented as a use case to show how type II and III tags that convey photoplethysmogram (PPG) statistics and usability specifiers can be calculated and embedded into the data stream without degrading performance. PMID:22254768

  1. A cleavable biotin tagging reagent that enables the enrichment and identification of carbonylation sites in proteins.

    PubMed

    Coffey, Chelsea M; Gronert, Scott

    2016-01-01

    The utility of a new, cleavable tag for identifying and enriching protein carbonyls is examined. Using a model system, human serum albumin modified with acrolein, the EZ-Link alkoxyamine-PEG4-SS-PEG4-biotin affinity tag, was tested for its ability to label protein carbonyls in proteomic analyses of protein carbonylation. The efficiency of the labeling was assayed and compared to standard biotin hydrazide reagents. The label was also tested in liquid chromatography-tandem mass spectrometry (LC/MS/MS) experiments. The quality of the fragmentation spectra was assessed and the relative detection efficiency of various modification sites was compared to standard biotin hydrazide reagents. Finally, the viability of using the label with streptavidin bead enrichment protocols in a standard proteomics workflow was probed. PMID:26613796

  2. Rapid tagging of endogenous mouse genes by recombineering and ES cell complementation of tetraploid blastocysts

    PubMed Central

    Zhou, Dewang; Ren, Jin-Xiang; Ryan, Thomas M.; Higgins, N. Patrick; Townes, Tim M.

    2004-01-01

    The construction of knockin vectors designed to modify endogenous genes in embryonic stem (ES) cells and the generation of mice from these modified cells is time consuming. The timeline of an experiment from the conception of an idea to the availability of mature mice is at least 9 months. We describe a method in which this timeline is typically reduced to 3 months. Knockin vectors are rapidly constructed from bacterial artificial chromosome clones by recombineering followed by gap-repair (GR) rescue, and mice are rapidly derived by injecting genetically modified ES cells into tetraploid blastocysts. We also describe a tandem affinity purification (TAP)/floxed marker gene plasmid and a GR rescue plasmid that can be used to TAP tag any murine gene. The combination of recombineering and tetraploid blastocyst complementation provides a means for large-scale TAP tagging of mammalian genes. PMID:15356288

  3. Selective binding and magnetic separation of histidine-tagged proteins using Fe3O4/Cu-apatite nanoparticles.

    PubMed

    Li, Ping; Li, Liangliang; Zhao, Yanbao; Sun, Lei; Zhang, Yu

    2016-03-01

    Hierarchical Fe3O4@Cu-apatite nanoparticles (NPs) were synthesized via a facile hydrothermal method. The Fe3O4 cores present spherical shape and have a mean diameter of 300nm, and the Cu-apatite shell with thickness of about 50nm is composed of a large number of sheets. Using the high affinity of Cu(2+) on the surface toward histidine tags, the Fe3O4@Cu-apatite NPs can be applied to enrich and magnetically separate histidine tagged (His-tagged) proteins directly from the mixture of lysed cells. Research results indicated that the Fe3O4@Cu-apatite NPs presented negligible nonspecific protein adsorption and high protein binding ability. PMID:26773852

  4. Site-specific dual labeling of proteins by using small orthogonal tags at neutral pH.

    PubMed

    Grnewald, Jan; Jones, David H; Brock, Ansgar; Chiu, Hsien-Po; Bursulaya, Badry; Ng, Kenneth; Vo, Todd; Patterson, Paula; Uno, Tetsuo; Hunt, James; Spraggon, Glen; Geierstanger, Bernhard H

    2014-08-18

    To expand the utility of proteinaceous FRET biosensors, we have developed a dual-labeling approach based on two small bio-orthogonal tags: pyrroline-carboxy-lysine (Pcl) and the S6 peptide. The lack of cross-reactivity between those tags enables site-specific two-color protein conjugation in a one-pot reaction. Moreover, Pcl/S6 dual-tagged proteins can be produced in both bacterial and mammalian expression systems, as demonstrated for Z domain and IgE-Fc, respectively. Both proteins could be efficiently dual-labeled with FRET-compatible fluorescent dyes at neutral pH. In the case of IgE-Fc, the resulting conjugate enabled the monitoring of IgE binding to its high-affinity receptor Fc?RI, which is a key event in allergic disease. PMID:25044133

  5. A fluorogenic probe for SNAP-tagged plasma membrane proteins based on the solvatochromic molecule Nile Red.

    PubMed

    Prifti, Efthymia; Reymond, Luc; Umebayashi, Miwa; Hovius, Ruud; Riezman, Howard; Johnsson, Kai

    2014-03-21

    A fluorogenic probe for plasma membrane proteins based on the dye Nile Red and SNAP-tag is introduced. It takes advantage of Nile Red, a solvatochromic molecule highly fluorescent in an apolar environment, such as cellular membranes, but almost dark in a polar aqueous environment. The probe possesses a tuned affinity for membranes allowing its Nile Red moiety to insert into the lipid bilayer of the plasma membrane, becoming fluorescent, only after its conjugation to a SNAP-tagged plasma membrane protein. The fluorogenic character of the probe was demonstrated for different SNAP-tag fusion proteins, including the human insulin receptor. This work introduces a new approach for generating a powerful turn-on probe for "no-wash" labeling of plasma membrane proteins with numerous applications in bioimaging. PMID:24471525

  6. Co-evolution of affinity and stability of grafted amyloid-motif domain antibodies.

    PubMed

    Julian, Mark C; Lee, Christine C; Tiller, Kathryn E; Rabia, Lilia A; Day, Evan K; Schick, Arthur J; Tessier, Peter M

    2015-10-01

    An attractive approach for designing lead antibody candidates is to mimic natural protein interactions by grafting peptide recognition motifs into the complementarity-determining regions (CDRs). We are using this approach to generate single-domain (VH) antibodies specific for amyloid-forming proteins such as the Alzheimer's A? peptide. Here, we use random mutagenesis and yeast surface display to improve the binding affinity of a lead VH domain grafted with A? residues 33-42 in CDR3. Interestingly, co-selection for improved A? binding and VH display on the surface of yeast yields antibody domains with improved affinity and reduced stability. The highest affinity VH domains were strongly destabilized on the surface of yeast as well as unfolded when isolated as autonomous domains. In contrast, stable VH domains with improved affinity were reliably identified using yeast surface display by replacing the display antibody that recognizes a linear epitope tag at the terminus of both folded and unfolded VH domains with a conformational ligand (Protein A) that recognizes a discontinuous epitope on the framework of folded VH domains. Importantly, we find that selection for improved stability using Protein A without simultaneous co-selection for improved A? binding leads to strong enrichment for stabilizing mutations that reduce antigen binding. Our findings highlight the importance of simultaneously optimizing affinity and stability to improve the rapid isolation of well-folded and specific antibody fragments. PMID:26386257

  7. HaloTag is an effective expression and solubilisation fusion partner for a range of fibroblast growth factors

    PubMed Central

    Taylor, Sarah E.; Mao, Xianqing; Wilkinson, Mark C.

    2015-01-01

    The production of recombinant proteins such as the fibroblast growth factors (FGFs) is the key to establishing their function in cell communication. The production of recombinant FGFs in E. coli is limited, however, due to expression and solubility problems. HaloTag has been used as a fusion protein to introduce a genetically-encoded means for chemical conjugation of probes. We have expressed 11 FGF proteins with an N-terminal HaloTag, followed by a tobacco etch virus (TEV) protease cleavage site to allow release of the FGF protein. These were purified by heparin-affinity chromatography, and in some instances by further ion-exchange chromatography. It was found that HaloTag did not adversely affect the expression of FGF1 and FGF10, both of which expressed well as soluble proteins. The N-terminal HaloTag fusion was found to enhance the expression and yield of FGF2, FGF3 and FGF7. Moreover, whereas FGF6, FGF8, FGF16, FGF17, FGF20 and FGF22 were only expressed as insoluble proteins, their N-terminal HaloTag fusion counterparts (Halo-FGFs) were soluble, and could be successfully purified. However, cleavage of Halo-FGF6, -FGF8 and -FGF22 with TEV resulted in aggregation of the FGF protein. Measurement of phosphorylation of p42/44 mitogen-activated protein kinase and of cell growth demonstrated that the HaloTag fusion proteins were biologically active. Thus, HaloTag provides a means to enhance the expression of soluble recombinant proteins, in addition to providing a chemical genetics route for covalent tagging of proteins. PMID:26137434

  8. Engineering adeno-associated virus for one-step purification via immobilized metal affinity chromatography.

    PubMed

    Koerber, James T; Jang, Jae-Hyung; Yu, Julie H; Kane, Ravi S; Schaffer, David V

    2007-04-01

    Adeno-associated virus (AAV) is a promising vehicle for gene therapy, which will rely on the generation of high-titer, high-purity recombinant vectors. However, numerous purification protocols can involve challenging optimization or scalability issues, and most AAV serotypes do not bind heparin or sialic acid, used for AAV2/3 or AAV4/5 purification, requiring the development of new chromatography strategies. Immobilized metal affinity chromatography (IMAC) allows for robust protein purification via affinity tags such as the hexahistidine (His(6)) sequence. Through the combination of a diverse AAV2 library and rational peptide insertions, we have located an optimal His(6) tag insertion site within the viral capsid. This mutant and a related AAV8 variant can be purified from clarified cell lysate in a single gravity column step at infectious particle yields exceeding 90%. Furthermore, injection of IMAC-purified vector into the brain demonstrates that it mediates high-efficiency gene delivery in vivo, equivalent to that of wild-type capsid, with minimal immune cell activation. This affinity chromatography method may offer advantages in ease of purification, final vector purity, and process scalability. Moreover, a combined rational design and high-throughput library selection approach can aid in the design of enhanced viral gene delivery vectors. PMID:17437357

  9. Antisymmetric tensor generalizations of affine vector fields

    NASA Astrophysics Data System (ADS)

    Houri, Tsuyoshi; Morisawa, Yoshiyuki; Tomoda, Kentaro

    2016-02-01

    Tensor generalizations of affine vector fields called symmetric and antisymmetric affine tensor fields are discussed as symmetry of spacetimes. We review the properties of the symmetric ones, which have been studied in earlier works, and investigate the properties of the antisymmetric ones, which are the main theme in this paper. It is shown that antisymmetric affine tensor fields are closely related to one-lower-rank antisymmetric tensor fields which are parallelly transported along geodesics. It is also shown that the number of linear independent rank-p antisymmetric affine tensor fields in n-dimensions is bounded by (n + 1)!/p!(n - p)!. We also derive the integrability conditions for antisymmetric affine tensor fields. Using the integrability conditions, we discuss the existence of antisymmetric affine tensor fields on various spacetimes.

  10. Conformal field theory on affine Lie groups

    SciTech Connect

    Clubok, K.S.

    1996-04-01

    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.

  11. Purification of lysozyme by multistage affinity filtration.

    PubMed

    He, L Z; Sun, Y

    2002-09-01

    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 from a model system (a mixture of lysozyme and BSA) and a natural source (chicken egg white). From the chicken egg white, the three-stage affinity filtration increased the recovery yield of lysozyme from 61 to 96%, compared with the one-stage process. A mathematical model taking into account the film and interior diffusions of protein and eluant was developed for the modeling and analysis of the experimental data. Both the experimental and modeling results indicate that the multistage affinity filtration technique can be employed for the selective recovery of proteins. PMID:14508673

  12. Neural net controlled tag gas sampling system for nuclear reactors

    DOEpatents

    Gross, K.C.; Laug, M.T.; Lambert, J.B.; Herzog, J.P.

    1997-02-11

    A method and system are disclosed 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. 12 figs.

  13. Neural net controlled tag gas sampling system for nuclear reactors

    DOEpatents

    Gross, Kenneth C.; Laug, Matthew T.; Lambert, John D. B.; Herzog, James P.

    1997-01-01

    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.

  14. Associated Particle Tagging (APT) in Magnetic Spectrometers

    SciTech Connect

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

    2012-10-16

    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.

  15. Structural determinants of sigma receptor affinity

    SciTech Connect

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

    1987-12-01

    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.

  16. Maxwell-affine gauge theory of gravity

    NASA Astrophysics Data System (ADS)

    Cebecio?lu, O.; Kibaro?lu, S.

    2015-12-01

    The Maxwell extension of the affine algebra in four dimensions with additional tensor generator is given. Using the methods of nonlinear realizations, we find the transformation rules for the group parameters and the corresponding generators. Gauging the Maxwell-affine algebra we present two possible invariant actions for gravity: one is first order and the other one is second order in the affine curvature. We notice that equations of motion for the action, second order in the affine curvature, lead to the generalized Bianchi identities on the choice of appropriate coefficients for a particular solution of the constraint equation.

  17. A Novel Vertex Affinity for Community Detection

    SciTech Connect

    Yoo, Andy; Sanders, Geoffrey; Henson, Van; Vassilevski, Panayot

    2015-10-05

    We propose a novel vertex affinity measure in this paper. The new vertex affinity quantifies the proximity between two vertices in terms of their clustering strength and is ideal for such graph analytics applications as community detection. We also developed a framework that combines simple graph searches and resistance circuit formulas to compute the vertex affinity efficiently. We study the properties of the new affinity measure empirically in comparison to those of other popular vertex proximity metrics. Our results show that the existing metrics are ill-suited for community detection due to their lack of fundamental properties that are essential for correctly capturing inter- and intra-cluster vertex proximity.

  18. A brief examination of optical tagging technologies.

    SciTech Connect

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

    2003-07-01

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2011-10-01

    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.

  20. Tags to Track Illicit Uranium and Plutonium

    SciTech Connect

    Haire, M. Jonathan; Forsberg, Charles W.

    2007-07-01

    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)

  1. Structure of classical affine and classical affine fractional W-algebras

    SciTech Connect

    Suh, Uhi Rinn

    2015-01-15

    We introduce a classical BRST complex (See Definition 3.2.) and show that one can construct a classical affine W-algebra via the complex. This definition clarifies that classical affine W-algebras can be considered as quasi-classical limits of quantum affine W-algebras. We also give a definition of a classical affine fractional W-algebra as a Poisson vertex algebra. As in the classical affine case, a classical affine fractional W-algebra has two compatible λ-brackets and is isomorphic to an algebra of differential polynomials as a differential algebra. When a classical affine fractional W-algebra is associated to a minimal nilpotent, we describe explicit forms of free generators and compute λ-brackets between them. Provided some assumptions on a classical affine fractional W-algebra, we find an infinite sequence of integrable systems related to the algebra, using the generalized Drinfel’d and Sokolov reduction.

  2. Loop realizations of quantum affine algebras

    SciTech Connect

    Cautis, Sabin; Licata, Anthony

    2012-12-15

    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.

  3. Ultrafast tissue staining with chemical tags

    PubMed Central

    Kohl, Johannes; Ng, Julian; Cachero, Sebastian; Ciabatti, Ernesto; Dolan, Michael-John; Sutcliffe, Ben; Tozer, Adam; Ruehle, Sabine; Krueger, Daniel; Frechter, Shahar; Branco, Tiago; Tripodi, Marco; Jefferis, Gregory S. X. E.

    2014-01-01

    Genetically encoded fluorescent proteins and immunostaining are widely used to detect cellular and subcellular structures in fixed biological samples. However, for thick or whole-mount tissue, each approach suffers from limitations, including limited spectral flexibility and lower signal or slow speed, poor penetration, and high background labeling, respectively. We have overcome these limitations by using transgenically expressed chemical tags for rapid, even, high-signal and low-background labeling of thick biological tissues. We first construct a platform of widely applicable transgenic Drosophila reporter lines, demonstrating that chemical labeling can accelerate staining of whole-mount fly brains by a factor of 100. Using viral vectors to deliver chemical tags into the mouse brain, we then demonstrate that this labeling strategy works well in mice. Thus this tag-based approach drastically improves the speed and specificity of labeling genetically marked cells in intact and/or thick biological samples. PMID:25157152

  4. Molecular tagging velocimetry in turbulence using biacetyl.

    PubMed

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

    2012-10-01

    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

  5. Smart tagged composite using piezoelectric particles

    NASA Astrophysics Data System (ADS)

    White, Scott R.

    1995-05-01

    A new class of materials called smart tagged composites has great potential for use in structural health monitoring. This paper introduces concepts and interrogation schemes for piezoelectric tagged composite materials. Experiments were undertaken to demonstrate the feasibility of piezoelectric tagging by incorporating PZT-5A particles into a polyester matrix. Several types of diametral compression specimens were fabricated and tested to failure while monitoring the induced charge across electrodes placed on the front and back faces of the specimens. The effect of volume fraction of piezo particles and glass reinforcement was investigated along with connectivity of the piezo phase. Small amounts of graphite were added to some specimens to aid in the poling process, however this proved to be of little benefit. The greatest sensitivity and highest induced fields were obtained for a pseudo 1 - 3 piezocomposite system with 6% volume fraction loading of the piezo phase.

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

    NASA Astrophysics Data System (ADS)

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

    2011-02-01

    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, α- and β-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.

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

    NASA Astrophysics Data System (ADS)

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

    2003-06-01

    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.

  8. Does a tag system effectively support emerging cooperation?

    PubMed

    Tanimoto, Jun

    2007-08-21

    This paper investigates whether the so-called Tag Systems support emerging cooperation with respect to 2x2 games. The Tag System, initially proposed by Riolo et al. [2001. Evolution of cooperation without reciprocity. Nature 414, 441-443], gives each agent both a Tag and Tol defined by [0,1] real numbers. Tol is a tolerance for recognizing an opponent as a company. Both Tag and Tol are assumed to be evolving. Results show that the tag's effectiveness depends on whether the AllD strategy is allowed in the system. Allowing AllD implies that green beard effect does not work in the system. Thus, (1) the tag's effectiveness is more meager than that reported by Riolo et al., (2) the Tag System can use alternating reciprocity more effectively than the analytic solution in a Hero game; (3) a system using a 2D tag space supports cooperation more effectively than the usual Tag System. PMID:17507033

  9. Improving image segmentation by learning region affinities

    SciTech Connect

    Prasad, Lakshman; Yang, Xingwei; Latecki, Longin J

    2010-11-03

    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.

  10. Tags to Track Illicit Uranium and Plutonium

    SciTech Connect

    Haire, Marvin Jonathan

    2007-01-01

    With the expansion of nuclear power, it is essential to avoid diversion of nuclear materials into the hands of 'rogue nations,' terrorists, and other opportunists. This paper describes (1) the use of a detection tag to make it easier to detect smuggled material by creating a nuclear fingerprint and (2) the use of attribution tags to enable law enforcement to determine where any recovered stolen nuclear materials came from, identify the individuals responsible for the unlawful diversion, and reduce future loss of nuclear materials.

  11. B mixing and flavor tagging at CDF

    SciTech Connect

    Russ, James S.; /Carnegie Mellon U.

    2004-12-01

    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.

  12. Photon-tagged and B-meson-tagged b-jet production at the LHC

    NASA Astrophysics Data System (ADS)

    Huang, Jinrui; Kang, Zhong-Bo; Vitev, Ivan; Xing, Hongxi

    2015-11-01

    Tagged jet measurements in high energy hadronic and nuclear reactions provide constraints on the energy and parton flavor origin of the parton shower that recoils against the tagging particle. Such additional insight can be especially beneficial in illuminating the mechanisms of heavy flavor production in proton-proton collisions at the LHC and their modification in the heavy ion environment, which are not fully understood. With this motivation, we present theoretical results for isolated-photon-tagged and B-meson-tagged b-jet production at ?{sNN} = 5.1TeV for comparison to the upcoming lead-lead data. We find that photon-tagged b-jets exhibit smaller momentum imbalance shift in nuclear matter, and correspondingly smaller energy loss, than photon-tagged light flavor jets. Our results show that B-meson tagging is most effective in ensuring that the dominant fraction of recoiling jets originate from prompt b-quarks. Interestingly, in this channel the large suppression of the cross section is not accompanied by a significant momentum imbalance shift.

  13. Double-Lanthanide-Binding Tags for Macromolecular Crystallographic Structure Determination

    SciTech Connect

    Silvaggi,N.; Martin, L.; Schwalbe, H.; Imperiali, B.; Allen, K.

    2007-01-01

    A double-lanthanide-binding tag (dLBT), a small peptide sequence engineered to bind two lanthanide ions (e.g., Tb{sup 3+}) with high affinity, was used to solve the phase problem for the structure determination of ubiquitin by the single-wavelength anomalous diffraction (SAD) method. Since the dLBT is comprised exclusively of encoded amino acids, the necessity for the incorporation of unnatural amino acids or chemical modification of the protein as a prerequisite for X-ray structure determination is eliminated. A construct encoding the dLBT as an N-terminal fusion with ubiquitin provides for facile expression and purification using standard methods. Phasing of the single-wavelength X-ray data (at 2.6 {angstrom} resolution) using only the anomalous signal from the two tightly bound Tb{sup 3+} ions in the dLBT led to clear electron-density maps. Nearly 75% of the ubiquitin structure was built using automated model-building software without user intervention. It is anticipated that this technique will be broadly applicable, complementing existing macromolecular phasing methodologies. The dLBT should be particularly useful in cases where protein derivatization with heavy atoms proves to be problematic or synchrotron facilities are unavailable.

