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1

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

Microsoft Academic Search

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

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

1999-01-01

2

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

Microsoft Academic Search

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

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

2001-01-01

3

Trypsin-catalyzed N-terminal labeling of peptides with stable isotope-coded affinity tags for proteome analysis.  

PubMed

An enzymatic approach to label peptide N-termini with isotope-coded affinity tags is presented. This method exploits the high activity of trypsin for peptide synthesis in organic solvents. A cosubstrate containing a stable isotope-coded Arg residue and a biotin tag was synthesized. When the cosubstrate was incubated with tryptic peptides and trypsin in ethanol solution, the stable isotope-coded affinity tag was specifically coupled onto the N-termini of peptides via the formation of new peptide bonds. The labeled peptides were specifically enriched by avidin affinity chromatography and then were submitted to liquid chromatography-tandem mass spectrometry (LC/MS/MS) for quantification. This enrichment step effectively reduced the interference by unlabeled peptides. The excellent performance of this approach was demonstrated by labeling standard peptides as well as a mouse liver digest. In addition to one amino acid residue, a few dipeptide tags were also introduced to the N-termini of peptides successfully by this enzymatic approach. It was found that the identifications for samples labeled with these tags were highly complementary. Coupling a short sequence tag onto peptides could be an effective approach to improve the coverage for proteome analysis. PMID:24354301

Pan, Yanbo; Ye, Mingliang; Zheng, Hao; Cheng, Kai; Sun, Zhen; Liu, Fangjie; Liu, Jing; Wang, Keyun; Qin, Hongqiang; Zou, Hanfa

2014-01-21

4

Thiol metabolomics of endothelial cells using capillary liquid chromatography mass spectrometry with isotope coded affinity tags.  

PubMed

Thiol and disulfide levels are critical to maintaining the redox potential of a cell. Perturbations of these levels are important in disease pathogenesis. To improve endogenous mammalian metabolome quantitation, thiol specific tagging, extraction and relative quantitation were undertaken. Reduced and oxidized thiol (disulfide) metabolites from endothelial cells were tagged and extracted using cleavable isotope coded affinity tags (cICAT). Extracted cICAT labeled thiols were analyzed using capillary reverse phase liquid chromatography coupled to mass spectrometry (capLC-MS) with positive mode electrospray ionization. Reactions between thiol metabolite standards and the reactive group of cICAT indicate completion by 8h at pH 9 with no apparent disulfide formation. cICAT labeled reduced thiols from endothelial cells showed 1-5% RSD using ratiometric quantitation of isotopes and 6-17% RSD based on signal intensity alone. Sample injection was optimized to 16 pmol. Using high mass accuracy MS, 75 putative thiol metabolites were detected in all experimental samples. Treatment of endothelial cells with 2,3-dimethoxy-5-methyl-1,4-benzoquinone (BQ) shows decreased levels in 28 putative reduced thiols and increased levels of 27 putative disulfides. Treatment of endothelial cells with 30 mM glucose resulted in 22 putative reduced thiols with decreased levels and 7 putative disulfides with increased concentration. Thiols were identified based on accurate mass within 3 ppm and analysis of fragmentation patterns. Using higher collision induced dissociation (HCD), shared product ions between different thiols led to the analysis of thiols from the cysteine-glutathione (Cys-GSH) pathway. Specific reduced thiols and disulfides in this pathway revealed changes different from the overall trends of thiols/disulfides. This suggests varying regulation of the Cys-GSH pathway distinct from other thiol-containing pathways and dependence on the type of environmental stimulus. These results indicate the utility of analyzing reduced thiols and disulfides in eukaryotic samples. PMID:21420094

Yuan, Wei; Edwards, James L

2011-05-01

5

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

PubMed Central

Shrimp subcuticular epithelial cells are the initial and major targets of white spot syndrome virus (WSSV) infection. Proteomic studies of WSSV-infected subcuticular epithelium of Penaeus monodon were performed through two approaches, namely, subcellular fractionation coupled with shotgun proteomics to identify viral and host proteins and a quantitative time course proteomic analysis using cleavable isotope-coded affinity tags (cICATs) to identify differentially expressed cellular proteins. Peptides were analyzed by offline coupling of two-dimensional liquid chromatography with matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry. We identified 27, 20, and 4 WSSV proteins from cytosolic, nuclear, and membrane fractions, respectively. Twenty-eight unique WSSV proteins with high confidence (total ion confidence interval percentage [CI%], >95%) were observed, 11 of which are reported here for the first time, and 3 of these novel proteins were shown to be viral nonstructural proteins by Western blotting analysis. A first shrimp protein data set containing 1,999 peptides (ion score, ?20) and 429 proteins (total ion score CI%, >95%) was constructed via shotgun proteomics. We also identified 10 down-regulated proteins and 2 up-regulated proteins from the shrimp epithelial lysate via cICAT analysis. This is the first comprehensive study of WSSV-infected epithelia by proteomics. The 11 novel viral proteins represent the latest addition to our knowledge of the WSSV proteome. Three proteomic data sets consisting of WSSV proteins, epithelial cellular proteins, and differentially expressed cellular proteins generated in the course of WSSV infection provide a new resource for further study of WSSV-shrimp interactions.

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

2007-01-01

6

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

SciTech Connect

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

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

2003-10-15

7

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

PubMed

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 synthesized in 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 headgroup zone of the lipid bilayer was labeled rapidly. Peptide for which the cysteine was positioned below the headgroup 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 analysis to the presence of lipids will enable future facile investigation of membrane proteins in a cellular context. PMID:23725486

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

2013-07-17

8

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

PubMed Central

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.

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

2013-01-01

9

Overview of affinity tags for protein purification.  

PubMed

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

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

2013-01-01

10

Comparison of affinity tags for protein purification  

Microsoft Academic Search

Affinity tags are highly efficient tools for purifying proteins from crude extracts. To facilitate the selection of affinity tags for purification projects, we have compared the efficiency of eight elutable affinity tags to purify proteins from Escherichia coli, yeast, Drosophila, and HeLa extracts. Our results show that the HIS, CBP, CYD (covalent yet dissociable NorpD peptide), Strep II, FLAG, HPC

Jordan J. Lichty; Joshua L. Malecki; Heather D. Agnew; Daniel J. Michelson-Horowitz; Song Tan

2005-01-01

11

Quantification of Tryptic Peptides in Quadrupole Ion Trap Using High-Mass Signals Derived from Isotope-Coded N-Acetyl Dipeptide Tags  

NASA Astrophysics Data System (ADS)

Isotope-labeled N-acetyl dipeptides (Ac-Xxx-Ala) are coupled to the primary amines of tryptic peptides and then analyzed by tandem mass spectrometry. Amide bond cleavage between Xxx and Ala provides both low- and high-mass isotope-coded signals for quantification of peptides. Especially, facile cleavage at the modified lysine side chain yields very strong high-mass quantitation signals in a noise-free region. Tagging tryptic peptides with isobaric N-acetyl dipeptides is a viable strategy for accurate quantification of proteins, which can be used with most quadrupole ion trap mass spectrometers carrying the 1/3 mass cut-off problem.

Seo, Jongcheol; Yoon, Hye-Joo; Shin, Seung Koo

2011-09-01

12

Quantification of tryptic peptides in quadrupole ion trap using high-mass signals derived from isotope-coded N-acetyl dipeptide tags.  