  14. Stability of the Neurotensin Receptor NTS1 Free in Detergent Solution and Immobilized to Affinity Resin

    PubMed Central

    White, Jim F.; Grisshammer, Reinhard

    2010-01-01

    Background Purification of recombinant membrane receptors is commonly achieved by use of an affinity tag followed by an additional chromatography step if required. This second step may exploit specific receptor properties such as ligand binding. However, the effects of multiple purification steps on protein yield and integrity are often poorly documented. We have previously reported a robust two-step purification procedure for the recombinant rat neurotensin receptor NTS1 to give milligram quantities of functional receptor protein. First, histidine-tagged receptors are enriched by immobilized metal affinity chromatography using Ni-NTA resin. Second, remaining contaminants in the Ni-NTA column eluate are removed by use of a subsequent neurotensin column yielding pure NTS1. Whilst the neurotensin column eluate contained functional receptor protein, we observed in the neurotensin column flow-through misfolded NTS1. Methods and Findings To investigate the origin of the misfolded receptors, we estimated the amount of functional and misfolded NTS1 at each purification step by radio-ligand binding, densitometry of Coomassie stained SDS-gels, and protein content determination. First, we observed that correctly folded NTS1 suffers damage by exposure to detergent and various buffer compositions as seen by the loss of [3H]neurotensin binding over time. Second, exposure to the neurotensin affinity resin generated additional misfolded receptor protein. Conclusion Our data point towards two ways by which misfolded NTS1 may be generated: Damage by exposure to buffer components and by close contact of the receptor to the neurotensin affinity resin. Because NTS1 in detergent solution is stabilized by neurotensin, we speculate that the occurrence of aggregated receptor after contact with the neurotensin resin is the consequence of perturbations in the detergent belt surrounding the NTS1 transmembrane core. Both effects reduce the yield of functional receptor protein. PMID:20830205

  15. Evaluation of Intercontinental Transport of Ozone Using Full-tagged, Tagged-N and Sensitivity Methods

    NASA Astrophysics Data System (ADS)

    Guo, Y.; Liu, J.; Mauzerall, D. L.; Emmons, L. K.; Horowitz, L. W.; Fan, S.; Li, X.; Tao, S.

    2014-12-01

    Long-range transport of ozone is of great concern, yet the source-receptor relationships derived previously depend strongly on the source attribution techniques used. Here we describe a new tagged ozone mechanism (full-tagged), the design of which seeks to take into account the combined effects of emissions of ozone precursors, CO, NOx and VOCs, from a particular source, while keeping the current state of chemical equilibrium unchanged. We label emissions from the target source (A) and background (B). When two species from A and B sources react with each other, half of the resulting products are labeled A, and half B. Thus the impact of a given source on downwind regions is recorded through tagged chemistry. We then incorporate this mechanism into the Model for Ozone and Related chemical Tracers (MOZART-4) to examine the impact of anthropogenic emissions within North America, Europe, East Asia and South Asia on ground-level ozone downwind of source regions during 1999-2000. We compare our results with two previously used methods -- the sensitivity and tagged-N approaches. The ozone attributed to a given source by the full-tagged method is more widely distributed spatially, but has weaker seasonal variability than that estimated by the other methods. On a seasonal basis, for most source/receptor pairs, the full-tagged method estimates the largest amount of tagged ozone, followed by the sensitivity and tagged-N methods. In terms of trans-Pacific influence of ozone pollution, the full-tagged method estimates the strongest impact of East Asian (EA) emissions on the western U.S. (WUS) in MAM and JJA (~3 ppbv), which is substantially different in magnitude and seasonality from tagged-N and sensitivity studies. This difference results from the full-tagged method accounting for the maintenance of peroxy radicals (e.g., CH3O2, CH3CO3, and HO2), in addition to NOy, as effective reservoirs of EA source impact across the Pacific, allowing for a significant contribution to ozone formation over WUS (particularly in summer). Thus, the full-tagged method, with its clear discrimination of source and background contributions on a per-reaction basis, provides unique insights into the critical role of VOCs (and additional reactive nitrogen species) in determining the nonlinear inter-continental influence of ozone pollution.

  16. Applicability of tandem affinity purification MudPIT to pathway proteomics in yeast.

    PubMed

    Graumann, Johannes; Dunipace, Leslie A; Seol, Jae Hong; McDonald, W Hayes; Yates, John R; Wold, Barbara J; Deshaies, Raymond J

    2004-03-01

    A combined multidimensional chromatography-mass spectrometry approach known as "MudPIT" enables rapid identification of proteins that interact with a tagged bait while bypassing some of the problems associated with analysis of polypeptides excised from SDS-polyacrylamide gels. However, the reproducibility, success rate, and applicability of MudPIT to the rapid characterization of dozens of proteins have not been reported. We show here that MudPIT reproducibly identified bona fide partners for budding yeast Gcn5p. Additionally, we successfully applied MudPIT to rapidly screen through a collection of tagged polypeptides to identify new protein interactions. Twenty-five proteins involved in transcription and progression through mitosis were modified with a new tandem affinity purification (TAP) tag. TAP-MudPIT analysis of 22 yeast strains that expressed these tagged proteins uncovered known or likely interacting partners for 21 of the baits, a figure that compares favorably with traditional approaches. The proteins identified here comprised 102 previously known and 279 potential physical interactions. Even for the intensively studied Swi2p/Snf2p, the catalytic subunit of the Swi/Snf chromatin remodeling complex, our analysis uncovered a new interacting protein, Rtt102p. Reciprocal tagging and TAP-MudPIT analysis of Rtt102p revealed subunits of both the Swi/Snf and RSC complexes, identifying Rtt102p as a common interactor with, and possible integral component of, these chromatin remodeling machines. Our experience indicates it is feasible for an investigator working with a single ion trap instrument in a conventional molecular/cellular biology laboratory to carry out proteomic characterization of a pathway, organelle, or process (i.e. "pathway proteomics") by systematic application of TAP-MudPIT. PMID:14660704

  17. Cobalt carbonyl complexes as probes for alkyne-tagged lipids[S

    PubMed Central

    Tallman, Keri A.; Armstrong, Michelle D.; Milne, Stephen B.; Marnett, Lawrence J.; Brown, H. Alex; Porter, Ned A.

    2013-01-01

    Monitoring lipid distribution and metabolism in cells and biological fluids poses many challenges because of the many molecular species and metabolic pathways that exist. This study describes the synthesis and study of molecules that contain an alkyne functional group as surrogates for natural lipids in cultured cells. Thus, hexadec-15-ynoic and hexadec-7-ynoic acids were readily incorporated into RAW 264.7 cells, principally as phosphocholine esters; the alkyne was used as a “tag” that could be transformed to a stable dicobalt-hexacarbonyl complex; and the complex could then be detected by HPLC/MS or HPLC/UV349nm. The 349 nm absorbance of the cobalt complexes was used to provide qualitative and quantitative information about the distribution and cellular concentrations of the alkyne lipids. The alkyne group could also be used as an affinity tag for the lipids by a catch-and-release strategy on phosphine-coated silica beads. Lipid extracts were enriched in the tagged lipids in this way, making the approach of potential utility to study lipid transformations in cell culture. Both terminal alkynes and internal alkynes were used in this affinity “pull-down” strategy. This method facilitates measuring lipid species that might otherwise fall below limits of detection. PMID:23307946

  18. Beliefs and Uses of Tagging among Undergraduates

    ERIC Educational Resources Information Center

    Kramer-Duffield, Jacob

    2010-01-01

    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. …

  19. Novel and efficient tag SNPs selection algorithms.

    PubMed

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

    2014-01-01

    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:24212035

  20. Beliefs and Uses of Tagging among Undergraduates

    ERIC Educational Resources Information Center

    Kramer-Duffield, Jacob

    2010-01-01

    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.

  1. Imaging mass spectrometer with mass tags

    DOEpatents

    Felton, James S.; Wu, Kuang Jen J.; Knize, Mark G.; Kulp, Kristen S.; Gray, Joe W.

    2013-01-29

    A method of analyzing biological material by exposing the biological material to a recognition element, that is coupled to a mass tag element, directing an ion beam of a mass spectrometer to the biological material, interrogating at least one region of interest area from the biological material and producing data, and distributing the data in plots.

  2. Imaging mass spectrometer with mass tags

    DOEpatents

    Felton, James S.; Wu, Kuang Jen; Knize, Mark G.; Kulp, Kristen S.; Gray, Joe W.

    2010-06-01

    A method of analyzing biological material by exposing the biological material to a recognition element, that is coupled to a mass tag element, directing an ion beam of a mass spectrometer to the biological material, interrogating at least one region of interest area from the biological material and producing data, and distributing the data in plots.

  3. Semantic Enhancement of Social Tagging Systems

    NASA Astrophysics Data System (ADS)

    Abel, Fabian; Henze, Nicola; Krause, Daniel; Kriesell, Matthias

    Social tagging systems have shown an impressive potential for information discovery and exploration. Enriched with Semantic Web technologies, they enable to tap valuable metadata about Web resources and to detect hidden relations, thus, to capture information about both content and context of the resources. In this article, we propose a novel way to combine semantic technologies with Web 2.0 paradigms. We introduce the GroupMe! system, which extends current social tagging systems by giving users more flexibility in organizing and maintaining Web content. In GroupMe!, users can create groups of Web resources they consider relevant by simple drag & drop operations. They can tag and share their groups and Web content with fellow users and benefit from improved search and retrieval capabilities. We evaluate the GroupMe! approach and investigate on the effect of grouping resources for search in tag-based social systems. Our experiments show that the quality of search result ranking can be significantly improved by introducing and exploiting the grouping of resources.

  4. Automated Data Tagging in the HLA

    NASA Astrophysics Data System (ADS)

    Gaffney, N. I.; Miller, W. W.

    2008-08-01

    One of the more powerful and popular forms of data organization implemented in most popular information sharing web applications is data tagging. With a rich user base from which to gather and digest tags, many interesting and often unanticipated yet very useful associations are revealed. With regard to an existing information, the astronomical community has a rich pool of existing digitally stored and searchable data than any of the currently popular web community, such as You Tube or My Space, had when they started. In initial experiments with the search engine for the Hubble Legacy Archive, we have created a simple yet powerful scheme by which the information from a footprint service, the NED and SIMBAD catalog services, and the ADS abstracts and keywords can be used to initially tag data with standard keywords. By then ingesting this into a public ally available information search engine, such as Apache Lucene, one can create a simple and powerful data tag search engine and association system. By then augmenting this with user provided keys and usage pattern analysis, one can produce a powerful modern data mining system for any astronomical data warehouse.

  5. Krypton tagging velocimetry of an underexpanded jet.

    PubMed

    Parziale, N J; Smith, M S; Marineau, E C

    2015-06-01

    In this work, we present the excitation/emission strategy, experimental setup, and results of an implementation of krypton tagging velocimetry (KTV). KTV is performed as follows: (i) seed a base flow with krypton; (ii) photosynthesize metastable krypton atoms with a frequency-doubled dye laser to form the tagged tracer; (iii) record the translation of the tagged metastable krypton by imaging the laser-induced fluorescence (LIF) that is produced with an additional dye laser. The principle strength of KTV, relative to other tagging velocimetry techniques, is the use of a chemically inert tracer. KTV results are presented for an underexpanded jet of three mixtures of varying Kr/N2 concentration. It is demonstrated that KTV can be used in gas mixtures of relatively low krypton mole fraction (0.5% Kr/99.5% N2), and the KTV data from that experiment are found to be in good agreement with an empirical fit found in the literature. We find that KTV is useful to perform instantaneous velocity measurements with metastable krypton as a chemically inert, dilute, long-lifetime tracer in gas-phase flows. PMID:26192670

  6. GHRSST-14 DAS-TAG Report

    NASA Technical Reports Server (NTRS)

    Armstrong, Edward; Piolle, Jean Francois

    2013-01-01

    The DAS-TAG provides the informatics and data management expertise in emerging information technologies for the GHRSST community. It provides expertise in data and metadata formats and standards, fosters improvements for GHRSST data curation, experiments with new data processing paradigms, and evaluates services and tools for data usage. It provides a forum for producer and distributor data management issues and coordination.

  7. Aptamers in Affinity Separations: Stationary Separation

    NASA Astrophysics Data System (ADS)

    Ravelet, Corinne; Peyrin, Eric

    The use of DNA or RNA aptamers as tools in analytical chemistry is a very promising field of research because of their capabilities to bind specifically the target molecules with an affinity similar to that of antibodies. Notably, they appear to be of great interest as target-specific ligands for the separation and capture of various analytes in affinity chromatography and related affinity-based methods such as magnetic bead technology. In this chapter, the recent developments of these aptamer-based separation/capture approaches are addressed.

  8. Visualizing antibody affinity maturation in germinal centers.

    PubMed

    Tas, Jeroen M J; Mesin, Luka; Pasqual, Giulia; Targ, Sasha; Jacobsen, Johanne T; Mano, Yasuko M; Chen, Casie S; Weill, Jean-Claude; Reynaud, Claude-Agnès; Browne, Edward P; Meyer-Hermann, Michael; Victora, Gabriel D

    2016-03-01

    Antibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with nonimmunodominant specificities must be elicited, as is the case for HIV-1 and influenza. PMID:26912368

  9. Protein purification using PDZ affinity chromatography.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2015-01-01

    PDZ domains function in nature as protein-binding domains within scaffold and membrane-associated proteins. They comprise approximately 90 residues and undergo specific, high-affinity interactions with complementary C-terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands. 2015 by John Wiley & Sons, Inc. PMID:25829303

  10. Vertex representations of quantum affine algebras.

    PubMed

    Frenkel, I B; Jing, N

    1988-12-01

    We construct vertex representations of quantum affine algebras of ADE type, which were first introduced in greater generality by Drinfeld and Jimbo. The limiting special case of our construction is the untwisted vertex representation of affine Lie algebras of Frenkel-Kac and Segal. Our representation is given by means of a new type of vertex operator corresponding to the simple roots and satisfying the defining relations. In the case of the quantum affine algebra of type A, we introduce vertex operators corresponding to all the roots and determine their commutation relations. This provides an analogue of a Chevalley basis of the affine Lie algebra [unk](n) in the basic representation. PMID:16594004

  11. Hylleraas hydride binding energy: diatomic electron affinities.

    PubMed

    Chen, Edward S; Keith, Herman; Lim, Tristan; Pham, Dang; Rosenthal, Reece; Herder, Charles; Pai, Sunil; Flores, R A; Chen, Edward C M

    2015-04-01

    Theoretical adiabatic electron affinities are often considered inaccurate because they are referenced to only a single value. Ground state electron affinities for all the main group elements and homonuclear diatomics were identified recently using the normalized binding energy of the hydrogen atom: [0.75420375(3)/2?=?0.37710187(1) eV]. Here we revisit experimental values and extend the identifications to diatomics in the G2-1 set. We assign new ground state electron affinities: (eV) Cl2, 3.2(2); Br2, 2.87(14); CH, 2.1(2); H2, 0.6 ; NH, 1.1, SiH, 1.90. Anion Morse potentials are calculated for H2 and N2 from positive electron affinities and for hyperfine superoxide states for the first time. PMID:25758340

  12. Vertex representations of quantum affine algebras

    PubMed Central

    Frenkel, Igor B.; Jing, Naihuan

    1988-01-01

    We construct vertex representations of quantum affine algebras of ADE type, which were first introduced in greater generality by Drinfeld and Jimbo. The limiting special case of our construction is the untwisted vertex representation of affine Lie algebras of Frenkel-Kac and Segal. Our representation is given by means of a new type of vertex operator corresponding to the simple roots and satisfying the defining relations. In the case of the quantum affine algebra of type A, we introduce vertex operators corresponding to all the roots and determine their commutation relations. This provides an analogue of a Chevalley basis of the affine Lie algebra [unk](n) in the basic representation. PMID:16594004

  13. 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...

  14. Affinity Electrophoresis Using Ligands Attached To Polymers

    NASA Technical Reports Server (NTRS)

    Van Alstine, James M.; Snyder, Robert S.; Harris, J. M.; Brooks, D. E.

    1990-01-01

    In new technique, reduction of electrophoretic mobilities by addition of polyethylene glycol to ligands increases electrophoretic separabilities. In immuno-affinity electrophoresis, modification of ligands extends specificity of electrophoretic separation to particles having surface electric-charge structures otherwise making them electrophoretically inseparable. Modification of antibodies by polyethylene glycol greatly reduces ability to aggregate while enhancing ability to affect electrophoretic mobilities of cells. In hydrophobic-affinity electrophoresis, addition of polyethylene glycol reduces tendency toward aggregation of cells or macromolecules.

  15. Proton affinities of simple organic compounds

    NASA Astrophysics Data System (ADS)

    Wrblewski, T.; Ziemczonek, L.; Szerement, K.; Karwasz, G. P.

    2006-10-01

    The Restricted Hatree-Fock method with 6-311G** split-valence molecular orbitals basis sets has been applied to geometrical optimizations and calculations of total electronic, zero point vibrational energies and proton affinities at 298 K for small neutral and protonated alkanes, alcohols, acetic acid, methyl and ethyl acetate, acetone, and acetaldehyde. Calculated values of proton affinities are compared with experimental data.

  16. An amperometric affinity penicillin-binding protein magnetosensor for the detection of ?-lactam antibiotics in milk.

    PubMed

    Gamella, M; Campuzano, S; Conzuelo, F; Esteban-Torres, M; de las Rivas, B; Reviejo, A J; Muoz, R; Pingarrn, J M

    2013-04-01

    The preparation, characterization and performance evaluation of an amperometric affinity disposable magnetosensor, based on the use of a recombinant penicillin-binding protein (PBP) and screen-printed carbon electrodes (SPCEs), for the specific detection and quantification of ?-lactam antibiotic residues in milk are reported. The PBP was immobilized onto His-Tag-Isolation-modified magnetic beads (His-Tag-Isolation-MBs), and a direct competitive assay using a tracer with horseradish peroxidase (HRP) for the enzymatic labeling was performed. The amperometric response obtained at -0.20 V vs. the Ag pseudo-reference electrode of the SPCE after the addition of H2O2 in the presence of hydroquinone (HQ) was used as the transduction signal. The developed methodology showed very low detection limits (in the low ppb level) for the 6 antibiotics tested in untreated milk samples, and a good selectivity against other antibiotic residues frequently detected in milk and dairy products. Due to the bioreceptor employed, this methodology was able to detect only the active form of ?-lactam antibiotics with high affinities for both penicillins and cephalosporins. Moreover, the analysis took only 30 min. PMID:23420036

  17. The Effects of Target Audience on Social Tagging

    ERIC Educational Resources Information Center

    Alsarhan, Hesham

    2013-01-01

    Online social bookmarking systems allow users to assign tags (i.e., keywords) to represent the content of resources. Research on the effects of target audience on social tagging suggests that taggers select different tags for themselves, their community (e.g., family, friends, colleagues), and the general public (Panke & Gaiser, 2009; Pu &…

  18. 9 CFR 2.53 - Use of tags.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Use of tags. 2.53 Section 2.53 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.53 Use of tags. Official tags obtained by a...

  19. 9 CFR 2.53 - Use of tags.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Use of tags. 2.53 Section 2.53 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.53 Use of tags. Official tags obtained by a...

  20. 9 CFR 2.53 - Use of tags.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Use of tags. 2.53 Section 2.53 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.53 Use of tags. Official tags obtained by a...

  1. 9 CFR 2.53 - Use of tags.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Use of tags. 2.53 Section 2.53 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.53 Use of tags. Official tags obtained by a...

  2. 9 CFR 2.53 - Use of tags.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Use of tags. 2.53 Section 2.53 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.53 Use of tags. Official tags obtained by a...

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

    ERIC Educational Resources Information Center

    Papper, Marc; Kempter, Richard; Leibold, Christian

    2011-01-01

    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…

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

    ERIC Educational Resources Information Center

    Papper, Marc; Kempter, Richard; Leibold, Christian

    2011-01-01

    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

  5. 29 CFR 1926.417 - Lockout and tagging of circuits.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 8 2010-07-01 2010-07-01 false Lockout and tagging of circuits. 1926.417 Section 1926.417... Practices 1926.417 Lockout and tagging of circuits. (a) Controls. Controls that are to be deactivated during the course of work on energized or deenergized equipment or circuits shall be tagged....

  6. 49 CFR 236.76 - Tagging of wires and interference of wires or tags with signal apparatus.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... with signal apparatus. 236.76 Section 236.76 Transportation Other Regulations Relating to... wires and interference of wires or tags with signal apparatus. Each wire shall be tagged or otherwise so... apparatus. Inspections and Tests; All Systems...

  7. 49 CFR 236.76 - Tagging of wires and interference of wires or tags with signal apparatus.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... with signal apparatus. 236.76 Section 236.76 Transportation Other Regulations Relating to... wires and interference of wires or tags with signal apparatus. Each wire shall be tagged or otherwise so... apparatus. Inspections and Tests; All Systems...