PubMed

Isotope-labeled N-acetyl dipeptides (Ac-Xxx-Ala) are coupled to the primary amines of tryptic peptides and then analyzed by tandem mass spectrometry. Amide bond cleavage between Xxx and Ala provides both low- and high-mass isotope-coded signals for quantification of peptides. Especially, facile cleavage at the modified lysine side chain yields very strong high-mass quantitation signals in a noise-free region. Tagging tryptic peptides with isobaric N-acetyl dipeptides is a viable strategy for accurate quantification of proteins, which can be used with most quadrupole ion trap mass spectrometers carrying the 1/3 mass cut-off problem. PMID:21953270

Seo, Jongcheol; Yoon, Hye-Joo; Shin, Seung Koo

2011-09-01

13

Several Affinity Tags Commonly Used in Chromatographic Purification  

PubMed Central

Affinity tags have become powerful tools from basic biological research to structural and functional proteomics. They were widely used to facilitate the purification and detection of proteins of interest, as well as the separation of protein complexes. Here, we mainly discuss the benefits and drawbacks of several affinity or epitope tags frequently used, including hexahistidine tag, FLAG tag, Strep II tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tag, HA tag, and c-Myc tag. In some cases, a large-size affinity tag, such as GST or MBP, can significantly impact on the structure and biological activity of the fusion partner protein. So it is usually necessary to excise the tag by protease. The most commonly used endopeptidases are enterokinase, factor Xa, thrombin, tobacco etch virus, and human rhinovirus 3C protease. The proteolysis features of these proteases are described in order to provide a general guidance on the proteolytic removal of the affinity tags.

Zhao, Xinyu; Liang, Shufang

2013-01-01

14

The Dac-tag, an affinity tag based on penicillin-binding protein 5.  

PubMed

Penicillin-binding protein 5 (PBP5), a product of the Escherichia coli gene dacA, possesses some ?-lactamase activity. On binding to penicillin or related antibiotics via an ester bond, it deacylates and destroys them functionally by opening the ?-lactam ring. This process takes several minutes. We exploited this process and showed that a fragment of PBP5 can be used as a reversible and monomeric affinity tag. At ambient temperature (e.g., 22°C), a PBP5 fragment binds rapidly and specifically to ampicillin Sepharose. Release can be facilitated either by eluting with 10mM ampicillin or in a ligand-free manner by incubation in the cold (1-10°C) in the presence of 5% glycerol. The "Dac-tag", named with reference to the gene dacA, allows the isolation of remarkably pure fusion protein from a wide variety of expression systems, including (in particular) eukaryotic expression systems. PMID:22705378

Lee, David Wei; Peggie, Mark; Deak, Maria; Toth, Rachel; Gage, Zoe Olivia; Wood, Nicola; Schilde, Christina; Kurz, Thimo; Knebel, Axel

2012-09-01

15

A tailor-made "tag-receptor" affinity pair for the purification of fusion proteins.  

PubMed

A novel affinity "tag-receptor" pair was developed as a generic platform for the purification of fusion proteins. The hexapeptide RKRKRK was selected as the affinity tag and fused to green fluorescent protein (GFP). The DNA fragments were designed, cloned in Pet-21c expression vector and expressed in E. coli host as soluble protein. A solid-phase combinatorial library based on the Ugi reaction was synthesized: 64 affinity ligands displaying complementary functionalities towards the designed tag. The library was screened by affinity chromatography in a 96-well format for binding to the RKRKRK-tagged GFP protein. Lead ligand A7C1 was selected for the purification of RKRKRK fusion proteins. The affinity pair RKRKRK-tagged GFP with A7C1 emerged as a promising solution (Ka of 2.45×10(5) ?M(-1) ). The specificity of the ligand towards the tag was observed experimentally and theoretically through automated docking and molecular dynamics simulations. PMID:24903894

Pina, Ana S; Guilherme, Márcia; Pereira, Alice S; Fernandes, Cláudia S F M; Branco, Ricardo J F; El Khoury, Graziella; Lowe, Christopher R; Roque, A Cecília A

2014-07-01

16

Purification of a recombinant protein with cellulose-binding module 3 as the affinity tag.  

PubMed

Easy-to-perform and low-cost protein purification methods are in high demand for the mass production of commonly used enzymes that play an important role in bioeconomy. A low-cost and rapid recombinant protein purification system was developed using CBM3 (family 3 cellulose-binding module) as affinity tag. This protocol describes the purification of CBM3-fusion protein and tag-free protein expressed in Pichia pastoris using CBM3 as an affinity tag. PMID:24943312

Wang, Dongmei; Hong, Jiong

2014-01-01

17

Comparative analysis of cleavable azobenzene-based affinity tags for bioorthogonal chemical proteomics.  

PubMed

The advances in bioorthogonal ligation methods have provided new opportunities for proteomic analysis of newly synthesized proteins, posttranslational modifications, and specific enzyme families using azide/alkyne-functionalized chemical reporters and activity-based probes. Efficient enrichment and elution of azide/alkyne-labeled proteins with selectively cleavable affinity tags are essential for protein identification and quantification applications. Here, we report the synthesis and comparative analysis of Na?S?O?-cleavable azobenzene-based affinity tags for bioorthogonal chemical proteomics. We demonstrated that ortho-hydroxyl substituent is required for efficient azobenzene-bond cleavage and show that these cleavable affinity tags can be used to identify newly synthesized proteins in bacteria targeted by amino acid chemical reporters as well as their sites of modification on endogenously expressed proteins. The azobenzene-based affinity tags are compatible with in-gel, in-solution, and on-bead enrichment strategies and should afford useful tools for diverse bioorthogonal proteomic applications. PMID:21095571

Yang, Yu-Ying; Grammel, Markus; Raghavan, Anuradha S; Charron, Guillaume; Hang, Howard C

2010-11-24

18

Molecular Interaction Between the Strep-tag Affinity Peptide and its Cognate Target, Streptavidin  

Microsoft Academic Search

TheStrep-tag is a selected nine-amino acid peptide (AWRHPQFGG) that displays intrinsic binding affinity towards streptavidin and has been used as an affinity tag for recombinant proteins. In order to elucidate the molecular mechanism underlying this type of artificial protein-peptide recognition, X-ray crystallographic analyses and binding measurements were carried out. The crystal structure of the complex between recombinant core streptavidin and

Thomas G. M. Schmidt; Jürgen Koepke; Ronald Frank; Arne Skerra

1996-01-01

19

DIFFERENTIAL EFFECTS OF SHORT AFFINITY TAGS ON THE CRYSTALLIZATION OF PYROCOCCUS FURIOSUS MALTODEXTRIN-BINDING PROTEIN  

Microsoft Academic Search

Pyrococcus furiosus maltodextrin-binding protein readily forms large orthorhombic crystals that diffract to high resolution. This protein was used as a model system to investigate the influence of five short affinity tags (His6, Arg5, Strep tag II, FLAG tag and the biotin acceptor peptide) on the formation of protein crystals and their ability to diffract X-rays. The results indicate that the

Matthew H. Bucher; A. EVDOKIMOV; David S. Waugh

2002-01-01

20

Histidine affinity tags affect MSP1(42) structural stability and immunodominance in mice.  

PubMed

Inclusion of affinity tags has greatly facilitated process development for protein antigens, primarily for their recovery from complex mixtures. Although generally viewed as supportive of product development, affinity tags may have unintended consequences on protein solubility, susceptibility to aggregation, and immunogenicity. Merozoite surface protein 1 (MSP1), an erythrocytic stage protein of Plasmodium falciparum and a candidate malaria vaccine, was used to evaluate the impact of a metal ion affinity-tag on both protein structure and the induction of immunity. To this end, codon harmonized gene sequences from the P. falciparum MSP1(42) of FVO and 3D7 parasites were cloned and purified with and without a histidine (His) tag. We report on the influence of His-affinity tags on protein expression levels, solubility, secondary structure, thermal denaturation, aggregation and the impact on humoral and cellular immune responses in mice. While the overall immunogenicity induced by His-tagged MSP1(42) proteins is greater, the fine specificity of the humoral and cellular immune responses is altered relative to anti-parasitic antibody activity and the breadth of T-cell responses. Thus, the usefulness of protein tags may be outweighed by their potential impact on structure and function, stressing the need for caution in their use. See accompanying commentary by Randolph DOI: 10.1002/biot.201100459. PMID:22076863

Khan, Farhat; Legler, Patricia M; Mease, Ryan M; Duncan, Elizabeth H; Bergmann-Leitner, Elke S; Angov, Evelina

2012-01-01

21

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

PubMed Central

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

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

2009-01-01

22

Copurification of the Lac Repressor with Polyhistidine-Tagged Proteins in Immobilized Metal Affinity Chromatography  

Microsoft Academic Search

One of the commonly used resins for immobilized metal affinity purification of polyhistidine-tagged recombinant proteins is TALON resin, a cobalt (II)-carboxymethylaspartate-based matrix linked to Sepharose CL-6B. Here, we show that TALON resin efficiently purifies the native form of Lac repressor, which represents the major contaminant when (His)6- tagged proteins are isolated from Escherichia coli host cells carrying the lacIq gene.

Róis??n M. Owens; Andrew Grant; Nicholas Davies; C. David O'Connor

2001-01-01

23

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

NASA Astrophysics Data System (ADS)

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.

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

24

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

SciTech Connect

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

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

2009-01-01

25

Immobilized metal affinity chromatography of histidine-tagged lentiviral vectors using monolithic adsorbents  

Microsoft Academic Search

Histidine-tagged lentiviral vectors were separated from crude cell culture supernatant using labscale monolithic adsorbents by immobilized metal affinity chromatography. The capture capacity, concentration factor, purification factor, and elution efficiency of a supermacroporous cryogel monolith were evaluated against the BIA Separations convective interaction media (CIM) disc, which is a commercial macroporous monolith. The morphology of the polymeric cryogel material was characterised

M. C. Cheeks; N. Kamal; A. Sorrell; D. Darling; F. Farzaneh; N. K. H. Slater

2009-01-01

26

Proteomic analysis of opsins and thyroid hormone-induced retinal development using isotope-coded affinity tags (ICAT) and mass spectrometry  

Microsoft Academic Search

Purpose: Analyses that reveal the relative abundance of proteins are informative in elucidating mechanisms of retinal development and disease progression. However, popular high-throughput proteomic methods do not reliably detect opsin protein abundance, which serve as markers of photoreceptor differentiation. We utilized thyroid-hormone (TH) treatment of rainbow trout (Oncorhynchus mykiss) as a model of cone apoptosis and cone regeneration. We used

W. Ted Allison; Kathy M. Veldhoen; Craig W. Hawryshyn

2006-01-01

27

Preparation of immobilized enzyme with high activity using affinity tag based on proteins A and G.  

PubMed

Affinity tag AG consisting of immunoglobulin G (lgG)-binding domains of protein A from Staphylococcus aureus (EDABC) and those of protein G from Streptococcus strain G148 (C2C3) were used to facilitate immobilization of beta-galactosidase (betagal) from Escherichia coli. Poly(methylmethacrylate/N-isopropylacrylamide/methacrylic acid) [P(MMA/NIPAM/MAA)] and poly(styrene/N-isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] latex particles, which show thermosensitivity, were used as support materals to prepare affinity adsorbents. Human gamma-globulin (HgammaGb), whose major fraction is lgG, was used as an affinity ligand and was covalently immobilized onto the both latex particles by the carbodiimide method under various conditions. A fusion protein, AGbetagal, was immobilized at pH 7.3 by the specific binding of affinity tag to these affinity adsorbents. The amount of adsorbed AGbetagal per unit amount of immobilized HgammaGb, namely, efficiency of ligand utilization, was strongly affected by the type of latex particles and pH value for HgammaGb immobilization. The efficiency of ligand utilization was maximum in the affinity adsorbents prepared at pH 6.0 to 7.0, and that in the HgammaGb-P(MMA/NIPAM/MAA) latex particles was high. This result could be explained by the conformation and orientation of immobilized HgammaGb molecules. Immobilized AGbetagal retained approximately 75% of its activity in solution and the binding is stable enough to allow repeated use. These results clearly demonstrate that combination of the affinity tag AG and the affinity adsorbents, based on the thermosensitive latex particles, offers a simple and widely applicable method for preparation of immobilized enzyme with high activity. (c) 1995 John Wiley & Sons, Inc. PMID:18623333

Kondo, A; Teshima, T

1995-06-01

28

Generation of Affinity-Tagged Fluoromycobacteriophages by Mixed Assembly of Phage Capsids  

PubMed Central

Addition of affinity tags to bacteriophage particles facilitates a variety of applications, including vaccine construction and diagnosis of bacterial infections. Addition of tags to phage capsids is desirable, as modification of the tails can lead to poor adsorption and loss of infectivity. Although tags can readily be included as fusions to head decoration proteins, many phages do not have decoration proteins as virion components. The addition of a small (10-amino-acid) Strep-tag II (STAG II) to the mycobacteriophage TM4 capsid subunit, gp9, was not tolerated as a genetically homogenous recombinant phage but could be incorporated into the head by growth of wild-type phage on a host expressing the capsid-STAG fusion. Particles with capsids composed of wild-type and STAG-tagged subunit mixtures could be grown to high titers, showed good infectivities, and could be used to isolate phage-bacterium complexes. Preparation of a STAG-labeled fluoromycobacteriophage enabled capture of bacterial complexes and identification of infected bacteria by fluorescence.

Piuri, Mariana; Rondon, Liliana; Urdaniz, Estefania

2013-01-01

29

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

Microsoft Academic Search

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

Thomas GM Schmidt; Arne Skerra

2007-01-01

30

Affinity purification of recombinant proteins using a novel silica-binding peptide as a fusion tag.  

PubMed

We recently reported that silica is deposited on the coat of Bacillus cereus spores as a layer of nanometer-sized particles (Hirota et al. 2010 J Bacteriol 192: 111-116). Gene disruption analysis revealed that the spore coat protein CotB1 mediates the accumulation of silica (our unpublished results). Here, we report that B. cereus CotB1 (171 amino acids [aa]) and its C-terminal 14-aa region (corresponding to residues 158-171, designated CotB1p) show strong affinity for silica particles, with dissociation constants at pH 8.0 of 2.09 and 1.24 nM, respectively. Using CotB1 and CotB1p as silica-binding tags, we developed a silica-based affinity purification method in which silica particles are used as an adsorbent for CotB1/CotB1p fusion proteins. Small ubiquitin-like modifier (SUMO) technology was employed to release the target proteins from the adsorbed fusion proteins. SUMO-protease-mediated site-specific cleavage at the C-terminus of the fused SUMO sequence released the tagless target proteins into the liquid phase while leaving the tag region still bound to the solid phase. Using the fluorescent protein mCherry as a model, our purification method achieved 85 % recovery, with a purity of 95 % and yields of 0.60?±?0.06 and 1.13?±?0.13 mg per 10-mL bacterial culture for the CotB1-SUMO-mCherry and CotB1p-SUMO-mCherry fusions, respectively. CotB1p, a short 14-aa peptide, which demonstrates high affinity for silica, could be a promising fusion tag for both affinity purification and enzyme immobilization on silica supports. PMID:24756322

Abdelhamid, Mohamed A A; Motomura, Kei; Ikeda, Takeshi; Ishida, Takenori; Hirota, Ryuichi; Kuroda, Akio

2014-06-01

31

A novel gigaporous GSH affinity medium for high-speed affinity chromatography of GST-tagged proteins.  