  8. Regulation of integrin affinity on cell surfaces

    PubMed Central

    Schrpf, Thomas; Springer, Timothy A

    2011-01-01

    Lymphocyte activation triggers adhesiveness of lymphocyte function-associated antigen-1 (LFA-1; integrin ?L?2) for intercellular adhesion molecules (ICAMs) on endothelia or antigen-presenting cells. Whether the activation signal, after transmission through multiple domains to the ligand-binding ?I domain, results in affinity changes for ligand has been hotly debated. Here, we present the first comprehensive measurements of LFA-1 affinities on T lymphocytes for ICAM-1 under a broad array of activating conditions. Only a modest increase in affinity for soluble ligand was detected after activation by chemokine or T-cell receptor ligation, conditions that primed LFA-1 and robustly induced lymphocyte adhesion to ICAM-1 substrates. By stabilizing well-defined LFA-1 conformations by Fab, we demonstrate the absolute requirement of the open LFA-1 headpiece for adhesiveness and high affinity. Interaction of primed LFA-1 with immobilized but not soluble ICAM-1 triggers energy-dependent affinity maturation of LFA-1 to an adhesive, high affinity state. Our results lend support to the traction or translational motion dependence of integrin activation. PMID:21946563

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

    PubMed Central

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

    2009-01-01

    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 ribosomal protein L12) at the chromosomal site of the wild-type strain MG1655. As a result, JE28 produces a homogeneous population of ribosomes (His)6-tagged at the C-termini of all four L12 proteins. Furthermore, we have developed a single-step, high-throughput method for purification of tetra-(His)6-tagged 70S ribosomes from this strain using affinity chromatography. These ribosomes, when compared with the conventionally purified ones in sucrose gradient centrifugation, 2D-gel, dipeptide formation and a full-length protein synthesis assay showed higher yield and activity. We further describe how this method can be adapted for purification of ribosomal subunits and mutant ribosomes. These methodologies could, in principle, also be used to purify any functional multimeric complex from the bacterial cell. PMID:19074194

  10. Expression and purification of recombinant proteins in Escherichia coli tagged with a small metal-binding protein from Nitrosomonas europaea.

    PubMed

    Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

    2016-02-01

    Escherichia coli is still the preferred organism for large-scale production of recombinant proteins. The use of fusion proteins has helped considerably in enhancing the solubility of heterologous proteins and their purification with affinity chromatography. Here, the use of a small metal-binding protein (SmbP) from Nitrosomonas europaea is described as a new fusion protein for protein expression and purification in E.coli. Fluorescent proteins tagged at the N-terminal with SmbP showed high levels of solubility, compared with those of maltose-binding protein and glutathione S-transferase, and low formation of inclusion bodies. Using commercially available IMAC resins charged with Ni(II), highly pure recombinant proteins were obtained after just one chromatography step. Proteins may be purified from the periplasm of E.coli if SmbP contains the signal sequence at the N-terminal. After removal of the SmbP tag from the protein of interest, high-yields are obtained since SmbP is a protein of just 9.9kDa. The results here obtained suggest that SmbP is a good alternative as a fusion protein/affinity tag for the production of soluble recombinant proteins in E.coli. PMID:26494603

  11. Survival, growth, and tag retention in age-0 Chinook Salmon implanted with 8-, 9-, and 12-mm PIT tags

    USGS Publications Warehouse

    Tiffan, Kenneth F.; Perry, Russell W.; Connor, William P.; Mullins, Frank L; Rabe, Craig; Nelson, Doug D

    2015-01-01

    The ability to represent a population of migratory juvenile fish with PIT tags becomes difficult when the minimum tagging size is larger than the average size at which fish begin to move downstream. Tags that are smaller (e.g., 8 and 9 mm) than the commonly used 12-mm PIT tags are currently available, but their effects on survival, growth, and tag retention in small salmonid juveniles have received little study. We evaluated growth, survival, and tag retention in age-0 Chinook Salmon Oncorhynchus tshawytscha of three size-groups: 40–49-mm fish were implanted with 8- and 9-mm tags, and 50– 59-mm and 60–69-mm fish were implanted with 8-, 9-, and 12-mm tags. Survival 28 d after tagging ranged from 97.8% to 100% across all trials, providing no strong evidence for a fish-size-related tagging effect or a tag size effect. No biologically significant effects of tagging on growth in FL (mm/d) or weight (g/d) were observed. Although FL growth in tagged fish was significantly reduced for the 40–49-mm and 50–59-mm groups over the first 7 d, growth rates were not different thereafter, and all fish were similar in size by the end of the trials (day 28). Tag retention across all tests ranged from 93% to 99%. We acknowledge that actual implantation of 8- or 9-mm tags into small fish in the field will pose additional challenges (e.g., capture and handling stress) beyond those observed in our laboratory. However, we conclude that experimental use of the smaller tags for small fish in the field is supported by our findings.

  12. Isolation of transcription factor complexes from Arabidopsis cell suspension cultures by tandem affinity purification.

    PubMed

    Van Leene, Jelle; Eeckhout, Dominique; Persiau, Geert; Van De Slijke, Eveline; Geerinck, Jan; Van Isterdael, Gert; Witters, Erwin; De Jaeger, Geert

    2011-01-01

    Defining protein complexes is critical to virtually all aspects of cell biology because most cellular processes are regulated by stable or more dynamic protein interactions. Elucidation of the protein-protein interaction network around transcription factors is essential to fully understand their function and regulation. In the last decade, new technologies have emerged to study protein-protein interactions under near-physiological conditions. We have developed a high-throughput tandem affinity purification (TAP)/mass spectrometry (MS) platform for cell suspension cultures to analyze protein complexes in Arabidopsis thaliana. This streamlined platform follows an integrated approach comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, TAP adapted for plant cells, and tandem matrix-assisted laser desorption ionization MS for the identification of purified proteins. Recently, we evaluated the GS tag, originally developed to study mammalian protein complexes, that combines two IgG-binding domains of protein G with a streptavidin-binding peptide, separated by two tobacco etch virus cleavage sites. We found that this GS tag outperforms the traditional TAP tag in plant cells, regarding both specificity and complex yield. Here, we provide detailed protocols of the GS-based TAP platform that allowed us to characterize transcription factor complexes involved in signaling in response to the plant phytohormone jasmonate. PMID:21720954

  13. Affinity-enhanced protein partitioning in decyl beta-D-glucopyranoside two-phase aqueous micellar systems.

    PubMed

    Lam, Henry; Kavoosi, Mojgan; Haynes, Charles A; Wang, Daniel I C; Blankschtein, Daniel

    2005-02-20

    Liquid-liquid extraction in two-phase aqueous complex-fluid systems has been proposed as a scalable, versatile, and cost-effective purification method for the downstream processing of biotechnological products. In the case of two-phase aqueous micellar systems, careful choices of the phase-forming surfactants or surfactant mixtures allow these systems to separate biomolecules based on size, hydrophobicity, charge, or specific affinity. In this article, we investigate the affinity-enhanced partitioning of a model affinity-tagged protei--green fluorescent protein fused to a family 9 carbohydrate-binding module (CBM9-GFP)--in a two-phase aqueous micellar system generated from the nonionic surfactant n-decyl beta-D-glucopyranoside (C10G1), which acts simultaneously as the phase-former and the affinity ligand. In this simple system, CBM9-GFP was extracted preferentially into the micelle-rich phase, despite the opposing tendency of the steric, excluded-volume interactions operating between the protein and the micelles. We obtained more than a sixfold increase (from 0.47 to 3.1) in the protein partition coefficient (Kp), as compared to a control case where the affinity interactions were "turned off" by the addition of a competitive inhibitor (glucose). It was demonstrated conclusively that the observed increase in Kp can be attributed to the specific affinity between the CBM9 domain and the affinity surfactant C10G1, suggesting that the method can be generally applied to any CBM9-tagged protein. To rationalize the observed phenomenon of affinity-enhanced partitioning in two-phase aqueous micellar systems, we formulated a theoretical framework to model the protein partition coefficient. The modeling approach accounts for both the excluded-volume interactions and the affinity interactions between the protein and the surfactants, and considers the contributions from the monomeric and the micellar surfactants separately. The model was shown to be consistent with the experimental data, as well as with our current understanding of the CBM9 domain. PMID:15643631

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

    USGS Publications Warehouse

    Burdick, Summer M.

    2011-01-01

    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.

  15. 49 CFR 234.239 - Tagging of wires and interference of wires or tags with signal apparatus.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... with signal apparatus. 234.239 Section 234.239 Transportation Other Regulations Relating to... Tagging of wires and interference of wires or tags with signal apparatus. Each wire shall be tagged or... of the apparatus. This requirement applies to each wire at each terminal in all housings...

  16. 49 CFR 234.239 - Tagging of wires and interference of wires or tags with signal apparatus.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... with signal apparatus. 234.239 Section 234.239 Transportation Other Regulations Relating to... Tagging of wires and interference of wires or tags with signal apparatus. Each wire shall be tagged or... of the apparatus. This requirement applies to each wire at each terminal in all housings...

  17. Affinity-based release of chondroitinase ABC from a modified methylcellulose hydrogel.

    PubMed

    Pakulska, Malgosia M; Vulic, Katarina; Shoichet, Molly S

    2013-10-10

    Chondroitinase ABC (ChABC) is a promising therapeutic for spinal cord injury as it can degrade the glial scar that is detrimental to regrowth and repair. However, the sustained delivery of bioactive ChABC is a challenge requiring highly invasive methods such as intra-spinal injections, insertion of intrathecal catheters, or implantation of delivery vehicles directly into the tissue. ChABC is thermally unstable, further complicating its delivery. Moreover, there are no commercial antibodies available for its detection. To achieve controlled release, we designed an affinity-based system that sustained the release of bioactive ChABC for at least 7days. ChABC was recombinantly expressed as a fusion protein with Src homology domain 3 (SH3) with an N-terminal histidine (HIS) tag and a C-terminal FLAG tag (ChABC-SH3). Protein purification was achieved using a nickel affinity column and, for the first time, direct quantification of ChABC down to 0.1nM was attained using an in-house HIS/FLAG double tag ELISA. The release of active ChABC-SH3 was sustained from a methylcellulose hydrogel covalently modified with an SH3 binding peptide. The rate of release was tunable by varying either the binding strength of the SH3-protein/SH3-peptide pair or the SH3-peptide to SH3-protein ratio. This innovative system has the potential to be used as a platform technology for the release and detection of other proteins that can be expressed using a similar construct. PMID:23831055

  18. Localization and tracking of passive RFID tags

    NASA Astrophysics Data System (ADS)

    Zhang, Yimin; Amin, Moeness G.

    2006-05-01

    Radio frequency identification (RFID) is poised for growth as businesses and governments explore applications implementing RFID. The RFID technology will continue to evolve to meet new demands for human and target location and tracking. In particular, there are increasing needs to find and track the positions of multiple RFID tagged items that are closely spaced. As a result, localization and tracking techniques with higher accuracy, yet low implementation complexity are required. This paper examines the applicability of direction-of-arrival (DOA) estimation methods to the localization and tracking problems of passive RFID tags. Different scenarios of stationary and moving targets are considered. It is shown through performance analysis and simulations that simple DOA estimation methods can be used to provide satisfactory localization performance.

  19. RFID Label Tag Design for Metallic Surface Environments

    PubMed Central

    Park, Chong Ryol; Eom, Ki Hwan

    2011-01-01

    This paper describes a metal mount RFID tag that works reliably on metallic surfaces. The method proposes the use of commercial label type RFID tags with 2.5 mm thick Styrofoam103.7 with a relative permittivity of 1.03 attached on the back of the tag. In order to verify the performance of the proposed method, we performed experiments on an electric transformer supply chain system. The experimental results showed that the proposed tags can communicate with readers from a distance of 2 m. The recognition rates are comparable to those of commercial metallic mountable tags. PMID:22346612

  20. RFID label tag design for metallic surface environments.

    PubMed

    Park, Chong Ryol; Eom, Ki Hwan

    2011-01-01

    This paper describes a metal mount RFID tag that works reliably on metallic surfaces. The method proposes the use of commercial label type RFID tags with 2.5 mm thick Styrofoam103.7 with a relative permittivity of 1.03 attached on the back of the tag. In order to verify the performance of the proposed method, we performed experiments on an electric transformer supply chain system. The experimental results showed that the proposed tags can communicate with readers from a distance of 2 m. The recognition rates are comparable to those of commercial metallic mountable tags. PMID:22346612

  1. Scanning Cargo Containers with Tagged Neutrons

    NASA Astrophysics Data System (ADS)

    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

    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.

  2. Scanning Cargo Containers with Tagged Neutrons

    SciTech Connect

    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.

    2007-10-26

    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.

  3. A fractal circular polarized RFID tag antenna

    NASA Astrophysics Data System (ADS)

    Chaouki, Guesmi; Ferchichi, Abdelhak; Gharsallah, Ali

    2013-09-01

    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.

  4. The tagged RIBs facility of LNS

    SciTech Connect

    De Napoli, M.; Raciti, G.; Rapisarda, E.; Cardella, G.; Amorini, F.; Calabretta, L.; Sfienti, C.

    2007-11-30

    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.

  5. Physics with tagged forward protons at RHIC

    SciTech Connect

    Yip,K.

    2009-08-30

    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.

  6. Single step purification of recombinant proteins using the metal ion-inducible autocleavage (MIIA) domain as linker for tag removal.

    PubMed

    Ibe, Susan; Schirrmeister, Jana; Zehner, Susanne

    2015-08-20

    For fast and easy purification, proteins are typically fused with an affinity tag, which often needs to be removed after purification. Here, we present a method for the removal of the affinity tag from the target protein in a single step protocol. The protein VIC_001052 of the coral pathogen Vibrio coralliilyticus ATCC BAA-450 contains a metal ion-inducible autocatalytic cleavage (MIIA) domain. Its coding sequence was inserted into an expression vector for the production of recombinant fusion proteins. Following, the target proteins MalE and mCherry were produced as MIIA-Strep fusion proteins in Escherichia coli. The target proteins could be separated from the MIIA-Strep part simply by the addition of calcium or manganese(II) ions within minutes. The cleavage is not affected in the pH range from 5.0 to 9.0 or at low temperatures (6C). Autocleavage was also observed with immobilized protein on an affinity column. The protein yield was similar to that achieved with a conventional purification protocol. PMID:26026704

  7. Gas-phase nitronium ion affinities.

    PubMed Central

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

    1995-01-01

    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. PMID:11607578

  8. Signature-tagged mutagenesis of Vibrio vulnificus

    PubMed Central

    YAMAMOTO, Mai; KASHIMOTO, Takashige; TONG, Ping; XIAO, Jianbo; SUGIYAMA, Michiko; INOUE, Miyuki; MATSUNAGA, Rie; HOSOHARA, Kohei; NAKATA, Kazue; YOKOTA, Kenji; OGUMA, Keiji; YAMAMOTO, Koichiro

    2015-01-01

    Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus. PMID:25755021

  9. The accelerating growth of online tagging systems

    NASA Astrophysics Data System (ADS)

    Wu, L. F.

    2011-09-01

    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.

  10. Signature-tagged mutagenesis of Vibrio vulnificus.

    PubMed

    Yamamoto, Mai; Kashimoto, Takashige; Tong, Ping; Xiao, Jianbo; Sugiyama, Michiko; Inoue, Miyuki; Matsunaga, Rie; Hosohara, Kohei; Nakata, Kazue; Yokota, Kenji; Oguma, Keiji; Yamamoto, Koichiro

    2015-07-01

    Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus. PMID:25755021

  11. An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes.

    PubMed

    Van Leene, Jelle; Eeckhout, Dominique; Cannoot, Bernard; De Winne, Nancy; Persiau, Geert; Van De Slijke, Eveline; Vercruysse, Leen; Dedecker, Maarten; Verkest, Aurine; Vandepoele, Klaas; Martens, Lennart; Witters, Erwin; Gevaert, Kris; De Jaeger, Geert

    2015-01-01

    Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most advanced methods to characterize protein complexes in plants, giving a comprehensive view on the protein-protein interactions (PPIs) of a certain protein of interest (bait). The bait protein is fused to a double affinity tag, which consists of a protein G tag and a streptavidin-binding peptide separated by a very specific protease cleavage site, allowing highly specific protein complex isolation under near-physiological conditions. Implementation of this optimized TAP tag, combined with ultrasensitive MS, means that these experiments can be performed on small amounts (25 mg of total protein) of protein extracts from Arabidopsis cell suspension cultures. It is also possible to use this approach to isolate low abundant protein complexes from Arabidopsis seedlings, thus opening perspectives for the exploration of protein complexes in a plant developmental context. Next to protocols for efficient biomass generation of seedlings (∼7.5 months), we provide detailed protocols for TAP (1 d), and for sample preparation and liquid chromatography-tandem MS (LC-MS/MS; ∼5 d), either from Arabidopsis seedlings or from cell cultures. For the identification of specific co-purifying proteins, we use an extended protein database and filter against a list of nonspecific proteins on the basis of the occurrence of a co-purified protein among 543 TAP experiments. The value of the provided protocols is illustrated through numerous applications described in recent literature. PMID:25521792

  12. Uncertainty of exploitation estimates made from tag returns

    USGS Publications Warehouse

    Miranda, L.E.; Brock, R.E.; Dorr, B.S.

    2002-01-01

    Over 6,000 crappies Pomoxis spp. were tagged in five water bodies to estimate exploitation rates by anglers. Exploitation rates were computed as the percentage of tags returned after adjustment for three sources of uncertainty: postrelease mortality due to the tagging process, tag loss, and the reporting rate of tagged fish. Confidence intervals around exploitation rates were estimated by resampling from the probability distributions of tagging mortality, tag loss, and reporting rate. Estimates of exploitation rates ranged from 17% to 54% among the five study systems. Uncertainty around estimates of tagging mortality, tag loss, and reporting resulted in 90% confidence intervals around the median exploitation rate as narrow as 15 percentage points and as broad as 46 percentage points. The greatest source of estimation error was uncertainty about tag reporting. Because the large investments required by tagging and reward operations produce imprecise estimates of the exploitation rate, it may be worth considering other approaches to estimating it or simply circumventing the exploitation question altogether.

  13. Using TAGs to speed up the ATLAS analysis process

    NASA Astrophysics Data System (ADS)

    Ehrenfeld, W.; Buckingham, R.; Cranshaw, J.; Cuhadar Donszelmann, T.; Doherty, T.; Gallas, E.; Hrivnac, J.; Malon, D.; Nowak, M.; Slater, M.; Viegas, F.; Vinek, E.; Zhang, Q.; ATLAS Collaboration

    2011-12-01

    In the ATLAS experiment, Tag Data, or short TAG, are event-level metadata -thumbnail information about events to support efficient identification and selection of events of interest to a given analysis. TAG quantities range from detector status and trigger information to basic physics quantities, e. g. the number of loose electrons candidates and kinematic information for a limited number of these candidates sorted by their transverse momentum. The average TAG size per event is around 1kB, which is a factor 100 smaller than the Analysis Object Data (AOD) used for physics analysis. TAGs are primarily produced from AODs and stored in ROOT files. For easier access and usability TAGs are also stored in a database. Queries to the database can produce again TAG files. In a standard ATLAS analysis job, TAGs can be used to preselect events based on the TAG quantities before accessing the full AOD content. This allows for a significant speed up of the processing time. This paper will discuss the different analysis work flows using TAGs and compare them with other analysis work flows within ATLAS. Further, the performance for preselecting events using either directly AODs or TAG files is measured and compared. Peak performance is estimated on a single machine with local disk access, while more realistic performance is estimated using Grid like data access.

  14. The use of tags in monitoring limits on mobile missiles

    SciTech Connect

    Fetter, S.

    1987-03-01

    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.

  15. Affinity Purification and Characterization of a G-Protein Coupled Receptor, Saccharomyces cerevisiae Ste2p

    SciTech Connect

    Lee, Byung-Kwon; Jung, Kyung-Sik; Son, Cagdas D; Kim, Heejung; Verberkmoes, Nathan C; Arshava, Boris; Naider, Fred; Becker, Jeffrey Marvin

    2007-01-01

    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.

  16. The affinity purification and characterization of ATP synthase complexes from mitochondria

    PubMed Central

    Runswick, Michael J.; Bason, John V.; Montgomery, Martin G.; Robinson, Graham C.; Fearnley, Ian M.; Walker, John E.