PubMed

Novel GSH-AP (phenoxyl agarose coated gigaporous polystyrene, Agap-co-PSt) microspheres were successfully prepared by introducing GSH ligand into hydrophilic AP microspheres pre-activated with 1,4-butanediol diglycidyl ether. The gigaporous structure and chromatographic properties of GSH-AP medium were evaluated and compared with commercial GSH Sepharose FF (GSH-FF) medium. The macropores (100-500nm) of gigaporous PSt microspheres were well maintained after coating with agarose and functionalized with GSH ligand. Hydrodynamic experiments showed that GSH-AP column had less backpressure and plate height than those of GSH-FF column at high flow velocity, which was beneficial for its use in high-speed chromatography. The presence of flow-through pores in GSH-AP microspheres also accelerated the mass transfer rate of biomolecules induced by convective flow, leading to high protein resolution and high dynamic binding capacity (DBC) of glutathione S-transferase (GST) at high flow velocity. High purity of GST and GST-tagged recombinant human interleukin-1 receptor antagonist (rhIL-1RA) were obtained from crude extract with an acceptable recovery yield within 1.5min at a velocity up to 1400cm/h. GSH-AP medium is promising for high-speed affinity chromatography for the purification of GST and GST-tagged proteins. PMID:24269760

Huang, Yongdong; Zhang, Rongyue; Li, Juan; Li, Qiang; Su, Zhiguo; Ma, Guanghui

2014-03-01

32

An automated method for high-throughput protein purification applied to a comparison of His-tag and GST-tag affinity chromatography  

PubMed Central

Background Functional Genomics, the systematic characterisation of the functions of an organism's genes, includes the study of the gene products, the proteins. Such studies require methods to express and purify these proteins in a parallel, time and cost effective manner. Results We developed a method for parallel expression and purification of recombinant proteins with a hexahistidine tag (His-tag) or glutathione S-transferase (GST)-tag from bacterial expression systems. Proteins are expressed in 96-well microplates and are purified by a fully automated procedure on a pipetting robot. Up to 90 microgram purified protein can be obtained from 1 ml microplate cultures. The procedure is readily reproducible and 96 proteins can be purified in approximately three hours. It avoids clearing of crude cellular lysates and the use of magnetic affinity beads and is therefore less expensive than comparable commercial systems. We have used this method to compare purification of a set of human proteins via His-tag or GST-tag. Proteins were expressed as fusions to an N-terminal tandem His- and GST-tag and were purified by metal chelating or glutathione affinity chromatography. The purity of the obtained protein samples was similar, yet His-tag purification resulted in higher yields for some proteins. Conclusion A fully automated, robust and cost effective method was developed for the purification of proteins that can be used to quickly characterise expression clones in high throughput and to produce large numbers of proteins for functional studies. His-tag affinity purification was found to be more efficient than purification via GST-tag for some proteins.

Scheich, Christoph; Sievert, Volker; Bussow, Konrad

2003-01-01

33

The esterase from Alicyclobacillus acidocaldarius as a reporter enzyme and affinity tag for protein biosynthesis.  

PubMed

Esterase from thermophilic bacteria Alicyclobacillus acidocaldarius can be produced up to 200 microg/ml by coupled in vitro transcription/translation system derived from Escherichia coli. The synthesized thermostable enzyme can be determined by photometrical and fluorescent assays at least up to 10(-8) M concentration or by activity staining in the polyacrylamide gels. Enhanced green fluorescence protein-esterase fusion protein was bound to a matrix with immobilized esterase inhibitor and purified by affinity chromatography. Thus, the esterase is suited as a reporter enzyme to monitor the expression of polypeptides coupled to its N-terminus and simultaneously, as a cleavable tag for polypeptide purification. PMID:15811322

Agafonov, Dmitry E; Rabe, Kersten S; Grote, Michael; Huang, Yiwei; Sprinzl, Mathias

2005-04-11

34

Measuring binding constants of His-tagged proteins using affinity chromatography and Ni-NTA-immobilized enzymes.  

PubMed

Affinity chromatography is one way to measure the binding constants of a protein-ligand interaction. Here we describe a method of measuring a binding constant using Ni-NTA resin to immobilize a His-tagged enzyme and the method of frontal analysis. While other methods of immobilization are possible, using the strong affinity interaction between His-tagged proteins and Ni-NTA supports results in a fast, easy, and gentle method of immobilization. Once the affinity support is created, frontal analysis can be used to measure the binding constant between the protein and various analytes. PMID:24648091

Moser, Annette C; White, Benjamin; Kovacs, Frank A

2014-01-01

35

A Bacillus megaterium Plasmid System for the Production, Export, and One-Step Purification of Affinity-Tagged Heterologous Levansucrase from Growth Medium  

Microsoft Academic Search

A multiple vector system for the production and export of recombinant affinity-tagged proteins in Bacillus megaterium was developed. Up to 1 mg\\/liter of a His6-tagged or Strep-tagged Lactobacillus reuteri levansucrase was directed into the growth medium, using the B. megaterium esterase LipA signal peptide, and recovered by one-step affinity chromatography.

Marco Malten; Rebekka Biedendieck; Martin Gamer; Ann-Christin Drews; Simon Stammen; Klaus Buchholz; Lubbert Dijkhuizen; Dieter Jahn

2006-01-01

36

Optimizing the bioavailability of small molecular optical imaging probes by conjugation to an albumin affinity tag.  

PubMed

Small molecular imaging probes are often found to be rapidly cleared from the circulation. In order to improve signal to noise ratio (SNR) by high probe accumulation in the target tissue we intended to prolong the presence of the probes in the circulation by exploiting inherent transport mechanisms. Human serum albumin (HSA) is playing an increasingly important role as a drug carrier in clinical settings and drugs directly bound to albumin or attached to albumin binding moieties have been successfully developed for treatment approaches. To optimize the bioavailability of existing fluorescent probes, a hydrophobic affinity tag is installed, which enhances albumin binding. In a first experiment an endothelin-A receptor (ETAR) probe is modified by inserting a trivalent linker, attaching an albumin affinity tag and labeling the conjugate with the fluorescent dye Cy 5.5. The spectroscopic properties of the conjugate are examined by photometer- and fluorometer measurements in comparison to a probe without albumin binding tag. Albumin binding was proven by agarose gel electrophoresis. The affinity towards ETAR was confirmed in vitro by cell binding assays on human fibrosarcoma cells (HT-1080) and in vivo by murine xenograft imaging studies. In vitro, the modified probe retains high target binding in the absence and presence of albumin. Binding could be blocked by predosing with ETAR antagonist atrasentan, proving specificity. The in vivo examinations in comparison to the established probe showed a reduced renal elimination and a prolonged circulation of the tracer resulting in significantly higher signal intensity (SI) at the target and a higher signal-to-noise ratio (SNR) between 3h and 96h after injection. In summary, we designed a small molecular, non-peptidic fluorescent probe which targets ETAR and reversibly binds to serum albumins. The reversible binding to albumin enhances the biological half-life of the probe substantially and enables near infrared optical imaging of subcutaneous tumors for several days. This approach of reversibly attaching probes to serum albumin may serve as a tool to optimize tracer distribution for more precise target characterization in molecular imaging experiments. PMID:24815420

Hahnenkamp, Anke; Alsibai, Wael; Bremer, Christoph; Höltke, Carsten

2014-07-28

37

Cancer cell sensing and therapy using affinity tag-conjugated gold nanorods  

PubMed Central

Through the developments in controlling the shape of gold nanoparticles, synthesis of gold nanorods (AuNRs) can be considered as a milestone discovery in the area of nanomaterial-based cancer treatments. Besides having tuneable absorption maxima at near infrared (NIR) range, AuNRs have superior absorption cross section at NIR frequencies compared with other gold nanoparticles. When this unique optical property is combined with the specificity against cancer cells used by affinity tag conjugations, AuNRs become one of the most important nanoparticles used in both cancer cell sensing and in therapy. In this review, the impact of size and shape control of nanoparticles, especially AuNRs, on cancer cell treatments and a range of aptamer-conjugated AuNR applications in this regard are reviewed.

Yasun, Emir; Kang, Huaizhi; Erdal, Huseyin; Cansiz, Sena; Ocsoy, Ismail; Huang, Yu-Fen; Tan, Weihong

2013-01-01

38

Effect of number of poly(His) tags on the adsorption of engineered proteins on immobilized metal affinity chromatography adsorbents  

Microsoft Academic Search

The equilibrium adsorption of four homo-oligomeric recombinant proteins containing up to eight poly(histidine) affinity tags on a silica-based Cu(II)-IDA immobilized metal affinity chromatography (IMAC) adsorbent is reported in this study. The equilibrium adsorption data are well fitted with the three-parameter Langmuir–Freundlich isotherm model, indicating the presence of positive cooperativity for the adsorption of these model proteins and the degree of

Sung-Yuan Tsai; Sung-Chyr Lin; Shing-Yi Suen; Wen-Hwei Hsu

2006-01-01

39

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

PubMed Central

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

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

2009-01-01

40

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

SciTech Connect

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.