    2013-01-01

    The mitochondrial F1-ATPase inhibitor protein, IF1, 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 IF1 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 IF1 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 160 of bovine IF1 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

  17. claMP Tag: A Versatile Inline Metal-Binding Platform Based on the Metal Abstraction Peptide

    PubMed Central

    2015-01-01

    Molecularly targeted research and diagnostic tools are essential to advancing understanding and detection of many diseases. Metals often impart the desired functionality to these tools, and conjugation of high-affinity chelators to proteins is carried out to enable targeted delivery of the metal. This approach has been much more effective with large lanthanide series metals than smaller transition metals. Because chemical conjugation requires additional processing and purification steps and yields a heterogeneous mixture of products, inline incorporation of a peptide tag capable of metal binding is a highly preferable alternative. Development of a transition metal binding tag would provide opportunity to greatly expand metal-based analyses. The metal abstraction peptide (MAP) sequence was genetically engineered into recombinant protein to generate the claMP Tag. The effects of this tag on recombinant epidermal growth factor (EGF) protein expression, disulfide bond formation, tertiary structural integrity, and transition metal incorporation using nickel were examined to confirm the viability of utilizing the MAP sequence to generate linker-less metal conjugates. PMID:24807049

  18. Facile purification of Escherichia coli expressed tag-free recombinant human tumor necrosis factor alpha from supernatant.

    PubMed

    Zhang, Chun; Liu, Yongdong; Zhao, Dawei; Li, Xiunan; Yu, Rong; Su, Zhiguo

    2014-03-01

    Fusing affinity tag at N-terminus was reported to decrease the biological activity of the recombinant human tumor necrosis factor alpha. Although preparation of tag-free rhTNF-? has already been achieved, the processes were yet laborious, especially in large scale. In this paper, tag-free rhTNF-? was almost equally synthesized by Escherichia coli in both soluble and insoluble forms. A two-step ion exchange chromatography, DEAE-Sepharose combined with CM-Sepharose, was developed to purify the soluble specie from supernatant after cell lysis. Native PAGE and HP-SEC showed the rhTNF-? extracted from supernatant existed in a homogeneous form. HP-SAX and SDS-PAGE analysis demonstrated the purity of the final fraction was over 98% with a very high recovery of 75%. Circular dichroism spectrum demonstrated that ?-sheet structure was dominant and fluorescence analysis suggested no dramatic exposure of aromatic amino acid residues on the protein surface. Bioassay indicated that purified rhTNF-? was biologically active with a specific activity of approximately 2.010(7)U/mg. All these results suggested that this two-step ion exchange chromatography is efficient for preparation of biologically active tag-free rhTNF-? from supernatant. PMID:24412132

  19. Classification of neocortical interneurons using affinity propagation

    PubMed Central

    Santana, Roberto; McGarry, Laura M.; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits. PMID:24348339

  20. Purification of antibodies using affinity chromatography.

    PubMed

    Darcy, Elaine; Leonard, Paul; Fitzgerald, Jenny; Danaher, Martin; O'Kennedy, Richard

    2011-01-01

    Affinity chromatography permits the isolation of a target analyte from a complex mixture and can be utilised to purify proteins, carbohydrates, drugs, haptens, or any analyte of interest once an affinity pair is available. It involves the exploitation of specific interactions between a binding affinity pair, such as those between an antibody and its associated antigen, or between any ligand and its associated binding receptor/protein. With the discovery of protein A in 1970, and, subsequently proteins G and L, immuno-affinity chromatography has grown in popularity and is now the standard methodology for the purification of antibodies which may be implemented for a selection of different applications such as immunodiagnostics. This chapter is designed to inform the researcher about the basic techniques involved in the affinity chromatography-based purification of monoclonal, polyclonal, and recombinant antibodies. Examples are provided for the use of proteins A and G. In addition, tables are provided that allow the reader to select the most appropriate protein for use in the isolation of their antibody. PMID:20978976

  1. Classification of neocortical interneurons using affinity propagation.

    PubMed

    Santana, Roberto; McGarry, Laura M; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits. PMID:24348339

  2. A His6-SUMO-eXact tag for producing human prepro-urocortin 2 in Escherichia coli for raising monoclonal antibodies.

    PubMed

    Liew, Oi Wah; Ang, Cui Xia; Peh, Yu Pei; Chong, Pek Ching Jenny; Ng, Yan Xia; Hwang, Le-Ann; Koh, Xin Yu; Yip, Yin Mun; Liu, Wei; Richards, A Mark

    2014-01-31

    This is a first report of recombinant production of human prepro-Urocortin 2 in Escherichia coli by N-terminal fusion with a triple His?-SUMO-eXact tag and its subsequent use as an antigen for the production and screening of very high affinity monoclonal antibodies. The rationale for this combinatorial construct is that the His tag allows first step protein purification of insoluble and soluble proteins, the SUMO tag enhances protein expression level and solubility, while the eXact tag facilitates anion-triggered on-column cleavage of the triple tag to recover pure native proteins in a simple two-step protein purification procedure. Compared with an eXact fusion alone, the presence of the SUMO moiety enhanced overall expression levels by 4 to 10 fold but not the solubility of the highly basic prepro-Urocortin 2. Insoluble SUMO-eXact-preproUCN2 was purified in milligram quantities by denaturing IMAC and solubilized in native phosphate buffer by on-column refolding or step-wise dialysis. Only a small fraction of this solubilized protein was able to bind onto the eXact affinity column and cleaved by NaF treatment. To test whether binding and cleavage failure was due to improperly refolded SUMO-eXact-preproUCN2 or to the presence of N- and C-terminal sequences flanking the eXact moiety, we created a SUMO-eXact-thioredoxin construct which was overexpressed mainly in the soluble form. This protein bound to and was cleaved efficiently on the eXact column to yield native thioredoxin. Solubilized SUMO-eXact-preproUCN2 was used successfully to generate two high affinity mouse monoclonal antibodies (KD~10?? and 10? M) specific to the pro-region of Urocortin 2. PMID:24291344

  3. High-resolution structural insights on the sugar-recognition and fusion tag properties of a versatile ?-trefoil lectin domain from the mushroom Laetiporus sulphureus.

    PubMed

    Angulo, Ivn; Acebrn, Ivn; de las Rivas, Blanca; Muoz, Rosario; Rodrguez-Crespo, I; Menndez, Margarita; Garca, Pedro; Tateno, Hiroaki; Goldstein, Irwin J; Prez-Agote, Begoa; Mancheo, Jos M

    2011-10-01

    In this work, we analyzed at high resolution the sugar-binding mode of the recombinant N-terminal ricin-B domain of the hemolytic protein LSLa (LSL(150)) from the mushroom Laetiporus sulphureus and also provide functional in vitro evidence suggesting that, together with its putative receptor-binding role, this module may also increase the solubility of its membrane pore-forming partner. We first demonstrate that recombinant LSL(150) behaves as an autonomous folding unit and an active lectin. We have determined its crystal structure at 1.47 resolution and also that of the [LSL(150):(lactose)?, ?)] binary complex at 1.67 resolution. This complex reveals two lactose molecules bound to the ? and ? sites of LSL(150), respectively. Isothermal titration calorimetry indicates that LSL(150) binds two lactoses in solution with highly different affinities. Also, we test the working hypothesis that LSL(150) exhibits in vivo properties typical of solubility tags. With this aim, we have fused an engineered version of LSL(150) (LSL(t)) to the N-terminal end of various recombinant proteins. All the designed LSL(150)-tagged fusion proteins were successfully produced at high yield, and furthermore, the target proteins were purified by a straightforward affinity procedure on agarose-based matrices due to the excellent properties of LSL(150) as an affinity tag. An optimized protocol for target protein purification was devised, which involved removal of the LSL(150) tag through in-column cleavage of the fusion proteins with His(6)-tagged TEV endoprotease. These results permitted to set up a novel, lectin-based system for production and purification of recombinant proteins in E. coli cells with attractive biotechnological applications. PMID:21632870

  4. Modeling data from double-tagging experiments to estimate heterogeneous rates of tag shedding in lake trout (Salvelinus namaycush)

    USGS Publications Warehouse

    Fabrizio, Mary C.; Nichols, James D.; Hines, James E.; Swanson, Bruce L.; Schram, Stephen T.

    1999-01-01

    Data from mark-recapture studies are used to estimate population rates such as exploitation, survival, and growth. Many of these applications assume negligible tag loss, so tag shedding can be a significant problem. Various tag shedding models have been developed for use with data from double-tagging experiments, including models to estimate constant instantaneous rates, time-dependent rates, and type I and II shedding rates. In this study, we used conditional (on recaptures) multinomial models implemented using the program SURVIV (G.C. White. 1983. J. Wildl. Manage. 47: 716-728) to estimate tag shedding rates of lake trout (Salvelinus namaycush) and explore various potential sources of variation in these rates. We applied the models to data from several long-term double-tagging experiments with Lake Superior lake trout and estimated shedding rates for anchor tags in hatchery-reared and wild fish and for various tag types applied in these experiments. Estimates of annual tag retention rates for lake trout were fairly high (80-90%), but we found evidence (among wild fish only) that retention rates may be significantly lower in the first year due to type I losses. Annual retention rates for some tag types varied between male and female fish, but there was no consistent pattern across years. Our estimates of annual tag retention rates will be used in future studies of survival rates for these fish.

  5. Modeling data from double-tagging experiments to estimate heterogeneous rates of tag-shedding in lake trout (Salvelinus namaycush)

    USGS Publications Warehouse

    Fabrizio, M.C.; Nichols, J.D.; Hines, J.E.; Swanson, B.L.; Schram, S.T.

    1999-01-01

    Data from mark-recapture studies are used to estimate population rates such as exploitation, survival, and growth. Many of these applications assume negligible tag loss, so tag shedding can be a significant problem. Various tag shedding models have been developed for use with data from double-tagging experiments, including models to estimate constant instantaneous rates, time-dependent rates, and type I and II shedding rates. I n this study, we used conditional (on recaptures) multinomial models implemented using the program SURVIV (G.C. White. 1983. J. Wildl. Manage. 47: 716-728) to estimate tag shedding rates of lake trout (Salvelinus namaycush) and explore various potential sources of variation in these rates. We applied the models to data from several long-term double-tagging experiments with Lake Superior lake trout and estimated shedding rates for anchor tags in hatchery-reared and wild fish and for various tag types applied in these experiments. Estimates of annual tag retention rates for lake trout were fairly high (80-90%), but we found evidence (among wild fish only) that retention rates may be significantly lower in the first year due to type I losses. Annual retention rates for some tag types varied between male and female fish, but there was no consistent pattern across years. Our estimates of annual tag retention rates will be used in future studies of survival rates for these fish.

  6. Chromatographic purification of an insoluble histidine tag recombinant Ykt6p SNARE from Arabidopsis thaliana over-expressed in E. coli.

    PubMed

    Vincent, Patrick; Dieryck, Wilfrid; Maneta-Peyret, Lilly; Moreau, Patrick; Cassagne, Claude; Santarelli, Xavier

    2004-08-25

    In order to undertake in plant cell the study of the endoplasmic reticulum (ER)-Golgi apparatus (GA) protein and/or lipid vesicular transport pathway, expressed sequence tag (EST) coding for a homologue to the yeast soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Ykt6p has been cloned in Arabidopsis thaliana by reverse transcription polymerase chain reaction (RT-PCR). The corresponding protein was over-expressed as a recombinant histidine-tag (his-tag) protein in E. coli. Starting from one litter of culture, an ultrasonic homogenization was performed for cell disruption and after centrifugation the Arabidopsis Ykt6p SNARE present in inclusion bodies in the pellet was solubilized. After centrifugation, the clarified feedstock obtained was injected onto an immobilized metal affinity chromatography (IMAC) in presence of 6 M guanidine and on-column refolding was performed. Folded and subsequently purified (94% purity) recombinant protein was obtained with 82% of recovery. PMID:15236690

  7. Identity, Affinity, Reality: Making the Case for Affinity Groups in Elementary School

    ERIC Educational Resources Information Center

    Parsons, Julie; Ridley, Kimberly

    2012-01-01

    Affinity groups are places where students build connections and process "ouch" moments from their classes. Children talk about the isolation they sometimes feel. The relationships students gain through race-based affinity groups enable them to feel less alone with their emotions and help them build a stronger sense of self. At the same time,

  8. High affinity capture surface for matrix-assisted laser desorption/ionisation compatible protein microarrays.

    PubMed

    Koopmann, Jens-Oliver; Blackburn, Jonathan

    2003-01-01

    A surface for the capture of biotin-tagged proteins on matrix-assisted laser desorption/ionisation (MALDI) targets has been investigated. Binding of a poly-L-lysine poly(ethylene glycol)-biotin polymer to glass and gold surfaces has been demonstrated using dual wavelength interferometry. Biotinylated proteins were captured onto this surface using tetrameric neutravidin as a multivalent bridging molecule. Biotin tagging of proteins was achieved by chemical biotinylation or by expressing a protein with a biotinylation consensus sequence in E. coli. The specificity of the surface for biotin-tagged proteins allowed the purification of biotin-tagged glutathione-S-transferase from a bacterial lysate directly onto a MALDI target. Subsequently, the protein was digested on the MALDI target and a protein fingerprint analysis confirmed its presence directly, but no E. coli proteins were detected. Therefore, we conclude that this surface is highly specific for the capture of biotin-labelled proteins and has low non-specific binding properties for non-biotinylated proteins. Furthermore, protein-protein interactions using biotinylated lectins were investigated, and the selective capture of the glycoprotein fetuin with wheat germ agglutinin was demonstrated. Also, immobilised Arachis hypogea agglutinin recognised a minor asialo component of this glycoprotein on the array. The high affinity immobilisation of proteins onto this surface allowed effective desalting procedures to be used which improved the desorption of high molecular weight proteins. Another aspect of this surface is that a highly ordered coupling of the analyte can be achieved which eliminates the search for the sweet spot and allows the creation of densely packed protein microarrays for use in mass spectrometry. PMID:12590394

  9. Addition of six-His-tagged peptide to the C terminus of adeno-associated virus VP3 does not affect viral tropism or production.

    PubMed

    Zhang, Huang-Ge; Xie, Jinfu; Dmitriev, Igor; Kashentseva, Elena; Curiel, David T; Hsu, Hui-Chen; Mountz, John D

    2002-12-01

    Production of large quantities of recombinant adeno-associated virus (AAV) is difficult and not cost-effective. To overcome this problem, we have explored the feasibility of creating a recombinant AAV encoding a 6xHis tag on the VP3 capsid protein. We generated a plasmid vector containing a six-His (6xHis)-tagged AAV VP3. A second plasmid vector was generated that contained the full-length AAV capsid capable of producing VP1 and VP2, but not VP3 due to a mutation at position 2809 that encodes the start codon for VP3. These plasmids, necessary for production of AAV, were transfected into 293 cells to generate a 6xHis-tagged VP3mutant recombinant AAV. The 6xHis-tagged VP3 did not affect the formation of AAV virus, and the physical properties of the 6xHis-modified AAV were equivalent to those of wild-type particles. The 6xHis-tagged AAV did not affect the production titer of recombinant AAV and could be used to purify the recombinant AAV using an Ni-nitrilotriacetic acid column. Addition of the 6xHis tag did not alter the viral tropism compared to wild-type AAV. These observations demonstrate the feasibility of producing high-titer AAV containing a 6xHis-tagged AAV VP3 capsid protein and to utilize the 6xHis-tagged VP3 capsid to achieve high-affinity purification of this recombinant AAV. PMID:12414944

  10. Tags, wireless communication systems, tag communication methods, and wireless communications methods

    DOEpatents

    Scott; Jeff W. (Pasco, WA), Pratt; Richard M. (Richland, WA)

    2006-09-12

    Tags, wireless communication systems, tag communication methods, and wireless communications methods are described. In one aspect, a tag includes a plurality of antennas configured to receive a plurality of first wireless communication signals comprising data from a reader, a plurality of rectifying circuits coupled with. respective individual ones of the antennas and configured to provide rectified signals corresponding to the first wireless communication signals, wherein the rectified signals are combined to produce a composite signal, an adaptive reference circuit configured to vary a reference signal responsive to the composite signal, a comparator coupled with the adaptive reference circuit and the rectifying circuits and configured to compare the composite signal with respect to the reference signal and to output the data responsive to the comparison, and processing circuitry configured to receive the data from the comparator and to process the data.

  11. On the affine structure of perceptual space.

    PubMed

    Todd, J T; Oomes, A H; Koenderink, J J; Kappers, A M

    2001-05-01

    Affine geometry is a generalization of Euclidean geometry in which distance can be scaled along parallel directions, though relative distances in different directions may be incommensurable. This article presents a new procedure for testing the intrinsic affine structure of a psychological space by having subjects perform bisection judgments over multiple directions. If those judgments are internally consistent with one another, they must satisfy a theorem first proved by Pierre Varignon around 300 years ago. In the experiment reported here, this procedure was employed to measure the perceived structure of a visual ground surface. The results revealed that observers' judgments were systematically distorted relative to the physical environment, but that the judged bisections in different directions had an internally consistent affine structure. Implications of these findings for other possible response tasks are considered. PMID:11437300

  12. Displacement phenomena in lectin affinity chromatography.

    PubMed

    Cho, Wonryeon

    2015-10-01

    The work described here examines displacement phenomena that play a role in lectin affinity chromatography and their potential to impact reproducibility. This was achieved using Lycopersicon esculentum lectin (LEL), a lectin widely used in monitoring cancer. Four small identical LEL columns were coupled in series to form a single affinity chromatography system with the last in the series connected to an absorbance detector. The serial affinity column set (SACS) was then loaded with human plasma proteins. At the completion of loading, the column set was disassembled, the four columns were eluted individually, the captured proteins were trypsin digested, the peptides were deglycosylated with PNGase F, and the parent proteins were identified through mass spectral analyses. Significantly different sets of glycoproteins were selected by each column, some proteins appearing to be exclusively bound to the first column while others were bound further along in the series. Clearly, sample displacement chromatography (SDC) occurs. Glycoproteins were bound at different places in the column train, identifying the presence of glycoforms with different affinity on a single glycoprotein. It is not possible to see these phenomena in the single column mode of chromatography. Moreover, low abundance proteins were enriched, which facilitates detection. The great advantage of this method is that it differentiates between glycoproteins on the basis of their binding affinity. Displacement phenomena are concluded to be a significant component of the separation mechanism in heavily loaded lectin affinity chromatography columns. This further suggests that care must be exercised in sample loading of lectin columns to prevent analyte displacement with nonretained proteins. PMID:26348026

  13. Fluorescent boronic acid polymer grafted on silica particles for affinity separation of saccharides.

    PubMed

    Xu, Zhifeng; Uddin, Khan Mohammad Ahsan; Kamra, Tripta; Schnadt, Joachim; Ye, Lei

    2014-02-12

    Boronic acid affinity gels are important for effective separation of biological active cis-diols, and are finding applications both in biotech industry and in biomedical research areas. To increase the efficacy of boronate affinity separation, it is interesting to introduce repeating boronic acid units in flexible polymer chains attached on solid materials. In this work, we synthesize polymer brushes containing boronic acid repeating units on silica gels using surface-initiated atom transfer radical polymerization (ATRP). A fluorescent boronic acid monomer is first prepared from an azide-tagged fluorogenic boronic acid and an alkyne-containing acrylate by Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction (the CuAAC click chemistry). The boronic acid monomer is then grafted to the surface of silica gel modified with an ATRP initiator. The obtained composite material contains boronic acid polymer brushes on surface and shows favorable saccharide binding capability under physiological pH conditions, and displays interesting fluorescence intensity change upon binding fructose and glucose. In addition to saccharide binding, the flexible polymer brushes on silica also enable fast separation of a model glycoprotein based on selective boronate affinity interaction. The synthetic approach and the composite functional material developed in this work should open new opportunities for high efficiency detection, separation, and analysis of not only simple saccharides, but also glycopeptides and large glycoproteins. PMID:24444898

  14. The CRAPome: a Contaminant Repository for Affinity Purification Mass Spectrometry Data

    PubMed Central

    Mellacheruvu, Dattatreya; Wright, Zachary; Couzens, Amber L.; Lambert, Jean-Philippe; St-Denis, Nicole; Li, Tuo; Miteva, Yana V.; Hauri, Simon; Sardiu, Mihaela E.; Low, Teck Yew; Halim, Vincentius A.; Bagshaw, Richard D.; Hubner, Nina C.; al-Hakim, Abdallah; Bouchard, Annie; Faubert, Denis; Fermin, Damian; Dunham, Wade H.; Goudreault, Marilyn; Lin, Zhen-Yuan; Badillo, Beatriz Gonzalez; Pawson, Tony; Durocher, Daniel; Coulombe, Benoit; Aebersold, Ruedi; Superti-Furga, Giulio; Colinge, Jacques; Heck, Albert J. R.; Choi, Hyungwon; Gstaiger, Matthias; Mohammed, Shabaz; Cristea, Ileana M.; Bennett, Keiryn L.; Washburn, Mike P.; Raught, Brian; Ewing, Rob M.; Gingras, Anne-Claude; Nesvizhskii, Alexey I.