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

2009-01-01

41

Vectors for expressing T7 epitope- and His6 affinity-tagged fusion proteins in S. cerevisiae.  

PubMed

We have constructed a series of vectors (YGALSETs) for the expression of epitope- and affinity-tagged fusion proteins in yeast cells using the regulated GAL10 promoter. Fusion proteins produced from YGALSET plasmids include a leader peptide at the N terminus that encodes both a T7 gene 10 epitope tag and a His6 affinity tag. The YGALSET vector series includes centromere plasmids for low-copy plasmid maintenance and 2 micron episomal plasmids for multicopy plasmid maintenance and four different selectable markers: TRP1, URA3, LEU2 and HIS3. We also provide a convenient approach for transferring cloned genes from a bacterial expression vector into YGALSET vectors by in vivo recombination and a rapid method to screen directly for clones that express the fusion protein of interest. PMID:9591127

Enomoto, S; Chen, G; Berman, J

1998-05-01

42

Rapid single-tube method for small-scale affinity purification of polyclonal antibodies using HaloTag Technology.  

PubMed

Even in this era of advanced biotechniques, specific antibodies against a protein still prove to be powerful tools to study proteins and their functions. The polyclonal antisera obtained from the immunized rabbits, however, are not always pure, high affinity, antigen-specific polyclonal antibodies. With our new rapid HaloTag-based procedure, specific antibodies are obtained in just two, short steps: (1) simultaneous purification and covalent coupling of the antigen to Sepharose resin via the HaloTag and HaloLink reaction, and (2) affinity column purification of the polyclonal serum (10 microl). The combined antigen purification and coupling step requires only 1 h of room-temperature incubation, plus successive washing steps. Because different regions of an antigen can elicit the production of low affinity antibodies with relatively high cross-reactivity, the best way to produce high affinity antibodies against a protein of interest is to survey all antigenic determinants of that protein and identify the epitopes that result in the production of antibodies with a high affinity and specificity for that protein. Because our HaloTag procedure is quite rapid and simple, potential epitopes can be assessed with relatively little effort for their ability to elicit the production of highly specific antibodies. PMID:17331584

Hata, Toshiyuki; Nakayama, Manabu

2007-06-10

43

The utility of affinity-tags for detection of a streptococcal protein from a variety of streptococcal species  

PubMed Central

There is no systematic examination of affinity tag utility in Gram-positive bacteria, which limits the investigation of protein function in this important group of bacteria as specific antibodies for many of native proteins are generally not available. In this study, we utilized an E. coli-streptococcal shuttle vector pVT1666 and constructed two sets of expression plasmids pVPT-CTag and pVPT-NTag, with each set containing five affinity tags (GST, GFP, HSV, T7 and Nano) that can be fused to either the C- or N-terminus of a target protein. A putative glycosyltransferase (Gtf2) essential for Fap1 glycosylation was used to demonstrate the utility of the cassettes in detection of Gtf2 fusion proteins, and the biological relevance of the proteins in our working strain Streptococcus parasanguinis. GFP and T7 tags were readily expressed in S. parasanguinis as either an N- or C-terminal fusion to Gtf2. Only the C- terminal fusion of GST and HSV were able to be identified in S. parasanguinis. The Nano tag was not detected in either E. coli or S. parasanguinis. Genetic complementation experiments indicated that all the tagged Gtf2 fusion proteins could restore the Gtf2 function in the null mutant except for the Nano-tagged Gtf2 at its N-terminal fusion. Using a T7-tagged Gtf2 fusion construct, we demonstrated that the fusion cassette is also useful in detection of the fusion tag expression in other streptococci including S. mutans, S. pneumoniae and S. sanguinis. Therefore, the expression cassettes we constructed will be a useful tool not only to investigate protein-protein interactions in Fap1 biogenesis in S. parasanguinis, but also to study protein functions in other gram-positive bacteria in which pVT1666 replicates.

Zhou, Meixian; Fives-Taylor, Paula; Wu, Hui

2008-01-01

44

Rapid single-tube method for small-scale affinity purification of polyclonal antibodies using HaloTag™ Technology  

Microsoft Academic Search

Even in this era of advanced biotechniques, specific antibodies against a protein still prove to be powerful tools to study proteins and their functions. The polyclonal antisera obtained from the immunized rabbits, however, are not always pure, high affinity, antigen-specific polyclonal antibodies. With our new rapid HaloTag-based procedure, specific antibodies are obtained in just two, short steps: (1) simultaneous purification

Toshiyuki Hata; Manabu Nakayama

2007-01-01

45

Immobilized metal affinity chromatography in open-loop simulated moving bed technology: Purification of a heat stable histidine tagged ?-glucosidase  

Microsoft Academic Search

Open-loop simulated moving bed (SMB) has been used for immobilized metal affinity chromatographic (IMAC) purification of his-tagged ?-glucosidase expressed in E. coli. A simplified approach based on an optimized single column protocol is used to design the open-loop SMB. A set of columns in the SMB represent one step in the chromatographic cycle i.e. there will be one set each

Deepti Sahoo; Jonatan Andersson; Bo Mattiasson

2009-01-01

46

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

PubMed Central

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

2012-01-01

47

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

PubMed

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 ((AuNP)tris-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 (AuNP)tris-NTA labeled proteins by electron microscopy is further ensured by the reagent's short conformationally restricted linker. We used (AuNP)tris-NTA to localize His-tagged proteins in an oligomeric ATPase and in the bacterial 50S ribosomal subunit. (AuNP)tris-NTA can specifically bind to the target proteins in these assemblies and is clearly discernible. Our labeling reagent should find broad application in noncovalent, site-specific labeling of protein termini to pinpoint their location in macromolecular assemblies. PMID:24560806

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

2014-04-01

48

Microarrays based on affinity-tagged single-chain Fv antibodies: sensitive detection of analyte in complex proteomes.  

PubMed

Protein-based microarrays are among the novel class of rapidly emerging proteomic technologies that will allow us to efficiently perform global proteome analysis. However, the process of designing adequate protein microarrays is a major inherent problem. In this study, we have evaluated a protein microarray platform based on nonpurified affinity-tagged single-chain (sc) Fv antibody fragments to generate proof-of-principle and to demonstrate the specificity and sensitivity of the array design. To this end, we used our human recombinant scFv antibody library genetically constructed around one framework, the n-CoDeR library containing 2 x 10(10) clones, as a source for our probes. The probes were immobilized via engineered C-terminal affinity tags, his- or myc-tags, to either Ni(2+)-coated slides or anti-tag antibody coated substrates. The results showed that highly functional microarrays were generated and that nonpurified scFvs readily could be applied as probes. Specific and sensitive microarrays were obtained, providing a limit of detection in the pM to fM range, using fluorescence as the mode of detection. Further, the results showed that spotting the analyte on top of the arrayed probes, instead of incubating the array with large sample volumes (333 pL vs. 40 microL), could reduce the amount of analyte required 4000 times, from 1200 attomole to 300 zeptomole. Finally, we showed that a highly complex proteome, such as human sera containing several thousand different proteins, could be directly fluorescently labeled and successfully analyzed without compromising the specificity and sensitivity of the antibody microarrays. This is a prerequisite for the design of high-density antibody arrays applied in high-throughput proteomics. PMID:15732136

Wingren, Christer; Steinhauer, Cornelia; Ingvarsson, Johan; Persson, Erik; Larsson, Katrin; Borrebaeck, Carl A K

2005-04-01

49

Application of a new dual localization-affinity purification tag reveals novel aspects of protein kinase biology in Aspergillus nidulans.  