    2013-01-01

    Affinity purification coupled with mass spectrometry (AP-MS) is now a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background contaminants (e.g. proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. While the standard approach is to identify nonspecific interactions using one or more negative controls, most small-scale AP-MS studies do not capture a complete, accurate background protein set. Fortunately, negative controls are largely bait-independent. Hence, aggregating negative controls from multiple AP-MS studies can increase coverage and improve the characterization of background associated with a given experimental protocol. Here we present the Contaminant Repository for Affinity Purification (the CRAPome) and describe the use of this resource to score protein-protein interactions. The repository (currently available for Homo sapiens and Saccharomyces cerevisiae) and computational tools are freely available online at www.crapome.org. PMID:23921808

  15. Genetic affinities of the Shetland islanders.

    PubMed

    Roberts, D F

    1990-01-01

    Phenotype and gene frequencies in blood group, red cell enzyme, and serum protein polymorphisms in a sample of Shetland islanders are reported. Analyses of genetic distance and relationship indicate that Shetland affinities are closer to nearby populations bordering the North Sea rather than to those of the North Atlantic. The closest affinities seem to be with populations in north Britain with a similar history of Norse influence. There is evidence that the Shetlanders retain an element of an ultra-European population extreme in some gene frequencies and so, like the Orcadians, may be regarded as much diluted relict of an ancient population. PMID:2334107

  16. New unitary affine-Virasoro constructions

    SciTech Connect

    Halpern, M.B.; Kiritsis, E.; Obers, N.A.; Poratti, M. ); Yamron, J.P. )

    1990-06-20

    This paper reports on a quasi-systematic investigation of the Virasoro master equation. The space of all affine-Virasoro constructions is organized by K-conjugation into affine-Virasoro nests, and an estimate of the dimension of the space shows that most solutions await discovery. With consistent ansatze for the master equation, large classes of new unitary nests are constructed, including quadratic deformation nests with continuous conformal weights, and unitary irrational central charge nests, which may dominate unitary rational central charge on compact g.

  17. Negative Electron Affinity Mechanism for Diamond Surfaces

    NASA Technical Reports Server (NTRS)

    Krainsky, I. L.; Asnin, V. M.

    1998-01-01

    The energy distribution of the secondary electrons for chemical vacuum deposited diamond films with Negative Electron Affinity (NEA) was investigated. It was found that while for completely hydrogenated diamond surfaces the negative electron affinity peak in the energy spectrum of the secondary electrons is present for any energy of the primary electrons, for partially hydrogenated diamond surfaces there is a critical energy above which the peak is present in the spectrum. This critical energy increases sharply when hydrogen coverage of the diamond surface diminishes. This effect was explained by the change of the NEA from the true type for the completely hydrogenated surface to the effective type for the partially hydrogenated surfaces.

  18. Structure and electron affinity of platinum fluorides.

    PubMed

    Wesendrup, R; Schwerdtfeger, P

    2001-07-01

    The structure, stability, and electron affinity of the even numbered molecular platinum fluorides PtF(2)(n) (n = 1-4) were studied by scalar relativistic density functional and coupled cluster methods. The di, tetra, and hexafluorides possess triplet ground states, while PtF(8) is a singlet. Formation of the latter from PtF(6) and F(2) is found to be endothermic. Differences between adiabiatic and vertical electron affinities are only significant for PtF(2). PMID:11421679

  19. Satake diagrams of affine Kac Moody algebras

    NASA Astrophysics Data System (ADS)

    Tripathy, L. K.; Pati, K. C.

    2006-02-01

    Satake diagrams of affine Kac-Moody algebras (untwisted and twisted) are obtained from their Dynkin diagrams. These diagrams give a classification of restricted root systems associated with these algebras. In the case of simple Lie algebras, these root systems and Satake diagrams correspond to symmetric spaces which have recently found many physical applications in quantum integrable systems, quantum transport problems, random matrix theories etc. We hope these types of root systems may have similar applications in theoretical physics in future and may correspond to symmetric spaces analogue of affine Kac-Moody algebras if they exist.

  20. Active sensor tags for global visibility of asset readiness

    NASA Astrophysics Data System (ADS)

    Burghard, B. J.; Silvers, K. L.; Skorpik, J. R.

    2005-05-01

    The era of wireless communication and discrete, autonomous sensors platforms is upon us. Advances in radio-frequency (RF) technology from simple two-way personal communications to smart, independent, sensor command, and control units has greatly expanded the applications domain. In the past four years, Pacific Northwest National Laboratory (PNNL) scientists and engineers have developed smart sensor tags (health tags) for the Army to monitor environmental conditions of high value assets over their lifetime (10 yrs). These field tested health tags uniquely identify individual assets, record and store data, run diagnostic and prognostic protocols, identify asset performance status (GO, CAUTION, NO-GO), and provide all this information over a wireless RF link to a portable, hand held reader. Leveraging the innovation achieved for health monitoring tags, the next generation active sensor tag has been developed (FlexiTag) providing reduced tag size and manufacturing cost, greater sensor interface capabilities, and a flexible substrate for surface mount conformity. The design has a greatly reduced part count due to the use of newly available, highly integrated RF chip sets. In addition to asset health monitoring, the new tag platform opens up additional application areas such as TTL (tagging, tracking, and locating), real-time machine fault monitoring, and ad-hoc sensor networking. This paper will compare and contrast the FlexiTag to its predecessors and discuss the current application areas it is being applied to.

  1. Integrated Management and Visualization of Electronic Tag Data with Tagbase

    PubMed Central

    Lam, Chi Hin; Tsontos, Vardis M.

    2011-01-01

    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

  2. Development of techniques for tagging precursor and essential chemicals

    SciTech Connect

    Swansiger, W.A.; Shepodd, T.J.; Phillips, M.L.F.

    1994-01-01

    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.

  3. A New Approach to Tagging Data in the Astronomical Literature

    NASA Astrophysics Data System (ADS)

    Alexov, A.; Good, J. C.

    2007-10-01

    Data Tags are strings used in journals to indicate the origin of the archival data and to enable the reader to recover the data. The NASA/IPAC Infrared Science Archive (IRSA) has recently introduced a new approach to production of data tags and recovery of data from them. Many of the data access services at the IRSA return filtered data sets (such as subsets of source catalogs) and dynamically created products (such as image cutouts); these dynamically created products are not saved permanently at the archive. Rather than tag the data sets from which the query result sets are drawn, the archive tags the query that generates the results. A single tag can, then, encode a complex dynamic data set and simplifies the embedding of tags in manuscripts and journals. By logging user queries and all the parameters for those query as Data Tags, IRSA can re-create the query and rerun the IRSA service using the same search parameters used when the Data Tag was created. At the same time, the logs give a simple count of the actual numbers of queries made to the archive, a powerful metric of archive usage unobtainable from the Apache web server logs. Currently, IRSA creates tags for queries to more than 20 data sets, including the Infrared Astronomical Satellite (IRAS), Cosmic Evolution Survey (COSMOS) and Spitzer Space Telescope Legacy Data Sets. These tags are returned by the spatial query engine, Atlas {http://irsa.ipac.caltech.edu/applications/Atlas/}. IRSA plans to create tags for queries to the rest of its services in late Spring 2007. The archive provides a simple web interface {http://irsa.ipac.caltech.edu/applications/DataTag/} which recovers a data set that corresponds to the input data tag. Archived data sets may evolve in time due to improved calibrations or augmentations to the data set. IRSA's query based approach guarantees that users always receive the best available data sets.

  4. Triosephosphate isomerase is a common crystallization contaminant of soluble His-tagged proteins produced in Escherichia coli

    PubMed Central

    Kozlov, Guennadi; Vinaik, Roohi; Gehring, Kalle

    2013-01-01

    Attempts to crystallize several mammalian proteins overexpressed in Escherichia coli revealed a common contaminant, triosephosphate isomerase, a protein involved in glucose metabolism. Even with triosephosphate isomerase present in very small amounts, similarly shaped crystals appeared in the crystallization drops in a number of polyethylene glycol-containing conditions. All of the target proteins were His-tagged and their purification involved immobilized metal-affinity chromatography (IMAC), a step that was likely to lead to triosephosphate isomerase contamination. Analysis of the triosephosphate isomerase crystals led to the structure of E. coli triosephosphate isomerase at 1.85? resolution, which is a significant improvement over the previous structure. PMID:23695562

  5. N2O molecular tagging velocimetry

    NASA Astrophysics Data System (ADS)

    ElBaz, A. M.; Pitz, R. W.

    2012-03-01

    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.

  6. Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step.

    PubMed

    Huang, Renhua; Gorman, Kevin T; Vinci, Chris R; Dobrovetsky, Elena; Grslund, Susanne; Kay, Brian K

    2015-01-01

    Often when generating recombinant affinity reagents to a target, one singles out an individual binder, constructs a secondary library of variants, and affinity selects a tighter or more specific binder. To enhance the throughput of this general approach, we have developed a more integrated strategy where the "affinity maturation" step is part of the phage-display pipeline, rather than a follow-on process. In our new schema, we perform two rounds of affinity selection, followed by error-prone PCR on the pools of recovered clones, generation of secondary libraries, and three additional rounds of affinity selection, under conditions of off-rate competition. We demonstrate the utility of this approach by generating low nanomolar fibronectin type III (FN3) monobodies to five human proteins: ubiquitin-conjugating enzyme E2 R1 (CDC34), COP9 signalosome complex subunit 5 (COPS5), mitogen-activated protein kinase kinase 5 (MAP2K5), Splicing factor 3A subunit 1 (SF3A1) and ubiquitin carboxyl-terminal hydrolase 11 (USP11). The affinities of the resulting monobodies are typically in the single-digit nanomolar range. We demonstrate the utility of two binders by pulling down the targets from a spiked lysate of HeLa cells. This integrated approach should be applicable to directed evolution of any phage-displayed affinity reagent scaffold. PMID:26437402

  7. Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step

    PubMed Central

    Huang, Renhua; Gorman, Kevin T.; Vinci, Chris R.; Dobrovetsky, Elena; Grslund, Susanne; Kay, Brian K.

    2015-01-01

    Often when generating recombinant affinity reagents to a target, one singles out an individual binder, constructs a secondary library of variants, and affinity selects a tighter or more specific binder. To enhance the throughput of this general approach, we have developed a more integrated strategy where the affinity maturation step is part of the phage-display pipeline, rather than a follow-on process. In our new schema, we perform two rounds of affinity selection, followed by error-prone PCR on the pools of recovered clones, generation of secondary libraries, and three additional rounds of affinity selection, under conditions of off-rate competition. We demonstrate the utility of this approach by generating low nanomolar fibronectin type III (FN3) monobodies to five human proteins: ubiquitin-conjugating enzyme E2 R1 (CDC34), COP9 signalosome complex subunit 5 (COPS5), mitogen-activated protein kinase kinase 5 (MAP2K5), Splicing factor 3A subunit 1 (SF3A1) and ubiquitin carboxyl-terminal hydrolase 11 (USP11). The affinities of the resulting monobodies are typically in the single-digit nanomolar range. We demonstrate the utility of two binders by pulling down the targets from a spiked lysate of HeLa cells. This integrated approach should be applicable to directed evolution of any phage-displayed affinity reagent scaffold. PMID:26437402

  8. A Lanthanum-Tagged Chemotherapeutic Agent HA-Pt to Track the In Vivo Distribution of Hyaluronic Acid Complexes

    PubMed Central

    Forrest, W.C.; Cai, Shuang; Aires, Daniel; Forrest, M. Laird

    2015-01-01

    Hyaluronic acid drug conjugates can target anti-cancer drugs directly to tumor tissue for loco-regional treatment with enhanced bioavailability, local efficacy and reduced toxicity. In this study, the distribution and pharmacokinetics of hyaluronic acid carrier and a conjugated cisplatin anti-cancer drug were tracked by lanthanum (III) [La(III)] affinity tagging of the nanocarrier. The strong binding affinity of La(III) to HA enabled the simple preparation of a physiologically stable complex HA-Pt-La and straightforward simultaneous detection of HA-La and Pt in biological matrices using inductively coupled plasma-mass spectrometry (ICP-MS). Consequently, after subcutaneous injection of HA-Pt-La nanoparticles in human head and neck squamous cell carcinoma (HNSCC) tumor-bearing mice, the HA and Pt content were detected and quantified simultaneously in the plasma, primary tumor, liver and spleen. PMID:26756040

  9. Two bradykinin binding sites with picomolar affinities

    SciTech Connect

    Manning, D.C.; Vavrek, R.; Stewart, J.M.; Snyder, S.H.

    1986-05-01

    Bradykinin (BK) and related peptides exert a wide range of effects on several organ systems. We have attempted to sort out these effects by studying the binding interaction of (/sup 3/H)BK at the membrane level with in vitro receptor binding techniques. High specific activity (/sup 3/H)BK and an enzyme inhibitor cocktail has enabled us to label two BK binding sites with different affinity and peptide specificity in several guinea-pig tissues. In the guinea-pig ileum the high-affinity site has an equilibrium dissociation constant (Kd) for (/sup 3/H)BK of 13 pM and a maximal number of binding sites of 8.3 pmol/g of tissue wet weight. The low-affinity guinea-pig ileum site displays a Kd of 910 pM, a maximum number of binding sites of 14 pmol/g of tissue wet weight and shows a greater selectivity for BK analogs over Lysyl-BK analogs. Two similar sites can also be discriminated in kidney and heart. The potencies of a series of BK analogs at the high-affinity guinea-pig ileum site correlate well with their potencies in contracting ileal smooth muscle. The binding of (/sup 3/H)BK in the guinea-pig ileum is inhibited by physiological concentrations of monovalent and divalent cations.

  10. Vygotsky's and Buber's Pedagogical Perspectives: Some Affinities

    ERIC Educational Resources Information Center

    Bartholo, Roberto; Tunes, Elizabeth; Tacca, Maria Carmen Villela Rosa

    2010-01-01

    The purpose of this paper is to examine the dialogical and creative character of pedagogic work by analyzing the affinities between Martin Buber's "I-Thou relation" and Lev Semenovich Vygotsky's "Zone of Proximal Development". Backed up by empirical studies on the teacher-student relation, we understand that education can only result in students'…

  11. Membrane strip affinity purification of autoantibodies.

    PubMed

    Kurien, Biji T

    2015-01-01

    A method for affinity purification of autoantibodies from a membrane strip using small volumes of human sera is described. The membrane strip is excised from a western blot containing a target antigen electrophoretically transferred from a sodium dodecyl sulfate (SDS) polyacrylamide gel. This method is a very useful alternative for affinity column chromatography, particularly when the antigen of interest is of low abundance in a HeLa cell extract. The protein mixture is resolved on a preparative SDS polyacrylamide gel and transferred to nitrocellulose membrane. A couple of strips are excised from either side of the blotted membrane and immunoblotted with specific antisera to identify the target band. Then the target band is excised horizontally and used for affinity purification. We have used this procedure to affinity purify antibodies to a 70,000 molecular weight protein derived from HeLa cell extract. A sham band, excised away from the target antigen, was used as a control for sham purification of autoantibodies. The autoantibodies purified in this manner reproduced the multiple nuclear dot anti-nuclear antibody pattern obtained using crude sera from 21 patients without primary biliary cirrhosis or anti-mitochondrial antibody. PMID:26044008

  12. Modern affinity reagents: Recombinant antibodies and aptamers.

    PubMed

    Groff, Katherine; Brown, Jeffrey; Clippinger, Amy J

    2015-12-01

    Affinity reagents are essential tools in both basic and applied research; however, there is a growing concern about the reproducibility of animal-derived monoclonal antibodies. The need for higher quality affinity reagents has prompted the development of methods that provide scientific, economic, and time-saving advantages and do not require the use of animals. This review describes two types of affinity reagents, recombinant antibodies and aptamers, which are non-animal technologies that can replace the use of animal-derived monoclonal antibodies. Recombinant antibodies are protein-based reagents, while aptamers are nucleic-acid-based. In light of the scientific advantages of these technologies, this review also discusses ways to gain momentum in the use of modern affinity reagents, including an update to the 1999 National Academy of Sciences monoclonal antibody production report and federal incentives for recombinant antibody and aptamer efforts. In the long-term, these efforts have the potential to improve the overall quality and decrease the cost of scientific research. PMID:26482034

  13. Fan Affinity Laws from a Collision Model

    ERIC Educational Resources Information Center

    Bhattacharjee, Shayak

    2012-01-01

    The performance of a fan is usually estimated using hydrodynamical considerations. The calculations are long and involved and the results are expressed in terms of three affinity laws. In this paper we use kinetic theory to attack this problem. A hard sphere collision model is used, and subsequently a correction to account for the flow behaviour

  14. Affinity-Based Purification of Dehydrogenase Subproteomes

    PubMed Central

    Ge, Xia; Sem, Daniel S.

    2014-01-01

    Summary The high cost of drug discovery and development requires more efficient approaches to the identification and inhibition of tractable protein targets. One strategy is to pursue families of proteins that already possess affinity for a drug lead scaffold, where that scaffold plays the dual role of serving: (a) when tethered to a resin, as a ligand to purify a subproteome of interest, and (b) as a lead molecule that has the potential for optimization for a given member of the subproteome. Here, we describe the former application, the purification of a subproteome using a scaffold tailored to the dehydrogenase family of enzymes. Combined with modern LC-MS/MS and subsequent searching of proteome databases, such affinity chromatography strategies can be used to purify and identify any proteins with affinity for the scaffold molecule. The method is exemplified using the CRAA (Catechol Rhodanine Acetic Acid) privileged scaffold, which is tailored to dehydrogenases. CRAA affinity column chromatography, combined with LC-MS/MS, is described as a method for profiling dehydrogenase subproteomes. PMID:22065224

  15. On affine extension of splint root systems

    NASA Astrophysics Data System (ADS)

    Lyakhovsky, V. D.; Nazarov, A. A.

    2012-09-01

    Splint of root system of simple Lie algebra appears naturally in the study of (regular) embeddings of reductive subalgebras. It can be used to derive branching rules. Application of splint properties drastically simplifies calculations of branching coefficients. We study affine extension of splint root system of simple Lie algebra and obtain relations on theta and branching functions.

  16. Vygotsky's and Buber's Pedagogical Perspectives: Some Affinities

    ERIC Educational Resources Information Center

    Bartholo, Roberto; Tunes, Elizabeth; Tacca, Maria Carmen Villela Rosa

    2010-01-01

    The purpose of this paper is to examine the dialogical and creative character of pedagogic work by analyzing the affinities between Martin Buber's "I-Thou relation" and Lev Semenovich Vygotsky's "Zone of Proximal Development". Backed up by empirical studies on the teacher-student relation, we understand that education can only result in students'

  17. Method for nonlinear optimization for gas tagging and other systems

    DOEpatents

    Chen, T.; Gross, K.C.; Wegerich, S.

    1998-01-06

    A method and system are disclosed for providing nuclear fuel rods with a configuration of isotopic gas tags. The method includes selecting a true location of a first gas tag node, selecting initial locations for the remaining n-1 nodes using target gas tag compositions, generating a set of random gene pools with L nodes, applying a Hopfield network for computing on energy, or cost, for each of the L gene pools and using selected constraints to establish minimum energy states to identify optimal gas tag nodes with each energy compared to a convergence threshold and then upon identifying the gas tag node continuing this procedure until establishing the next gas tag node until all remaining n nodes have been established. 6 figs.

  18. Tagging module for lesion localization in capsule endoscopy.

    PubMed

    Chandrappan, Jayakrishnan; Ruiqi, Lim; Su, Nandar; Qiang, Tanq Shao; Vaidyanathan, Kripesh

    2010-01-01

    Capsule endoscopes are effective diagnostic tools for the gastro intestinal tract disorders at patient's comfort. However the present capsule endoscopes lack efficient localization techniques to specify a pathological area that may require further diagnosis or treatment. This paper presents the development of a tagging module based novel method for the real-time localization of the site of interest. The tagging module consists of a bio compatible micro tag, compressed spring with a string latch and thermal igniter. The module can be integrated with the capsule endoscope and activated using an external trigger signal. On activation, the micro tag releases instantly and penetrates the mucosa layer of GI tract, region of interest. X-ray imaging is used to detect the location of micro tag embedded in GI tract wall. The radiopaque micro tags provide pre-operative valuable position information of the infected area to facilitate further clinical procedures. PMID:21096425

  19. An overview of radio frequency identification (RFID) tags technology

    NASA Astrophysics Data System (ADS)

    Falinski, Wojciech

    2006-10-01

    RFID (Radio Frequency Identification) is the technology of wireless identification of tagged products. It is one of the fastest developing technologies in electronic market and it is predicted to replace soon the barcodes which are in common usage in today's economy. There are several advantages of RFID tags over barcode. The main are reading without must of scanning the product and the possibility to keep much more information on chip of the tag. In the article there are introduced the possible applications of RFID technology. There are also presented the classification of the RFID tags and the difference between working frequency. It is introduced every steps of manufacturing RFID tags with focus on the technology aspects (technologies of producing antenna, attaching the chip and creation of electrical connection between antenna and chip). Tele and Radio Research Institute is now starting to realize the project of manufacturing the RFID tags antenna. There is presented our guideline of the project.

  20. ?x boosted-bottom-jet tagging and Z' boson searches

    NASA Astrophysics Data System (ADS)

    Pedersen, Keith; Sullivan, Zack

    2016-01-01

    We present a new technique for tagging heavy-flavor jets with pT>500 GeV called "?x tagging." Current track-based methods of b -jet tagging lose efficiency and experience a large rise in fake rate in the boosted regime. Using muons from B hadron decay, we combine angular information and jet substructure to tag b jets, c jets, light jets, and "light-heavy" jets (those containing B hadrons from gluon splitting). We find tagging efficiencies of ?b=14 %, ?c=6.5 %, ?light-light=0.14 % , and ?light-heavy=0.5 %, respectively, that are nearly independent of transverse momentum at high energy. We demonstrate the usefulness of this new scheme by examining the discovery potential for multi-TeV leptophobic Z' bosons in the boosted-b -tagged dijet channel at the Large Hadron Collider.

  1. Method for nonlinear optimization for gas tagging and other systems

    DOEpatents

    Chen, Ting (Chicago, IL); Gross, Kenny C. (Bolingbrook, IL); Wegerich, Stephan (Glendale Heights, IL)

    1998-01-01

    A method and system for providing nuclear fuel rods with a configuration of isotopic gas tags. The method includes selecting a true location of a first gas tag node, selecting initial locations for the remaining n-1 nodes using target gas tag compositions, generating a set of random gene pools with L nodes, applying a Hopfield network for computing on energy, or cost, for each of the L gene pools and using selected constraints to establish minimum energy states to identify optimal gas tag nodes with each energy compared to a convergence threshold and then upon identifying the gas tag node continuing this procedure until establishing the next gas tag node until all remaining n nodes have been established.

  2. Implementation of RFID Tag for Metal Surface Mount

    NASA Astrophysics Data System (ADS)

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

    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 proposed method, we evaluated the experiment on the supply chain system of electric transformers. The experimental results on supply chain of electric transformers show that the proposed tags can communicate with readers from a distance of 2 m. The results of recognition rates are comparable to commercial metallic mountable tags.

  3. Single-step Antibody-based Affinity Cryo-Electron Microscopy for Imaging and Structural Analysis of Macromolecular Assemblies

    PubMed Central

    Yu, Guimei; Vago, Frank; Zhang, Dongsheng; Snyder, Jonathan E.; Yan, Rui; Zhang, Ci; Benjamin, Christopher; Jiang, Xi; Kuhn, Richard J.; Serwer, Philip; Thompson, David H.; Jiang, Wen

    2014-01-01

    Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies (i.e., protein complexes and viruses). Although single particle cryo-EM requires less concentrated and smaller amounts of samples than X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step. Here we report a new design of affinity cryo-EM approach, cryo-SPIEM, that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method. This approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. In the present work, we extensively tested the feasibility of cryo-SPIEM with multiple samples including those of high or low molecular weight, macromolecules with low or high symmetry, His-tagged or native particles, and high- or low-yield macromolecules. Results for all these samples (nonpurified His-tagged bacteriophage T7, His-tagged E. coli ribosomes, native Sindbis virus, and purified but low-concentration native Tulane virus) demonstrated the capability of cryo-SPIEM approach in specifically trapping and concentrating target particles on TEM grids with minimal view constraints for cryo-EM imaging and determination of 3D structures. PMID:24780590

  4. An ecdysone receptor from the pentatomomorphan, Nezara viridula, shows similar affinities for moulting hormones makisterone A and 20-hydroxyecdysone.

    PubMed

    Tohidi-Esfahani, Donya; Graham, Lloyd D; Hannan, Garry N; Simpson, Ann M; Hill, Ronald J

    2011-02-01

    It has been suggested that Pentatomomorpha utilise the C(28) ecdysteroid, makisterone A (MakA), as the major moulting hormone rather than the more common C(27) hormone, 20-hydroxyecdsyone (20E). The present study is the first to examine this postulate at the level of the ecdysone receptor protein, a heterodimer of nuclear receptors EcR and USP. cDNAs encoding two alternatively spliced isoforms of EcR and a single USP were isolated from a high-quality cDNA library prepared from a representative pentatomomorphan, Nezara viridula (Nv). NvEcR and NvUSP were found to group phylogenetically with heteropteran and other insect EcRs and USP/RXRs, respectively. Sequence comparison and phylogenetic analysis of these proteins found them to be distinct from those belonging to other hemipteran ecdysone receptors characterised to date. Co-expression of the His(6)-tagged ligand binding regions (LBRs) of the two NvEcR variants with the FLAG-tagged LBR of NvUSP was achieved in insect cells employing appropriately constructed baculoviruses. The corresponding heterodimers, designated NvE10 and NvE11, were purified by affinity chromatography utilising the His(6) tags on their NvEcR subunits. The heterodimers displayed nanomolar affinity for [(3)H]ponasterone A (K(d) = 6.8-7.5 nM), characteristic of ecdysone receptors. MakA has a similar affinity to 20E for both NvE10 and NvE11, consistent with MakA being a major moulting hormone in N. viridula. PMID:21035548

  5. Behavioral Tagging: A Translation of the Synaptic Tagging and Capture Hypothesis

    PubMed Central

    Moncada, Diego; Ballarini, Fabricio; Viola, Haydée

    2015-01-01

    Similar molecular machinery is activated in neurons following an electrical stimulus that induces synaptic changes and after learning sessions that trigger memory formation. Then, to achieve perdurability of these processes protein synthesis is required for the reinforcement of the changes induced in the network. The synaptic tagging and capture theory provided a strong framework to explain synaptic specificity and persistence of electrophysiological induced plastic changes. Ten years later, the behavioral tagging hypothesis (BT) made use of the same argument, applying it to learning and memory models. The hypothesis postulates that the formation of lasting memories relies on at least two processes: the setting of a learning tag and the synthesis of plasticity related proteins, which once captured at tagged sites allow memory consolidation. BT explains how weak events, only capable of inducing transient forms of memories, can result in lasting memories when occurring close in time with other behaviorally relevant experiences that provide proteins. In this review, we detail the findings supporting the existence of BT process in rodents, leading to the consolidation, persistence, and interference of a memory. We focus on the molecular machinery taking place in these processes and describe the experimental data supporting the BT in humans. PMID:26380117

  6. The dynamics of metric-affine gravity

    NASA Astrophysics Data System (ADS)

    Vitagliano, Vincenzo; Sotiriou, Thomas P.; Liberati, Stefano

    2011-05-01

    Metric-affine theories of gravity provide an interesting alternative to general relativity: in such an approach, the metric and the affine (not necessarily symmetric) connection are independent quantities. Furthermore, the action should include covariant derivatives of the matter fields, with the covariant derivative naturally defined using the independent connection. As a result, in metric-affine theories a direct coupling involving matter and connection is also present. The role and the dynamics of the connection in such theories is explored. We employ power counting in order to construct the action and search for the minimal requirements it should satisfy for the connection to be dynamical. We find that for the most general action containing lower order invariants of the curvature and the torsion the independent connection does not carry any dynamics. It actually reduces to the role of an auxiliary field and can be completely eliminated algebraically in favour of the metric and the matter field, introducing extra interactions with respect to general relativity. However, we also show that including higher order terms in the action radically changes this picture and excites new degrees of freedom in the connection, making it (or parts of it) dynamical. Constructing actions that constitute exceptions to this rule requires significant fine tuned and/or extra a priori constraints on the connection. We also consider f(R) actions as a particular example in order to show that they constitute a distinct class of metric-affine theories with special properties, and as such they cannot be used as representative toy theories to study the properties of metric-affine gravity.

  7. Some Fundamental Limits on SAW RFID Tag Information Capacity and Collision Resolution

    NASA Technical Reports Server (NTRS)

    Barton, Richard J.

    2013-01-01

    In this paper, we apply results from multi-user information theory to study the limits of information capacity and collision resolution for SAW RFID tags. In particular, we derive bounds on the achievable data rate per tag as a function of fundamental parameters such as tag time-bandwidth product, tag signal-to-noise ratio (SNR), and number of tags in the environment. We also discuss the implications of these bounds for tag waveform design and tag interrogation efficiency

  8. Plasmonic Nanogels for Unclonable Optical Tagging.

    PubMed

    Tian, Limei; Liu, Keng-Ku; Fei, Max; Tadepalli, Sirimuvva; Cao, Sisi; Geldmeier, Jeffrey A; Tsukruk, Vladimir V; Singamaneni, Srikanth

    2016-02-17

    We demonstrate the fabrication of novel functional gel coatings with randomized physical and chemical patterns that enable dual encoding ability to realize unclonable optical tags. This design is based on swelling-mediated massive reconstruction of an ultrathin responsive gelatinous polymer film uniformly adsorbed with plasmonic nanostructures into a randomized network of interacting folds, resulting in bright electromagnetic hotspots within the folds. We reveal a strong correlation between the topology and near-field electromagnetic field enhancement due to the intimate contact between two plasmonic surfaces within the folds, each of them representing a unique combination of local topography and chemical distribution caused by the formation of electromagnetic hotspots. Because of the efficient trapping of the Raman reporters within the uniquely distributed electromagnetic hotspots, the surface enhanced Raman scattering enhancement from the morphed plasmonic gel was found to be nearly 40 times higher compared to that from the pristine plasmonic gel. Harnessing the nondeterministic nature of the folds, the folded plasmonic gel can be employed as a multidimensional (with dual topo-chemical encoding) optical taggant for prospective anticounterfeiting applications. Such novel optical tags based on the spontaneous folding process are virtually impossible to replicate because of the combination of nondeterministic physical patterns and chemical encoding. PMID:26812528

  9. Hypergraph topological quantities for tagged social networks

    NASA Astrophysics Data System (ADS)

    Zlatić, Vinko; Ghoshal, Gourab; Caldarelli, Guido

    2009-09-01

    Recent years have witnessed the emergence of a new class of social networks, which require us to move beyond previously employed representations of complex graph structures. A notable example is that of the folksonomy, an online process where users collaboratively employ tags to resources to impart structure to an otherwise undifferentiated database. In a recent paper, we proposed a mathematical model that represents these structures as tripartite hypergraphs and defined basic topological quantities of interest. In this paper, we extend our model by defining additional quantities such as edge distributions, vertex similarity and correlations as well as clustering. We then empirically measure these quantities on two real life folksonomies, the popular online photo sharing site Flickr and the bookmarking site CiteULike. We find that these systems share similar qualitative features with the majority of complex networks that have been previously studied. We propose that the quantities and methodology described here can be used as a standard tool in measuring the structure of tagged networks.

  10. Endogenous gene tagging with fluorescent proteins.

    PubMed

    Fetter, John; Samsonov, Andrey; Zenser, Nathan; Zhang, Fan; Zhang, Hongyi; Malkov, Dmitry

    2015-01-01

    Human genome manipulation has become a powerful tool for understanding the mechanisms of numerous diseases including cancer. Inserting reporter sequences in the desired locations in the genome of a cell can allow monitoring of endogenous activities of disease related genes. Native gene expression and regulation is preserved in these knock-in cells in contrast to cell lines with target overexpression under an exogenous promoter as in the case of transient transfection or stable cell lines with random integration. The fusion proteins created using the modern genome editing tools are expressed at their physiological level and thus are more likely to retain the characteristic expression profile of the endogenous proteins in the cell. Unlike biochemical assays or immunostaining, using a tagged protein under endogenous regulation avoids fixation artifacts and allows detection of the target's activity in live cells. Multiple gene targets could be tagged in a single cell line allowing for the creation of effective cell-based assays for compound screening to discover novel drugs. PMID:25408409

  11. The GORE(®) TAG(®) thoracic endoprosthesis.

    PubMed

    Vallabhaneni, Raghuveer; Farber, Mark A

    2013-03-01

    The GORE(®) TAG(®) (W.L. Gore and Associates, Inc., [AZ, USA]) has gone through many changes since it was first introduced in the late 1990s. The Conformable GORE TAG has recently become commercially available and underwent several changes from the original design to help increase compression resistance, expand treatment ranges and oversizing windows, and improve conformability. This article describes the GORE TAG and Conformable GORE TAG devices, their potential uses and the outcomes in treating various aortic pathologies. PMID:23480084

  12. Retention of internal anchor tags by juvenile striped bass

    USGS Publications Warehouse

    Van Den Avyle, M.J.; Wallin, J.E.

    2001-01-01

    We marked hatchery-reared striped bass Morone saxatilis (145-265 mm total length) with internal anchor tags and monitored retention for 28 months after stocking in the Savannah River, Georgia and South Carolina. Anchor tags (with an 18-mm, T-shaped anchor and 42-mm streamer) were surgically implanted ventrally, and coded wire tags (1 mm long and 0.25 mm in diameter) were placed into the cheek muscle to help identify subsequent recaptures. The estimated probability of retention (SD) of anchor tags was 0.94 (0.05) at 4 months, 0.64 (0.13) at 16 months, and 0.33 (0.19) at 28 months. Of 10 fish recaptured with only coded wire tags, 5 showed an externally visible wound or scar near the point of anchor tag insertion. The incidence of wounds or scars, which we interpreted as evidence of tag shedding, increased to 50% in recaptures taken at 28 months (three of six fish). Our estimates for retention of anchor tags were generally lower than those in other studies of striped bass, possibly because of differences in the style of anchor or sizes of fish used. Because of its low rate of retention, the type of anchor tag we used may not be suitable for long-term assessments of stock enhancement programs that use striped bass of the sizes we evaluated.

  13. Isolation of ubiquitinated substrates by tandem affinity purification of E3 ligase-polyubiquitin-binding domain fusions (ligase traps).

    PubMed

    Mark, Kevin G; Loveless, Theresa B; Toczyski, David P

    2016-02-01

    Ubiquitination is an essential protein modification that influences eukaryotic processes ranging from substrate degradation to nonproteolytic pathway alterations, including DNA repair and endocytosis. Previous attempts to analyze substrates via physical association with their respective ubiquitin ligases have had some success. However, because of the transient nature of enzyme-substrate interactions and rapid protein degradation, detection of substrates remains a challenge. Ligase trapping is an affinity purification approach in which ubiquitin ligases are fused to a polyubiquitin-binding domain, which allows the isolation of ubiquitinated substrates. Immunoprecipitation is first used to enrich for proteins that are bound to the ligase trap. Subsequently, affinity purification is used under denaturing conditions to capture proteins conjugated with hexahistidine-tagged ubiquitin. By using this protocol, ubiquitinated substrates that are specific for a given ligase can be isolated for mass spectrometry or western blot analysis. After cells have been collected, the described protocol can be completed in 2-3 d. PMID:26766115

  14. Tag Retention and Survivorship of Hatchery Rainbow Trout Marked with Large-Format Visible Implant Alphanumeric Tags

    USGS Publications Warehouse

    Isely, J.J.; Trested, D.G.; Grabowski, T.B.

    2004-01-01

    Large-format visible implant alphanumeric (VIalpha) tags were implanted into 15,400 rainbow trout Oncorhynchus mykiss. Tag retention after 25 d was 82.6%, and survivorship was 92.8%. The results of this study compare favorably with those of similar studies on other species and suggest that large-format VIalpha tags are an appropriate choice for studies requiring the individual identification of larger rainbow trout.

  15. Superior triacylglycerol (TAG) accumulation in starchless mutants of Scenedesmus obliquus: (II) evaluation of TAG yield and productivity in controlled photobioreactors

    PubMed Central

    2014-01-01

    Background Many microalgae accumulate carbohydrates simultaneously with triacylglycerol (TAG) upon nitrogen starvation, and these products compete for photosynthetic products and metabolites from the central carbon metabolism. As shown for starchless mutants of the non-oleaginous model alga Chlamydomonas reinhardtii, reduced carbohydrate synthesis can enhance TAG production. However, these mutants still have a lower TAG productivity than wild-type oleaginous microalgae. Recently, several starchless mutants of the oleaginous microalga Scenedesmus obliquus were obtained which showed improved TAG content and productivity. Results The most promising mutant, slm1, is compared in detail to wild-type S. obliquus in controlled photobioreactors. In the slm1 mutant, the maximum TAG content increased to 57 ± 0.2% of dry weight versus 45 ± 1% in the wild type. In the wild type, TAG and starch were accumulated simultaneously during initial nitrogen starvation, and starch was subsequently degraded and likely converted into TAG. The starchless mutant did not produce starch and the liberated photosynthetic capacity was directed towards TAG synthesis. This increased the maximum yield of TAG on light by 51%, from 0.144 ± 0.004 in the wild type to 0.217 ± 0.011 g TAG/mol photon in the slm1 mutant. No differences in photosynthetic efficiency between the slm1 mutant and the wild type were observed, indicating that the mutation specifically altered carbon partitioning while leaving the photosynthetic capacity unaffected. Conclusions The yield of TAG on light can be improved by 51% by using the slm1 starchless mutant of S. obliquus, and a similar improvement seems realistic for the areal productivity in outdoor cultivation. The photosynthetic performance is not negatively affected in the slm1 and the main difference with the wild type is an improved carbon partitioning towards TAG. PMID:24883102

  16. Proteomic profiling of cardiac tissue by isolation of nuclei tagged in specific cell types (INTACT).

    PubMed

    Amin, Nirav M; Greco, Todd M; Kuchenbrod, Lauren M; Rigney, Maggie M; Chung, Mei-I; Wallingford, John B; Cristea, Ileana M; Conlon, Frank L

    2014-02-01

    The proper dissection of the molecular mechanisms governing the specification and differentiation of specific cell types requires isolation of pure cell populations from heterogeneous tissues and whole organisms. Here, we describe a method for purification of nuclei from defined cell or tissue types in vertebrate embryos using INTACT (isolation of nuclei tagged in specific cell types). This method, previously developed in plants, flies and worms, utilizes in vivo tagging of the nuclear envelope with biotin and the subsequent affinity purification of the labeled nuclei. In this study we successfully purified nuclei of cardiac and skeletal muscle from Xenopus using this strategy. We went on to demonstrate the utility of this approach by coupling the INTACT approach with liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic methodologies to profile proteins expressed in the nuclei of developing hearts. From these studies we have identified the Xenopus orthologs of 12 human proteins encoded by genes, which when mutated in human lead to congenital heart disease. Thus, by combining these technologies we are able to identify tissue-specific proteins that are expressed and required for normal vertebrate organ development. PMID:24496632

  17. Preparation of silica coated cobalt ferrite magnetic nanoparticles for the purification of histidine-tagged proteins

    NASA Astrophysics Data System (ADS)

    Aygar, Gülfem; Kaya, Murat; Özkan, Necati; Kocabıyık, Semra; Volkan, Mürvet

    2015-12-01

    Surface modified cobalt ferrite (CoFe2O4) nanoparticles containing Ni-NTA affinity group were synthesized and used for the separation of histidine tag proteins from the complex matrices through the use of imidazole side chains of histidine molecules. Firstly, CoFe2O4 nanoparticles with a narrow size distribution were prepared in an aqueous solution using the controlled co-precipitation method. In order to obtain small CoFe2O4 agglomerates, oleic acid and sodium chloride were used as dispersants. The CoFe2O4 particles were coated with silica and subsequently the surface of these silica coated particles (SiO2-CoFe2O4) was modified by amine (NH2) groups in order to add further functional groups on the silica shell. Then, carboxyl (-COOH) functional groups were added to the SiO2-CoFe2O4 magnetic nanoparticles through the NH2 groups. After that Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (NTA) was attached to carboxyl ends of the structure. Finally, the surface modified nanoparticles were labeled with nickel (Ni) (II) ions. Furthermore, the modified SiO2-CoFe2O4 magnetic nanoparticles were utilized as a new system that allows purification of the N-terminal His-tagged recombinant small heat shock protein, Tpv-sHSP 14.3.

  18. Proteomic profiling of cardiac tissue by isolation of nuclei tagged in specific cell types (INTACT)

    PubMed Central

    Amin, Nirav M.; Greco, Todd M.; Kuchenbrod, Lauren M.; Rigney, Maggie M.; Chung, Mei-I; Wallingford, John B.; Cristea, Ileana M.; Conlon, Frank L.