PubMed

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

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

2014-01-01

50

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

PubMed

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

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

2007-01-01

51

Export, purification, and activities of affinity tagged Lactobacillus reuteri levansucrase produced by Bacillus megaterium  

Microsoft Academic Search

Fructosyltransferases, like the Lactobacillus reteri levansucrase, are important for the production of new fructosyloligosaccharides. Various His6- and Strep-tagged variants of this enzyme were recombinantly produced and exported into the growth medium using the Gram-positive\\u000a bacterium Bacillus megaterium. Nutrient-rich growth medium significantly enhanced levansucrase production and export. The B. megaterium signal peptide of the extracellular esterase LipA mediated better levansucrase export

Rebekka Biedendieck; Rafael Beine; Martin Gamer; Eva Jordan; Klaus Buchholz; Jürgen Seibel; Lubbert Dijkhuizen; Marco Malten; Dieter Jahn

2007-01-01

52

Generation of a histidine-tagged antibotulinum toxin antibody fragment in E. coli : effects of post-induction temperature on yield and IMAC binding-affinity  

Microsoft Academic Search

  Recombinant E. coli clones expressing a 50-kDa poly-histidine tail tagged antibody fragment against botulinum toxin (bt-Fab) were initially screened\\u000a for yield and binding affinity. One clone was selected for bioprocess development. The selected bt-Fab vector was induced\\u000a by addition of IPTG and the protein was targeted to the periplasm by inclusion of a pelB leader sequence. A histidine6 affinity ligand

W E Bentley; R D Madurawe; R T Gill; M Shiloach; T E Chase; T R Pulliam-Holoman; J J Valdes

1998-01-01

53

Inexpensive and generic affinity purification of recombinant proteins using a family 2a CBM fusion tag.  

PubMed

The selective binding of the family 2a carbohydrate binding module (CBM2a) of xylanase 10A of the soil bacterium Cellulomonas fimi to a variety of cellulosic substrates is shown to provide a new, cost-effective affinity chromatography system for purification of recombinant protein. Genetic linkage of CBM2a to a target protein, in this case protein A from Staphylococcus aureus, results in a fusion protein that binds strongly to the particulate-cellulose resin Avicel PH101 and retains the biological activity of the fusion partner. Affinity purification of protein A-CBM2a from the supernatant of a recombinant E. coli JM101 culture results in a product purity of greater than 95% and a product concentration factor of 34 +/- 3. Measured column parameters are combined with one-dimensional equations governing continuity and intraparticle diffusion to predict product breakthrough curves with good accuracy over the range of realistic operating conditions. Peak spreading within the column is controlled by intraparticle diffusion for CBM2a and by a combination of film mass transfer and intraparticle diffusion for the larger protein A-CBM2a fusion protein. PMID:15458333

Rodriguez, Beatriz; Kavoosi, Mojgan; Koska, Jürgen; Creagh, A Louise; Kilburn, Douglas G; Haynes, Charles A

2004-01-01

54

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

PubMed

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

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

2013-10-01

55

Studies with an immobilized metal affinity chromatography cassette system involving binuclear triazacyclononane-derived ligands: Automation of batch adsorption measurements with tagged recombinant proteins.  

PubMed

This study describes the determination of the adsorption isotherms and binding kinetics of tagged recombinant proteins using a recently developed IMAC cassette system and employing automated robotic liquid handling procedures for IMAC resin screening. These results confirm that these new IMAC resins, generated from a variety of different metal-charged binuclear 1,4,7-triaza-cyclononane (tacn) ligands, interact with recombinant proteins containing a novel N-terminal metal binding tag, NT1A, with static binding capacities similar to those obtained with conventional hexa-His tagged proteins, but with significantly increased association constants. In addition, higher kinetic binding rates were observed with these new IMAC systems, an attribute that can be positively exploited to increase process productivity. The results from this investigation demonstrate that enhancements in binding capacities and affinities were achieved with these new IMAC resins and chosen NT1A tagged protein. Further, differences in the binding performances of the bis(tacn) xylenyl-bridged ligands were consistent with the distance between the metal binding centres of the two tacn moieties, the flexibility of the ligand and the potential contribution from the aromatic ring of the xylenyl group to undergo ?/? stacking interactions with the tagged proteins. PMID:24891160

Petzold, Martin; Coghlan, Campbell J; Hearn, Milton T W

2014-07-18

56

Increased affinity and solubility of peptides used for direct peptide ELISA on polystyrene surfaces through fusion with a polystyrene-binding peptide tag.  

PubMed

Peptide reagents can serve as alternatives or replacements to antibodies in sensing or diagnostic applications. The passive adsorption of peptides onto polystyrene surfaces can limit the target binding capability, especially for short, positively charged, or hydrophobic sequences. In this report, we show that fusing a peptide with a previously characterized 12-amino acid polystyrene binding sequence (PS-tag) improves overall peptide solubility and enzyme-linked immunosorbent assay (ELISA) results using the peptide as a capture agent. Specific improvements for protective antigen (PA; Bacillus anthracis) protein binding peptides selected from bacterial surface display were compared with native or biotinylated peptides. The PS-tag was added to either peptide terminus, using a (Gly)(4) spacer, and comparable binding affinities were obtained. Fusion with the PS-tag did not have any negative impact on peptide secondary structure as measured by circular dichroism. The addition of the PS-tag provides a convenient method to utilize peptide reagents from peptide display libraries as capture agents in an ELISA format without the need for a biotin tag or concerns about passive adsorption of critical residues for target capture. PMID:22313407

Kogot, Joshua M; Sarkes, Deborah A; Val-Addo, Irene; Pellegrino, Paul M; Stratis-Cullum, Dimitra N

2012-02-01

57

Identifying Novel Protein Complexes in Cancer Cells Using Epitope-Tagging of Endogenous Human Genes and Affinity-Purification Mass Spectrometry  

PubMed Central

Affinity-purification mass spectrometry (AP-MS) is the preeminent technique for identification of eukaryotic protein complexes in vivo. AP-MS workflows typically express epitope-tagged bait proteins, immunopurify, and then identify associated protein complexes using mass spectrometry. However, challenges of existing strategies include the construction of expression vectors for large open reading frames and the possibility that overexpression of bait proteins may result in expression of nonphysiological levels of the bait protein with concomitant perturbation of endogenous protein complexes. To address these issues, we use human cell lines with epitope-tagged endogenous genes as AP-MS substrates to develop a platform that we call “knock-in AP-MS”, thereby avoiding the challenges of expression vector construction and ensuring that expression of tagged proteins is driven by endogenous regulatory mechanisms. Using three different bait genes (MRE11A, DNMT1 and APC), we show that cell lines expressing epitope-tagged endogenous genes make good substrates for sensitive and reproducible identification of protein interactions using AP-MS. In particular, we identify novel interactors of the important oncoprotein Adenomatous Polyposis Coli (APC), including an interaction with Flightless-1 homologue (FLII) that is enriched in nuclear fractions.

Song, Jing; Hao, Yujun; Du, Zhanwen; Wang, Zhenghe; Ewing, Rob M.

2013-01-01

58

Identifying novel protein complexes in cancer cells using epitope-tagging of endogenous human genes and affinity-purification mass spectrometry.  

PubMed

Affinity-purification mass spectrometry (AP-MS) is the preeminent technique for identification of eukaryotic protein complexes in vivo. AP-MS workflows typically express epitope-tagged bait proteins, immunopurify, and then identify associated protein complexes using mass spectrometry. However, challenges of existing strategies include the construction of expression vectors for large open reading frames and the possibility that overexpression of bait proteins may result in expression of nonphysiological levels of the bait protein with concomitant perturbation of endogenous protein complexes. To address these issues, we use human cell lines with epitope-tagged endogenous genes as AP-MS substrates to develop a platform that we call "knock-in AP-MS", thereby avoiding the challenges of expression vector construction and ensuring that expression of tagged proteins is driven by endogenous regulatory mechanisms. Using three different bait genes (MRE11A, DNMT1 and APC), we show that cell lines expressing epitope-tagged endogenous genes make good substrates for sensitive and reproducible identification of protein interactions using AP-MS. In particular, we identify novel interactors of the important oncoprotein Adenomatous Polyposis Coli (APC), including an interaction with Flightless-1 homologue (FLII) that is enriched in nuclear fractions. PMID:23106643

Song, Jing; Hao, Yujun; Du, Zhanwen; Wang, Zhenghe; Ewing, Rob M

2012-12-01

59

High-affinity labeling and tracking of individual histidine-tagged proteins in live cells using Ni2+ tris-nitrilotriacetic acid quantum dot conjugates.  