    2014-01-01

    The proper dissection of the molecular mechanisms governing the specification and differentiation of specific cell types requires isolation of pure cell populations from heterogeneous tissues and whole organisms. Here, we describe a method for purification of nuclei from defined cell or tissue types in vertebrate embryos using INTACT (isolation of nuclei tagged in specific cell types). This method, previously developed in plants, flies and worms, utilizes in vivo tagging of the nuclear envelope with biotin and the subsequent affinity purification of the labeled nuclei. In this study we successfully purified nuclei of cardiac and skeletal muscle from Xenopus using this strategy. We went on to demonstrate the utility of this approach by coupling the INTACT approach with liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic methodologies to profile proteins expressed in the nuclei of developing hearts. From these studies we have identified the Xenopus orthologs of 12 human proteins encoded by genes, which when mutated in human lead to congenital heart disease. Thus, by combining these technologies we are able to identify tissue-specific proteins that are expressed and required for normal vertebrate organ development. PMID:24496632

  19. A Novel Method for High-Level Production of TEV Protease by Superfolder GFP Tag

    PubMed Central

    Wu, Xudong; Wu, Di; Lu, Zhisheng; Chen, Wentao; Hu, Xiaojian; Ding, Yu

    2009-01-01

    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

  20. A versatile drug delivery system using streptavidin-tagged pegylated liposomes and biotinylated biomaterials.

    PubMed

    Chen, Ming-Han; Soda, Yasushi; Izawa, Kiyoko; Kobayashi, Seiichiro; Tani, Kenzaburo; Maruyama, Kazuo; Tojo, Arinobu; Asano, Shigetaka

    2013-09-15

    Here we have developed a versatile liposome-mediated drug delivery system (DDS) allowing a strong bridge between the streptavidin-tagged liposome (SAL) and biotin (Bi)-tagged biomaterials which has strong affinity to surface proteins expressed in restricted cell lineages. This DDS was effective and specific for many leukemia cells in vitro and in vivo. When examining 6 human leukemia cell lines using calcein-encapsulated SALs in combination with Bi-granulocyte colony-stimulating factor (G-CSF), Bi-anti-CD33 monoclonal antibody (MAb) or Bi-anti-CD7 MAb, the fluorescent positive rate of each cell line was in almost proportion to degree of G-CSF receptor, CD33 or CD7 expression, respectively. More importantly, the binding ability was shown to be well maintained in a mouse xenograft model. Furthermore the cytosine arabinoside (AraC)-encapsulated SALs could kill the corresponding cells much more effectively in combination with Bi-biomaterials than free AraC, as expected. These findings strongly indicate that our SAL/Bi-biomaterial system could allow various types of medical agents to be delivered reliably and stably to the cells targeted. PMID:23806815

  1. Using an ?-bungarotoxin binding site tag to study GABA A receptor membrane localization and trafficking.

    PubMed

    Brady, Megan L; Moon, Charles E; Jacob, Tija C

    2014-01-01

    It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility(1-7). To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent ?-bungarotoxin with cells expressing constructs containing an ?-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the ? subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity(8,9). Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors(2,10). In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP(11)) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)(12). The BBS is 3' of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments. PMID:24747556

  2. Surface Plasmon Resonance Analysis of Histidine-Tagged F1-ATPase Surface Adsorption

    NASA Astrophysics Data System (ADS)

    Tucker, Jenifer K.; Richter, Mark L.; Berrie, Cindy L.

    2015-11-01

    Studies of the rotational activity of the enzymatic core (α3β3γ) of the F1-ATPase motor protein have relied on binding the enzyme to NTA-coated glass surfaces via polyhistidine tags engineered into the C-termini of each of the three α or β subunits. Those studies revealed the rotational motion of the central γ subunit by monitoring the motion of attached micron-long actin filaments or spherical nanoparticles. However, only a small percentage of the attached filaments or particles were observed to rotate, likely due, at least in part, to non-uniform surface attachment of the motor proteins. In this study, we have applied surface plasmon resonance to monitor the kinetics and affinity of binding of the His-tagged motor protein to NTA-coated gold sensor surfaces. The binding data, when fit to a heterogeneous binding model, exhibit two sets of adsorption-desorption rate constants with two dissociation constants of 4.0 × 10-9 M and 8.6 × 10-11 M for 6His-α3β3γ binding to the nickel ion-activated NTA surface. The data are consistent with mixed attachment of the protein via two (bimodal) and three (trimodal) NTA/Ni2+-His-tag interactions, respectively, with the less stable bimodal interaction dominating. The results provide a partial explanation for the low number of surface-attached F1 motors previously observed in rotation studies and suggest alternative approaches to uniform F1 motor surface attachment for future fabrication of motor-based nanobiodevices and materials.

  3. Rapid large-scale purification of myofilament proteins using a cleavable His6-tag.

    PubMed

    Zhang, Mengjie; Martin, Jody L; Kumar, Mohit; Khairallah, Ramzi J; de Tombe, Pieter P

    2015-11-01

    With the advent of high-throughput DNA sequencing, the number of identified cardiomyopathy-causing mutations has increased tremendously. As the majority of these mutations affect myofilament proteins, there is a need to understand their functional consequence on contraction. Permeabilized myofilament preparations coupled with protein exchange protocols are a common method for examining into contractile mechanics. However, producing large quantities of myofilament proteins can be time consuming and requires different approaches for each protein of interest. In the present study, we describe a unified automated method to produce troponin C, troponin T, and troponin I as well as myosin light chain 2 fused to a His6-tag followed by a tobacco etch virus (TEV) protease site. TEV protease has the advantage of a relaxed P1' cleavage site specificity, allowing for no residues left after proteolysis and preservation of the native sequence of the protein of interest. After expression in Esherichia coli, cells were lysed by sonication in imidazole-containing buffer. The His6-tagged protein was then purified using a HisTrap nickel metal affinity column, and the His6-tag was removed by His6-TEV protease digestion for 4 h at 30C. The protease was then removed using a HisTrap column, and complex assembly was performed via column-assisted sequential desalting. This mostly automated method allows for the purification of protein in 1 day and can be adapted to most soluble proteins. It has the advantage of greatly increasing yield while reducing the time and cost of purification. Therefore, production and purification of mutant proteins can be accelerated and functional data collected in a faster, less expensive manner. PMID:26386113

  4. A heterogeneous tag-attachment to the homodimeric type 1 photosynthetic reaction center core protein in the green sulfur bacterium Chlorobaculum tepidum.

    PubMed

    Azai, Chihiro; Kim, Kwang; Kondo, Toru; Harada, Jiro; Itoh, Shigeru; Oh-oka, Hirozo

    2011-07-01

    The 6xHis-tag-pscA gene, which was genetically engineered to express N-terminally histidine (His)-tagged PscA, was inserted into a coding region of the recA gene in the green sulfur bacterium Chlorobaculum tepidum (C. tepidum). Although the inactivation of the recA gene strongly suppressed a homologous recombination in C. tepidum genomic DNA, the mutant grew well under normal photosynthetic conditions. The His-tagged reaction center (RC) complex could be obtained simply by Ni(2+)-affinity chromatography after detergent solubilization of chlorosome-containing membranes. The complex consisted of three subunits, PscA, PscB, and PscC, in addition to the Fenna-Matthews-Olson protein, but there was no PscD. Low-temperature EPR spectroscopic studies in combination with transient absorption measurements indicated that the complex contained all intrinsic electron transfer cofactors as detected in the wild-type strain. Furthermore, the LC/MS/MS analysis revealed that the core protein consisted of a mixture of a His-/His-tagged PscA homodimer and a non-/His-tagged PscA heterodimer. The development of the pscA gene duplication method presented here, thus, enables not only a quick and large-scale preparation of the RC complex from C. tepidum but also site-directed mutagenesis experiments on the artificially incorporated 6xHis-tag-pscA gene itself, since the expression of the authentic PscA/PscA homodimeric RC complex could complement any defect in mutated His-tagged PscA. This method would provide an invaluable tool for structural and functional analyses of the homodimeric type 1 RC complex. PMID:21420930

  5. [Archival tags and geolocation methods for marine animals: A review].

    PubMed

    Zhang, Tian-feng; Fan, Wei; Dai, Yang

    2015-11-01

    Archival tags, a group of data storable electronic tags, are widely used as strong tools for obtaining long term and large scale activity information of marine animals, specifically highly migratory oceanic fishes, and corresponding environmental data. Though retrieving tags is an indispensable step for obtaining data, which is a shortage of archival tags, a series of achievements have been made on marine animals by using archival tags since the 1990s. With the appearance of pop-up satellite tag, which solved the problem of data retrieving and was fully independent of the fishing, both breadth and depth of marine animals' studies are extended by the end of the 1990s. Geolocation based on light intensity is the key to estimate marine animals' movement and has achieved some progress in the past 20 years. However, the accuracy of geolocation for latitude is not high enough, and there is still much room for improvement. To date, most geolocation methods that use ambient daylight involve identifying the times when the sun is at a precisely known zenith angle (e.g., sunrise and sunset). The problem of estimating longitude has been proved easy to solve, but accurate latitude estimates remain elusive. This paper mainly introduced two tags, i. e., archival tags and pop-up tags, and three geolocation methods, i.e. , 1) the "fixed reference" method, 2) the "variable reference" method, and 3) the "reflection" method. We also presented a prospect analysis on archival tags and possible research direction of geolocation methods. We believed that miniaturization and multi-sensor integration are the trends for electronic tags while more environmental factors such as depth, SST (sea surface temperature) or magnetic field intensity, instead of single factor, as auxiliary parameters would be used for improving the geolocation accuracy in the future. PMID:26915216

  6. Analysis of antibody clonotype by affinity immunoblotting.

    PubMed

    Kurien, Biji T; Scofield, R Hal

    2015-01-01

    A sensitive and specific method to analyze specific antibody clonotype changes in a lupus patient who developed autoantibodies to the Ro 60 autoantigen under observation is described. Patient sera, collected over several years, were separated by flatbed isoelectric focusing (IEF) and analyzed by affinity immunoblotting utilizing Ro 60-coated nitrocellulose membrane. When the Ro 60-coated nitrocellulose was laid over the surface of the IEF gel, the antibodies present on the surface of the acrylamide gel bound the Ro antigen on the nitrocellulose. Tween-20 was used to prevent nonspecific binding. The bound IgG clonotypes were detected using alkaline phosphatase conjugated anti-IgG. The patient's sera demonstrated an oligoclonal response to the Ro 60 autoantigen that increased in complexity and affinity over time. PMID:26043996

  7. Optimal Affine-Invariant Point Matching

    NASA Astrophysics Data System (ADS)

    Costa, Mauro S.; Haralick, Robert M.; Phillips, Tsaiyun I.; Shapiro, Linda G.

    1989-03-01

    The affine-transformation matching scheme proposed by Hummel and Wolfson (1988) is very efficient in a model-based matching system, not only in terms of the computational complexity involved, but also in terms of the simplicity of the method. This paper addresses the implementation of the affine-invariant point matching, applied to the problem of recognizing and determining the pose of sheet metal parts. It points out errors that can occur with this method due to quantization, stability, symmetry, and noise problems. By beginning with an explicit noise model which the Hummel and Wolfson technique lacks, we can derive an optimal approach which overcomes these problems. We show that results obtained with the new algorithm are clearly better than the results from the original method.

  8. Artificial Affinity Proteins as Ligands of Immunoglobulins

    PubMed Central

    Mouratou, Barbara; Bhar, Ghislaine; Pecorari, Frdric

    2015-01-01

    A number of natural proteins are known to have affinity and specificity for immunoglobulins. Some of them are widely used as reagents for detection or capture applications, such as Protein G and Protein A. However, these natural proteins have a defined spectrum of recognition that may not fit specific needs. With the development of combinatorial protein engineering and selection techniques, it has become possible to design artificial affinity proteins with the desired properties. These proteins, termed alternative scaffold proteins, are most often chosen for their stability, ease of engineering and cost-efficient recombinant production in bacteria. In this review, we focus on alternative scaffold proteins for which immunoglobulin binders have been identified and characterized. PMID:25647098

  9. Boronate affinity nanoparticles for nucleoside separation.

    PubMed

    Okutucu, Burcu; Vurmaz, Deniz; Tuncal, Akile; Trkcan, Ceren; Akta? Uygun, Deniz; Akgl, Sinan

    2016-02-01

    Boronate affinity systems have been recently used for the specific isolation of cis-diol group carrying biomolecules such as glycoproteins, nucleosides, carbohydrates. Nanosized materials have been extremely used for the biotechnological purposes due to their unique properties and their high surface areas. The objective of this presented work was to develop a new boronate affinity system for the nucleoside adsorption. For this purpose, poly(HEMA) nanoparticles were synthesized by using surfactant free emulsion polymerization technique and then functionalized with phenylboronic acid. Synthesized nanoparticles were characterized with FTIR, SEM, and Zeta size analysis. Nucleic acid adsorption experiments were repeated for different medium pH values, for various nucleosides concentrations, for different temperatures and ionic strengths, in order to determine the optimum adsorption conditions. In the light of these studies, it can be concluded that this boronate ligand carrying nanoparticles were very valuable for the separation of nucleosides. PMID:25137488

  10. Constituents and tissue affinities in herbal medicine.

    PubMed

    Tillotson, Alan

    2008-01-01

    Western-trained researchers and clinicians can better understand herbal medicine if they master at least a basic set of essential concepts used by herbal practitioners to describe how herbs work. Constituents, such as carotenoids and flavonoids, can all be used to develop a basis for imputed herb actions. Tissue affinity is also an important concept shared by all herb traditions, which can enhance clinical results and illuminate traditional herbal use. PMID:22432461

  11. Wave scattering from self-affine surfaces

    PubMed

    Simonsen; Vandembroucq; Roux

    2000-05-01

    Electromagnetic wave scattering from a perfectly reflecting self-affine surface is considered. Within the framework of the Kirchhoff approximation, we show that the scattering cross section can be exactly written as a function of the scattering angle via a centered symmetric Levy distribution for the general roughness amplitude, Hurst exponent and wavelength of the incident wave. Our prediction is supported by direct numerical simulations. PMID:11031653

  12. Reliable Food Traceability Using RFID Tagging

    NASA Astrophysics Data System (ADS)

    Azuara, Guillermo; Salazar, Jos L.; Tornos, Jos L.; Piles, Joan J.

    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.

  13. New tools for in vivo fluorescence tagging.

    PubMed

    Chapman, Sean; Oparka, Karl J; Roberts, Alison G

    2005-12-01

    Engineering of fluorescent proteins continues to produce new tools for in vivo studies. The current selection contains brighter, monomeric, spectral variants that will facilitate multiplex imaging and FRET, and a collection of optical highlighter proteins that might replace photoactivatable-GFP. These new highlighter proteins, which include proteins that have photoswitchable fluorescence characteristics and a protein whose fluorescence can be repeatedly turned on and off, should simplify refined analyses of protein dynamics and kinetics. Fluorescent protein-based systems have also been developed to allow facile detection of protein-protein interactions in planta. In addition, new tags in the form of peptides that bind fluorescent ligands and quantum dots offer the prospect of overcoming some of the limitations of fluorescent proteins such as excessive size and insufficient brightness. PMID:16188488

  14. QUANTITY: An Isobaric Tag for Quantitative Glycomics

    PubMed Central

    Yang, Shuang; Wang, Meiyao; Chen, Lijun; Yin, Bojiao; Song, Guoqiang; Turko, Illarion V.; Phinney, Karen W.; Betenbaugh, Michael J.; Zhang, Hui; Li, Shuwei

    2015-01-01

    Glycan is an important class of macromolecules that play numerous biological functions. Quantitative glycomics - analysis of glycans at global level - however, is far behind genomics and proteomics owing to technical challenges associated with their chemical properties and structural complexity. As a result, technologies that can facilitate global glycan analysis are highly sought after. Here, we present QUANTITY (Quaternary Amine Containing Isobaric Tag for Glycan), a quantitative approach that can not only enhance detection of glycans by mass spectrometry, but also allow high-throughput glycomic analysis from multiple biological samples. This robust tool enabled us to accomplish glycomic survey of bioengineered Chinese Hamster Ovary (CHO) cells with knock-in/out enzymes involved in protein glycosylation. Our results demonstrated QUANTITY is an invaluable technique for glycan analysis and bioengineering. PMID:26616285

  15. Biocompatible coatings with high albumin affinity.

    PubMed

    Tsai, C C; Huo, H H; Kulkarni, P; Eberhart, R C

    1990-01-01

    A process was developed to coat complex medical devices with a thin, transparent, biocompatible film. The film is based on silicone rubber (SR) but has higher albumin affinity than SR. Two polymer forms have been developed: one substitutes hydroxyl groups (OH), the other, 16 carbon acyl groups (C16) in the siloxane side chains. Oxymercuration/demercuration or hydroboration reactions can be used. SEM reveals film surfaces are smooth, uniform, and featureless. ATR/FTIR spectra and advancing/receding water contact angle measurements confirm the presence of surface OH groups and suggest the presence of surface acyl groups. Albumin adsorption and retention are markedly enhanced for surface OH and C16 concentrations as low as 5% reaction yield. Kinetics, isotherm, and competitive albumin/fibrinogen adsorption studies suggest that surface hydroxylation, and perhaps C16 acylation as well, markedly improve the albumin affinity, but not the fibrinogen affinity, of this material. The SR film can be durably coated on several materials, making it possible to favorably treat many blood-contacting devices, using a simple immersion process. PMID:2252685

  16. Calculation of protein-ligand binding affinities.

    PubMed

    Gilson, Michael K; Zhou, Huan-Xiang

    2007-01-01

    Accurate methods of computing the affinity of a small molecule with a protein are needed to speed the discovery of new medications and biological probes. This paper reviews physics-based models of binding, beginning with a summary of the changes in potential energy, solvation energy, and configurational entropy that influence affinity, and a theoretical overview to frame the discussion of specific computational approaches. Important advances are reported in modeling protein-ligand energetics, such as the incorporation of electronic polarization and the use of quantum mechanical methods. Recent calculations suggest that changes in configurational entropy strongly oppose binding and must be included if accurate affinities are to be obtained. The linear interaction energy (LIE) and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) methods are analyzed, as are free energy pathway methods, which show promise and may be ready for more extensive testing. Ultimately, major improvements in modeling accuracy will likely require advances on multiple fronts, as well as continued validation against experiment. PMID:17201676

  17. Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography.

    PubMed

    Thingholm, Tine E; Larsen, Martin R

    2016-01-01

    Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10-11, EDTA, or phosphate-containing buffers.Here we describe a protocol for IMAC using Fe(3+) for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture. PMID:26584922

  18. A MEMS Dielectric Affinity Glucose Biosensor

    PubMed Central

    Huang, Xian; Li, Siqi; Davis, Erin; Li, Dachao; Wang, Qian; Lin, Qiao

    2013-01-01

    Continuous glucose monitoring (CGM) sensors based on affinity detection are desirable for long-term and stable glucose management. However, most affinity sensors contain mechanical moving structures and complex design in sensor actuation and signal readout, limiting their reliability in subcutaneously implantable glucose detection. We have previously demonstrated a proof-of-concept dielectric glucose sensor that measured pre-mixed glucose-sensitive polymer solutions at various glucose concentrations. This sensor features simplicity in sensor design, and possesses high specificity and accuracy in glucose detection. However, lack of glucose diffusion passage, this device is unable to fulfill real-time in-vivo monitoring. As a major improvement to this device, we present in this paper a fully implantable MEMS dielectric affinity glucose biosensor that contains a perforated electrode embedded in a suspended diaphragm. This capacitive-based sensor contains no moving parts, and enables glucose diffusion and real-time monitoring. The experimental results indicate that this sensor can detect glucose solutions at physiological concentrations and possesses good reversibility and reliability. This sensor has a time constant to glucose concentration change at approximately 3 min, which is comparable to commercial systems. The sensor has potential applications in fully implantable CGM that require excellent long-term stability and reliability. PMID:24511215

  19. Versatile protein tagging in cells with split fluorescent protein.

    PubMed

    Kamiyama, Daichi; Sekine, Sayaka; Barsi-Rhyne, Benjamin; Hu, Jeffrey; Chen, Baohui; Gilbert, Luke A; Ishikawa, Hiroaki; Leonetti, Manuel D; Marshall, Wallace F; Weissman, Jonathan S; Huang, Bo

    2016-01-01

    In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications. PMID:26988139

  20. Virtual tagging for laxative-free CT colonography: Pilot evaluation

    PubMed Central

    Näppi, Janne; Yoshida, Hiroyuki

    2009-01-01

    Laxative-free computed tomographic colonography (lfCTC) could significantly improve patient adherence to colorectal screening. However, the interpretation of lfCTC data is complicated by the presence of poorly tagged feces and partial-volume artifacts that imitate colorectal lesions. The authors developed a method for virtual tagging of such artifacts. A probabilistic model of colonic wall was developed, and virtual tagging was performed on artifacts that were identified by the model. The method was evaluated with 46 clinical lfCTC cases that were prepared with dietary fecal tagging only. Visual examples show that the method can label partial-volume artifacts, poorly tagged feces, nonadhering completely untagged feces, and artifacts such as rectal tubes. The effect of virtual tagging was evaluated by comparing the detection accuracy of a fully automated polyp detection scheme without and with the method. With virtual tagging, the per-lesion detection sensitivity was 100% for lesions ⩾10 mm (n=4) with 3.8 false positives per patient (per two CT scan volumes) and 90% for lesions ⩾6 mm (n=10) with 5.4 false positives per patient on average. The improvement in detection performance by virtual tagging was statistically significant (p=0.03; JAFROC and JAFROC-1). PMID:19544802

  1. 9 CFR 381.99 - Official retention and rejection tags.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 2 2011-01-01 2011-01-01 false Official retention and rejection tags. 381.99 Section 381.99 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF... Certificates; Export Certificates; Certification Procedures 381.99 Official retention and rejection tags....

  2. MARC/AACR2/Authority Control Tagging. Blitz Cataloging Workbook.

    ERIC Educational Resources Information Center

    Ferguson, Bobby

    This workbook is designed to help librarians learn the correct way of applying cataloging tools, identifying errors in both original and copy cataloging, and maintaining proper authority control for more complete access. Chapter 1, "MARC Format," includes: introduction; families of tags; families of tags exercises; 008 (header) information,

  3. Tagging fast neutrons from an (241)Am/(9)Be source.

    PubMed

    Scherzinger, J; Annand, J R M; Davatz, G; Fissum, K G; Gendotti, U; Hall-Wilton, R; Håkansson, E; Jebali, R; Kanaki, K; Lundin, M; Nilsson, B; Rosborge, A; Svensson, H

    2015-04-01

    Shielding, coincidence, and time-of-flight measurement techniques are employed to tag fast neutrons emitted from an (241)Am/(9)Be source resulting in a continuous polychromatic energy-tagged beam of neutrons with energies up to 7MeV. The measured energy structure of the beam agrees qualitatively with both previous measurements and theoretical calculations. PMID:25644080

  4. 9 CFR 2.55 - Removal and disposal of tags.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Removal and disposal of tags. 2.55 Section 2.55 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.55 Removal and disposal of tags....

  5. 9 CFR 2.52 - How to obtain tags.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false How to obtain tags. 2.52 Section 2.52 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.52 How to obtain tags. Dealers or exhibitors may...

  6. 9 CFR 2.55 - Removal and disposal of tags.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Removal and disposal of tags. 2.55 Section 2.55 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.55 Removal and disposal of tags....

  7. 9 CFR 2.52 - How to obtain tags.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false How to obtain tags. 2.52 Section 2.52 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.52 How to obtain tags. Dealers or exhibitors may...

  8. 9 CFR 2.55 - Removal and disposal of tags.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Removal and disposal of tags. 2.55 Section 2.55 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.55 Removal and disposal of tags....

  9. 9 CFR 2.51 - Form of official tag.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Form of official tag. 2.51 Section 2.51 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.51 Form of official tag. (a) The official...

  10. 9 CFR 2.55 - Removal and disposal of tags.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Removal and disposal of tags. 2.55 Section 2.55 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.55 Removal and disposal of tags....

  11. 9 CFR 2.51 - Form of official tag.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Form of official tag. 2.51 Section 2.51 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.51 Form of official tag. (a) The official...

  12. 9 CFR 2.51 - Form of official tag.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Form of official tag. 2.51 Section 2.51 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.51 Form of official tag. (a) The official...

  13. 9 CFR 2.52 - How to obtain tags.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false How to obtain tags. 2.52 Section 2.52 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.52 How to obtain tags. Dealers or exhibitors may...

  14. 9 CFR 2.55 - Removal and disposal of tags.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Removal and disposal of tags. 2.55 Section 2.55 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.55 Removal and disposal of tags....

  15. 9 CFR 2.51 - Form of official tag.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Form of official tag. 2.51 Section 2.51 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.51 Form of official tag. (a) The official...

  16. 9 CFR 2.52 - How to obtain tags.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false How to obtain tags. 2.52 Section 2.52 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.52 How to obtain tags. Dealers or exhibitors may...

  17. 9 CFR 2.52 - How to obtain tags.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false How to obtain tags. 2.52 Section 2.52 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.52 How to obtain tags. Dealers or exhibitors may...

  18. 9 CFR 2.51 - Form of official tag.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Form of official tag. 2.51 Section 2.51 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.51 Form of official tag. (a) The official...

  19. Gas tagging and cover gas combination for nuclear reactor

    DOEpatents

    Gross, Kenny C.; Laug, Matthew T.

    1985-01-01

    The invention discloses the use of stable isotopes of neon and argon, that are grouped in preselected different ratios one to the other and are then sealed as tags in different cladded nuclear fuel elements to be used in a liquid metal fast breeder reactor. Failure of the cladding of any fuel element allows fission gases generated in the reaction and these tag isotopes to escape and to combine with the cover gas held in the reactor over the fuel elements. The isotopes specifically are Ne.sup.20, Ne.sup.21 and Ne.sup.22 of neon and Ar.sup.36, Ar.sup.38 and Ar.sup.40 of argon, and the cover gas is helium. Serially connected cryogenically operated charcoal beds are used to clean the cover gas and to separate out the tags. The first or cover gas cleanup bed is held between approximately 0.degree. and -25.degree. C. operable to remove the fission gases from the cover gas and tags and the second or tag recovery system bed is held between approximately -170.degree. and -185.degree. C. operable to isolate the tags from the cover gas. Spectrometric analysis further is used to identify the specific tags that are recovered, and thus the specific leaking fuel element. By cataloging the fuel element tags to the location of the fuel elements in the reactor, the location of the leaking fuel element can then be specifically determined.

  20. Improved gas tagging and cover gas combination for nuclear reactor

    DOEpatents

    Gross, K.C.; Laug, M.T.

    1983-09-26

    The invention discloses the use of stable isotopes of neon and argon, sealed as tags in different cladding nuclear fuel elements to be used in a liquid metal fast breeder reactor. Cladding failure allows fission gases and these tag isotopes to escape and to combine with the cover gas. The isotopes are Ne/sup 20/, Ne/sup 21/ and Ne/sup 22/ and Ar/sup 36/, Ar/sup 38/ and Ar/sup 40/, and the cover gas is He. Serially connected cryogenically operated charcoal beds are used to clean the cover gas and to separate out the tags. The first or cover gas cleanup bed is held between 0 and -25/sup 0/C to remove the fission gases from the cover gas and tags, and the second or tag recovery system bed between -170 and -185/sup 0/C to isolate the tags from the cover gas. Spectrometric analysis is used to identify the specific tags that are recovered, and thus the specific leaking fuel element. By cataloging the fuel element tags to the location of the fuel elements in the reactor, the location of the leaking fuel element can then be determined.

  1. 9 CFR 381.100 - Official detention tag.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 2 2011-01-01 2011-01-01 false Official detention tag. 381.100 Section 381.100 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE... Certificates; Export Certificates; Certification Procedures 381.100 Official detention tag. The detention...

  2. Apoferritin-Templated Synthesis of Encoded Metallic Phosphate Nanoparticle Tags

    SciTech Connect

    Liu, Guodong; Wu, Hong; Dohnalkova, Alice; Lin, Yuehe

    2007-07-31

    Encoded metallic-phosphate nanoparticle tags, with distinct encoding patterns, have been prepared using an apoferritin template. A center-cavity structure as well as the disassociation and reconstructive characteristics of apoferritin at different pH environments provide a facile route for preparing such encoded nanoparticle tags. Encapsulation and diffusion approaches have been investigated during the preparation. The encapsulation approach, which is based on the dissociation and reconstruction of apoferritin at different pHs, exhibits an effective route to prepare such encoded metallic-phosphate nanoparticle tags. The compositionally encoded nanoparticle tag leads to a high coding capacity with a large number of distinguishable voltammetric signals, reflecting the predetermined composition of the metal mixture solution (and hence the nanoparticle composition). Releasing the metal components from the nanoparticle tags at pH 4.6 acetate buffer avoids harsh dissolution conditions, such as strong acids. Such a synthesis of encoded nanoparticle tags, including single-component and compositionally encoded nanoparticle tags, is substantially simple, fast, and convenient compared to that of encoded metal nanowires and semiconductor nanoparticle (CdS, PbS, and ZnS) incorporated polystyrene beads. The encoded metallic-phosphate nanoparticle tags thus show great promise for bioanalytical or product-tracking/identification/protection applications.

  3. 29 CFR 1915.91 - Accident prevention signs and tags.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 29 Labor 7 2014-07-01 2014-07-01 false Accident prevention signs and tags. 1915.91 Section 1915.91... Working Conditions 1915.91 Accident prevention signs and tags. The requirements applicable to shipyard employment under this section are identical to the requirements set forth at 29 CFR 1910.145 of this chapter....

  4. 29 CFR 1915.91 - Accident prevention signs and tags.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 29 Labor 7 2013-07-01 2013-07-01 false Accident prevention signs and tags. 1915.91 Section 1915.91... Working Conditions 1915.91 Accident prevention signs and tags. The requirements applicable to shipyard employment under this section are identical to the requirements set forth at 29 CFR 1910.145 of this chapter....

  5. 29 CFR 1915.91 - Accident prevention signs and tags.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 29 Labor 7 2012-07-01 2012-07-01 false Accident prevention signs and tags. 1915.91 Section 1915.91... Working Conditions 1915.91 Accident prevention signs and tags. The requirements applicable to shipyard employment under this section are identical to the requirements set forth at 29 CFR 1910.145 of this chapter....

  6. Preparing Research Boats to Track Tagged Pallid Sturgeon

    USGS Multimedia Gallery

    Biologist, Dave Combs prepares a tracking boat (foreground) and a DIDSON survey boat (background) to search the Yellowstone River for tagged pallid sturgeon, Near Fairview, Montana.  Pallid sturgeon in Montana are tagged with radio telemetry transmitters that are detected with large antennas mo...

  7. Radio Tagged Adult Female Walrus on Ice Floe

    USGS Multimedia Gallery

    Adult female walrus on ice floe photographed shortly after receiving a behavior monitoring satellite-linked radio tag from USGS researchers.  Data acquired from such radio-tags are providing insights on the distribution and behavior of Pacific walruses during a time when their summer sea ice h...

  8. Fully printed flexible and disposable wireless cyclic voltammetry tag

    NASA Astrophysics Data System (ADS)

    Jung, Younsu; Park, Hyejin; Park, Jin-Ah; Noh, Jinsoo; Choi, Yunchang; Jung, Minhoon; Jung, Kyunghwan; Pyo, Myungho; Chen, Kevin; Javey, Ali; Cho, Gyoujin

    2015-01-01

    A disposable cyclic voltammetry (CV) tag is printed on a plastic film by integrating wireless power transmitter, polarized triangle wave generator, electrochemical cell and signage through a scalable gravure printing method. By proximity of 13.56 MHz RF reader, the printed CV tag generates 320 mHz of triangular sweep wave from +500 mV to -500 mV which enable to scan a printed electrochemical cell in the CV tag. By simply dropping any specimen solution on the electrochemical cell in the CV tag, the presence of solutes in the solution can be detected and shown on the signage of the CV tag in five sec. 10 mM of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was used as a standard solute to prove the working concept of fully printed disposable wireless CV tag. Within five seconds, we can wirelessly diagnose the presence of TMPD in the solution using the CV tag in the proximity of the 13.56 MHz RF reader. This fully printed and wirelessly operated flexible CV tag is the first of its kind and marks the path for the utilization of inexpensive and disposable wireless electrochemical sensor systems for initial diagnose hazardous chemicals and biological molecules to improve public hygiene and health.

  9. Acoustic competition in the gulf toadfish Opsanus beta: acoustic tagging.

    PubMed

    Thorson, Robert F; Fine, Michael L

    2002-05-01

    Nesting male gulf toadfish Opsanus beta produce a boatwhistle advertisement call used in male-male competition and to attract females and an agonistic grunt call. The grunt is a short-duration pulsatile call, and the boatwhistle is a complex call typically consisting of zero to three introductory grunts, a long tonal boop note, and zero to three shorter boops. The beginning of the boop note is also gruntlike. Anomalous boatwhistles contain a short-duration grunt embedded in the tonal portion of the boop or between an introductory grunt and the boop. Embedded grunts have sound-pressure levels and frequency spectra that correspond with those of recognized neighbors, suggesting that one fish is grunting during another's call, a phenomenon here termed acoustic tagging. Snaps of nearby pistol shrimp may also be tagged, and chains of tags involving more than two fish occur. The stimulus to tag is a relatively intense sound with a rapid rise time, and tags are generally produced within 100 ms of a trigger stimulus. Time between the trigger and the tag decreases with increased trigger amplitude. Tagging is distinct from increased calling in response to natural calls or stimulatory playbacks since calls rarely overlap other calls or playbacks. Tagging is not generally reciprocal between fish, suggesting parallels to dominance displays. PMID:12051450

  10. Acoustic competition in the gulf toadfish Opsanus beta: Acoustic tagging

    NASA Astrophysics Data System (ADS)

    Thorson, Robert F.; Fine, Michael L.

    2002-05-01

    Nesting male gulf toadfish Opsanus beta produce a boatwhistle advertisement call used in male-male competition and to attract females and an agonistic grunt call. The grunt is a short-duration pulsatile call, and the boatwhistle is a complex call typically consisting of zero to three introductory grunts, a long tonal boop note, and zero to three shorter boops. The beginning of the boop note is also gruntlike. Anomalous boatwhistles contain a short-duration grunt embedded in the tonal portion of the boop or between an introductory grunt and the boop. Embedded grunts have sound-pressure levels and frequency spectra that correspond with those of recognized neighbors, suggesting that one fish is grunting during another's call, a phenomenon here termed acoustic tagging. Snaps of nearby pistol shrimp may also be tagged, and chains of tags involving more than two fish occur. The stimulus to tag is a relatively intense sound with a rapid rise time, and tags are generally produced within 100 ms of a trigger stimulus. Time between the trigger and the tag decreases with increased trigger amplitude. Tagging is distinct from increased calling in response to natural calls or stimulatory playbacks since calls rarely overlap other calls or playbacks. Tagging is not generally reciprocal between fish, suggesting parallels to dominance displays.

  11. Fully printed flexible and disposable wireless cyclic voltammetry tag

    PubMed Central

    Jung, Younsu; Park, Hyejin; Park, Jin-Ah; Noh, Jinsoo; Choi, Yunchang; Jung, Minhoon; Jung, Kyunghwan; Pyo, Myungho; Chen, Kevin; Javey, Ali; Cho, Gyoujin

    2015-01-01

    A disposable cyclic voltammetry (CV) tag is printed on a plastic film by integrating wireless power transmitter, polarized triangle wave generator, electrochemical cell and signage through a scalable gravure printing method. By proximity of 13.56 MHz RF reader, the printed CV tag generates 320 mHz of triangular sweep wave from +500 mV to −500 mV which enable to scan a printed electrochemical cell in the CV tag. By simply dropping any specimen solution on the electrochemical cell in the CV tag, the presence of solutes in the solution can be detected and shown on the signage of the CV tag in five sec. 10 mM of N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) was used as a standard solute to prove the working concept of fully printed disposable wireless CV tag. Within five seconds, we can wirelessly diagnose the presence of TMPD in the solution using the CV tag in the proximity of the 13.56 MHz RF reader. This fully printed and wirelessly operated flexible CV tag is the first of its kind and marks the path for the utilization of inexpensive and disposable wireless electrochemical sensor systems for initial diagnose hazardous chemicals and biological molecules to improve public hygiene and health. PMID:25630250

  12. Versatile protein tagging in cells with split fluorescent protein

    PubMed Central

    Kamiyama, Daichi; Sekine, Sayaka; Barsi-Rhyne, Benjamin; Hu, Jeffrey; Chen, Baohui; Gilbert, Luke A.; Ishikawa, Hiroaki; Leonetti, Manuel D.; Marshall, Wallace F.; Weissman, Jonathan S.; Huang, Bo

    2016-01-01

    In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications. PMID:26988139

  13. TagGD: Fast and Accurate Software for DNA Tag Generation and Demultiplexing

    PubMed Central

    Costea, Paul Igor; Lundeberg, Joakim; Akan, Pelin

    2013-01-01

    Multiplexing is of vital importance for utilizing the full potential of next generation sequencing technologies. We here report TagGD (DNA-based Tag Generator and Demultiplexor), a fully-customisable, fast and accurate software package that can generate thousands of barcodes satisfying user-defined constraints and can guarantee full demultiplexing accuracy. The barcodes are designed to minimise their interference with the experiment. Insertion, deletion and substitution events are considered when designing and demultiplexing barcodes. 20,000 barcodes of length 18 were designed in 5 minutes and 2 million barcoded Illumina HiSeq-like reads generated with an error rate of 2% were demultiplexed with full accuracy in 5 minutes. We believe that our software meets a central demand in the current high-throughput biology and can be utilised in any field with ample sample abundance. The software is available on GitHub (https://github.com/pelinakan/UBD.git). PMID:23469199

  14. Localization of Free Field Realizations of Affine Lie Algebras

    NASA Astrophysics Data System (ADS)

    Futorny, Vyacheslav; Grantcharov, Dimitar; Martins, Renato A.

    2015-04-01

    We use localization technique to construct new families of irreducible modules of affine Kac-Moody algebras. In particular, localization is applied to the first free field realization of the affine Lie algebra or, equivalently, to imaginary Verma modules.

  15. Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

    PubMed Central

    Nilvebrant, Johan; strand, Mikael; Georgieva-Kotseva, Maria; Bjrnmalm, Mattias; Lfblom, John; Hober, Sophia

    2014-01-01

    The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein. PMID:25089830

  16. Passive UHF RFID Tag with Multiple Sensing Capabilities

    PubMed Central

    Fernández-Salmerón, José; Rivadeneyra, Almudena; Martínez-Martí, Fernando; Capitán-Vallvey, Luis Fermín; Palma, Alberto J.; Carvajal, Miguel A.

    2015-01-01

    This work presents the design, fabrication, and characterization of a printed radio frequency identification tag in the ultra-high frequency band with multiple sensing capabilities. This passive tag is directly screen printed on a cardboard box with the aim of monitoring the packaging conditions during the different stages of the supply chain. This tag includes a commercial force sensor and a printed opening detector. Hence, the force applied to the package can be measured as well as the opening of the box can be detected. The architecture presented is a passive single-chip RFID tag. An electronic switch has been implemented to be able to measure both sensor magnitudes in the same access without including a microcontroller or battery. Moreover, the chip used here integrates a temperature sensor and, therefore, this tag provides three different parameters in every reading. PMID:26506353

  17. Passive UHF RFID tag with multiple sensing capabilities.

    PubMed

    Fernández-Salmerón, José; Rivadeneyra, Almudena; Martínez-Martí, Fernando; Capitán-Vallvey, Luis Fermín; Palma, Alberto J; Carvajal, Miguel A

    2015-01-01

    This work presents the design, fabrication, and characterization of a printed radio frequency identification tag in the ultra-high frequency band with multiple sensing capabilities. This passive tag is directly screen printed on a cardboard box with the aim of monitoring the packaging conditions during the different stages of the supply chain. This tag includes a commercial force sensor and a printed opening detector. Hence, the force applied to the package can be measured as well as the opening of the box can be detected. The architecture presented is a passive single-chip RFID tag. An electronic switch has been implemented to be able to measure both sensor magnitudes in the same access without including a microcontroller or battery. Moreover, the chip used here integrates a temperature sensor and, therefore, this tag provides three different parameters in every reading. PMID:26506353

  18. CDF b-tagging: Measuring efficiency and false positive rate

    SciTech Connect

    Neu, Christopher; /Pennsylvania U.

    2006-06-01

    The CDF experiment has developed several high p{sub T} b-jet identification tools for the Run II physics program at the Tevatron. Herein we describe in detail one such b-tagging tool that exploits the long- lifetime of the b quark by identifying decay vertices significantly displaced from the primary interaction point. The b-tag efficiency is extracted from a b enriched data sample; the method is described, including a discussion of the important systematic effects. The data-driven measurement of the false positive tag rate is also described, as well as an explanation of how the per-jet false positive rate is used to predict the background contribution to the selected sample. Finally we conclude with a discussion of issues that have proven critical for b-tagging at CDF and should be given attention as we prepare b-tagging tools for LHC experiments.

  19. From the Cover: Semiotic dynamics and collaborative tagging

    NASA Astrophysics Data System (ADS)

    Cattuto, C.; Loreto, V.; Pietronero, L.

    2007-01-01

    Collaborative tagging has been quickly gaining ground because of its ability to recruit the activity of web users into effectively organizing and sharing vast amounts of information. Here we collect data from a popular system and investigate the statistical properties of tag co-occurrence. We introduce a stochastic model of user behavior embodying two main aspects of collaborative tagging: (i) a frequency-bias mechanism related to the idea that users are exposed to each other's tagging activity; (ii) a notion of memory - or aging of resources - in the form of a heavy-tailed access to the past state of the system. Remarkably, our simple modeling is able to account quantitatively for the observed experimental features, with a surprisingly high accuracy. This points in the direction of a universal behavior of users, who - despite the complexity of their own cognitive processes and the uncoordinated and selfish nature of their tagging activity - appear to follow simple activity patterns.

  20. Perfluoro(Methylcyclohexane) Tracer Tagging Test and Demonstration

    SciTech Connect

    Sigman, M.E.

    2000-09-26

    On February 14 and 15, 2000, a demonstration of current perfluorocarbon tagging technology and the future potential of these methods was held at Oak Ridge National Laboratory (ORNL). The demonstration consisted of a brief technical discussion followed by a laboratory demonstration. The laboratory demonstrations included the detection of letters, parcels, briefcases and lockers containing perfluorocarbon-tagged papers. Discrimination between tagged and non-tagged items and between three perfluorocarbon tags was demonstrated along with the detection of perfluorocarbon in a background of non-fluorinated volatile organic solvent. All demonstrations involved real-time detection using a direct sampling ion trap mass spectrometer. The technical results obtained at ORNL during and in preparation for the demonstration are presented in Appendix 1 to assist Tracer Detection Technology Corp. in further evaluating their position on development and marketing of perfluorocarbon tracer technology.