PubMed

Investigation of many cellular processes using fluorescent quantum dots (QDs) is hindered by the nontrivial requirements for QD surface functionalization and targeting. To address these challenges, we designed, characterized and applied QD-trisNTA, which integrates tris-nitrilotriacetic acid, a small and high-affinity recognition unit for the ubiquitous polyhistidine protein tag. Using QD-trisNTA, we demonstrate two-color QD tracking of the type-1 interferon receptor subunits in live cells, potentially enabling direct visualization of protein-protein interactions at the single molecule level. PMID:19216518

Roullier, Victor; Clarke, Samuel; You, Changjiang; Pinaud, Fabien; Gouzer, G Géraldine; Schaible, Dirk; Marchi-Artzner, Valérie; Piehler, Jacob; Dahan, Maxime

2009-03-01

60

Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry.  

PubMed

The adaptation of sequences of chemical reactions to a solid-phase format has been essential to the automation, reproducibility, and efficiency of a number of biotechnological processes including peptide and oligonucleotide synthesis and sequencing. Here we describe a method for the site-specific, stable isotopic labeling of cysteinyl peptides in complex peptide mixtures through a solid-phase capture and release process, and the concomitant isolation of the labeled peptides. The recovered peptides were analyzed by microcapillary liquid chromatography and tandem mass spectrometry (microLC-MS/MS) to determine their sequences and relative quantities. The method was used to detect galactose-induced changes in protein abundance in the yeast Saccharomyces cerevisiae. A side-by-side comparison with the isotope-coded affinity tag (ICAT) method demonstrated that the solid-phase method for stable isotope tagging of peptides is comparatively simpler, more efficient, and more sensitive. PMID:11981568

Zhou, Huilin; Ranish, Jeffrey A; Watts, Julian D; Aebersold, Ruedi

2002-05-01

61

Immobilized-metal affinity chromatography adsorbent with paramagnetism and its application in purification of histidine-tagged proteins  

Microsoft Academic Search

A new method for synthesis of an Immobilized-Metal Affinity chromatography (IMAC) adsorbent with superparamagnetism (Fe3O4\\/SiO2-GPTMS-Asp-Co) was reported in this paper. Fe3O4 nanoparticles were first modified by SiO2 to form the core–shell Fe3O4\\/SiO2 with superparamagnetism, the core–shell microspheres were then successively treated by 3-glycidoxypropyltrimethoxysilane (GPTMS), l-aspartic acid (l-Asp) and 2-bromoacetic acid to form Fe3O4\\/SiO2-GPTMS-Asp nanoparticles with tetradentate ligands. Finally, the IMAC

Guodong Feng; Daodao Hu; Lei Yang; Yali Cui; Xin-ai Cui; Hong Li

2010-01-01

62

One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system.  

PubMed

The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stress responses. Although the importance of AP-1 has long been recognized, the biochemical characterization of AP-1 remains limited in part due to the difficulty in purifying full-length, reconstituted dimers with active DNA-binding and transcriptional activity. Using a combination of bacterial coexpression and epitope-tagging methods, we successfully purified all 12 heterodimers (3 Junx4 Fos) of full-length human AP-1 complexes as well as c-Jun/c-Jun, JunD/JunD, and c-Jun/JunD dimers from bacterial inclusion bodies using one-step nickel-NTA affinity tag purification following denaturation and renaturation of coexpressed AP-1 subunits. Coexpression of two constitutive components in a dimeric AP-1 complex helps stabilize the proteins when compared with individual protein expression in bacteria. Purified dimeric AP-1 complexes are functional in sequence-specific DNA binding, as illustrated by electrophoretic mobility shift assays and DNase I footprinting, and are also active in transcription with in vitro-reconstituted human papillomavirus (HPV) chromatin containing AP-1-binding sites in the native configuration of HPV nucleosomes. The availability of these recombinant full-length human AP-1 complexes has greatly facilitated mechanistic studies of AP-1-regulated gene transcription in many biological systems. PMID:18329890

Wang, Wei-Ming; Lee, A-Young; Chiang, Cheng-Ming

2008-05-01

63

Integrative refolding and purification of histidine-tagged protein by like-charge facilitated refolding and metal-chelate affinity adsorption.  

PubMed

This work proposed an integrative method of protein refolding and purification by like-charged resin facilitated refolding and metal-chelate affinity adsorption. Hexahistidine-tagged enhanced green fluorescence protein (EGFP) was overexpressed in Escherichia coli as inclusion bodies (IBs), and then the protein was refolded and purified from urea-solubilized IBs by this method. A metal-chelating resin was fabricated by coupling iminodiacetic acid (IDA) to agarose gel (Sepharose FF). The anionic resin was used to facilitate the refolding of like-charged EGFP from IBs. After refolding, nickel ions were introduced for the affinity purification of the target protein by metal-chelating adsorption. It was found that the resin was effective in facilitating EGFP refolding. For 0.1mg/mL EGFP IBs refolding, the fluorescence recovery (FR) by direct dilution was only 64%; addition of only 0.05 g/mL resin increased the FR to over 90%. Moreover, the FR increased with increasing resin concentration. Owning to the shielding effect of the oppositely charged impurities embedded in IBs on the surface charges of the IDA resin, more resin particles were required to exert an aggregation inhibition effect in the IBs protein refolding. Additionally, compared with direct-dilution refolding, inclusion of like-charged resins not only offered an enhanced FR of EGFP, but also bound some opposite-charged contaminant proteins, leading to a preliminary purification effect. Afterwards, the refolded EGFP was recovered by metal-chelating adsorption at an FR of 85% and purity of 93%. This work has thus extended the like-charge facilitated protein refolding strategy to the integrative protein refolding and purification. PMID:24768124

Liu, Hu; Du, Wen-Jie; Dong, Xiao-Yan; Sun, Yan

2014-05-30

64

Live cell interactome of the human voltage dependent anion channel 3 (VDAC3) revealed in HeLa cells by affinity purification tag technique.  

PubMed

In higher eukaryotes three different VDAC genes encode three homologous proteins which do not show the same activity. VDAC1 and VDAC2 isoforms have been characterized while VDAC3 isoform is still elusive. To explore VDAC3 protein interactions, we have established a stable cell line expressing a fluorescent and dual-tagged construct. This clone expresses a stable amount of VDAC3. Live cell imaging shows that fluorescent VDAC3 localizes in the mitochondria. Proteins interacting with VDAC3 have been separated by tandem-affinity purification and 2-D gel electrophoresis and identified by mass spectrometry. In the list of putative interacting proteins, there are cytosolic, mitochondrial, cytoskeletal and ER proteins. Coherent pathways like cell redox homeostasis, response to stress, formation/rearrangement of disulfide bonds, response to unfolded proteins or protein folding have been found to be related to clusters of proteins identified in this experiment. The list of associated proteins has been validated by immunoprecipitation experiments utilizing specific antibodies. Likely biological and pathological processes have been analyzed. Cytosolic proteins associated with VDAC3 include tubulins and cytoskeletal proteins, stress sensors, chaperones and proteasome components, redox-mediating enzymes such as protein disulphide isomerase. The overall picture points to a role for VDAC3 as mediator for the organization of protein complexes and regulator of the traffic of misfolded or non-folded proteins evoked from different stimuli. PMID:24865465

Messina, Angela; Reina, Simona; Guarino, Francesca; Magrì, Andrea; Tomasello, Flora; Clark, Richard E; Ramsay, Rona R; De Pinto, Vito

2014-07-01

65

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates  

PubMed Central

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell migration, gene expression, cell cycle progression and cell adhesion 1. Rho proteins are molecular switches that are active in GTP-bound and inactive in GDP-bound state. Their activation is mediated by a family of Guanine-nucleotide Exchange Factor (GEF) proteins. Rho-GEFs constitute a large family, with overlapping specificities 2. Although a lot of progress has been made in identifying the GEFs activated by specific signals, there are still many questions remaining regarding the pathway-specific regulation of these proteins. The number of Rho-GEFs exceeds 70, and each cell expresses more than one GEF protein. In addition, many of these proteins activate not only Rho, but other members of the family, contributing further to the complexity of the regulatory networks. Importantly, exploring how GEFs are regulated requires a method to follow the active pool of individual GEFs in cells activated by different stimuli. Here we provide a step-by-step protocol for a method used to assess and quantify the available active Rho-specific GEFs using an affinity precipitation assay. This assay was developed a few years ago in the Burridge lab 3,4 and we have used it in kidney tubular cell lines 5,6,7. The assay takes advantage of a "nucleotide free" mutant RhoA, with a high affinity for active GEFs. The mutation (G17A) renders the protein unable to bind GDP or GTP and this state mimics the intermediate state that is bound to the GEF. A GST-tagged version of this mutant protein is expressed and purified from E. coli, bound to glutathione sepharose beads and used to precipitate active GEFs from lysates of untreated and stimulated cells. As most GEFs are activated via posttranslational modifications or release from inhibitory bindings, their active state is preserved in cell lysates, and they can be detected by this assay8. Captured proteins can be probed for known GEFs by detection with specific antibodies using Western blotting, or analyzed by Mass Spectrometry to identify unknown GEFs activated by certain stimuli.

Waheed, Faiza; Speight, Pamela; Dan, Qinghong; Garcia-Mata, Rafael; Szaszi, Katalin

2012-01-01

66

Affinity precipitation of active Rho-GEFs using a GST-tagged mutant Rho protein (GST-RhoA(G17A)) from epithelial cell lysates.  

PubMed

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell migration, gene expression, cell cycle progression and cell adhesion. Rho proteins are molecular switches that are active in GTP-bound and inactive in GDP-bound state. Their activation is mediated by a family of Guanine-nucleotide Exchange Factor (GEF) proteins. Rho-GEFs constitute a large family, with overlapping specificities. Although a lot of progress has been made in identifying the GEFs activated by specific signals, there are still many questions remaining regarding the pathway-specific regulation of these proteins. The number of Rho-GEFs exceeds 70, and each cell expresses more than one GEF protein. In addition, many of these proteins activate not only Rho, but other members of the family, contributing further to the complexity of the regulatory networks. Importantly, exploring how GEFs are regulated requires a method to follow the active pool of individual GEFs in cells activated by different stimuli. Here we provide a step-by-step protocol for a method used to assess and quantify the available active Rho-specific GEFs using an affinity precipitation assay. This assay was developed a few years ago in the Burridge lab and we have used it in kidney tubular cell lines. The assay takes advantage of a "nucleotide free" mutant RhoA, with a high affinity for active GEFs. The mutation (G17A) renders the protein unable to bind GDP or GTP and this state mimics the intermediate state that is bound to the GEF. A GST-tagged version of this mutant protein is expressed and purified from E. coli, bound to glutathione sepharose beads and used to precipitate active GEFs from lysates of untreated and stimulated cells. As most GEFs are activated via posttranslational modifications or release from inhibitory bindings, their active state is preserved in cell lysates, and they can be detected by this assay. Captured proteins can be probed for known GEFs by detection with specific antibodies using Western blotting, or analyzed by Mass Spectrometry to identify unknown GEFs activated by certain stimuli. PMID:22491204

Waheed, Faiza; Speight, Pamela; Dan, Qinghong; Garcia-Mata, Rafael; Szaszi, Katalin

2012-01-01

67

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

PubMed Central

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 organisms—including humans—are 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.

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

2013-01-01

68

Functional expression of hexahistidine-tagged ?-subunit of yeast F 1ATPase and isolation of the enzyme by immobilized metal affinity chromatography  

Microsoft Academic Search

Mitochondrial ATP synthase (F1Fo-ATPase) catalyzes the terminal step of oxidative phosphorylation. In this paper, we demonstrate the functional expression of the hexahistidine-tagged ?-subunit of yeast ATP synthase and the purification of the F1-ATPase from yeast cells. A gene encoding the ?-subunit from Saccharomyces cerevisiae was modified to encode a protein of which the original N-terminus import signal sequence was replaced

Naoki Ichikawa; Miki Mizuno

2004-01-01

69

Isotope and affinity tags in photoreactive substance P analogues to identify the covalent linkage within the NK-1 receptor by MALDI-TOF analysis.  

PubMed

Photoreactive analogues of substance P (biotin sulfone-spacer (amino pentanoic or Gly(3))-Arg-Pro-Lys-Pro-(pBzl)Phe-Gln-Phe-Phe-Gly-Leu-Met(O(2))NH(2)) with or without isotope (deuterium) labeling have been synthesized. Deuteriums were present on (d)-biotin or epibiotin sulfone (D(3)), on the Gly(3) spacer linker (D(6)), or on the Gly in position 9 of SP (D(2)). Therefore, peptide analogues could be either unlabeled or tri-, penta-, or hexadeuterated. Results obtained with the use of these peptide analogues show that (d)-biotin sulfone and epibiotin sulfone are not recognized with the same affinity by streptavidin, with (d)-biotin sulfone displaying better affinity for the protein. Photolabeling of the human NK-1 receptor with a 1:1 molar ratio of nondeuterated and deuterated photoreactive substance P (SP) analogues in position 5, followed by combined digestions, purification, and MALDI-TOF mass spectrometry analysis, made the identification of the domain of the receptor covalently linked by the photoreactive SP analogue easier. Indeed, doublets in mass spectra were specific for the covalent complex whereas single peaks could be attributed to contaminating species. This method is particularly suitable when minute amounts of complex have to be analyzed, as in the case of highly hydrophobic G-protein coupled receptors. PMID:14640725

Sachon, Emmanuelle; Tasseau, Olivier; Lavielle, Solange; Sagan, Sandrine; Bolbach, Gérard

2003-12-01

70

Method for Purifying and Recovering Silk Proteins Using Magnetic Affinity Separation.  

National Technical Information Service (NTIS)

A method for the purification of recombinant silk proteins from a sample using magnetic affinity separation is described. The recombinant silk protein is expressed with an affinity tag which has a high binding affinity for an affinity ligand immobilized o...

C. Hoffmann K. Keller

2005-01-01

71

Challenges and opportunities in the purification of recombinant tagged proteins.  

PubMed

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

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

2014-01-01

72

BiPS, a photocleavable, isotopically coded, fluorescent cross-linker for structural proteomics.  

PubMed

Cross-linking combined with mass spectrometry is an emerging approach for studying protein structure and protein-protein interactions. However, unambiguous mass spectrometric identification of cross-linked peptides derived from proteolytically digested cross-linked proteins is still challenging. Here we describe the use of a novel cross-linker, bimane bisthiopropionic acid N-succinimidyl ester (BiPS), that overcomes many of the challenges associated with other cross-linking reagents. BiPS is distinguished from other cross-linkers by a unique combination of properties: it is photocleavable, fluorescent, homobifunctional, amine-reactive, and isotopically coded. As demonstrated with a model protein complex, RNase S, the fluorescent moiety of BiPS allows for sensitive and specific monitoring of the different cross-linking steps, including detection and isolation of cross-linked proteins by gel electrophoresis, determination of in-gel digestion completion, and fluorescence-based separation of cross-linked peptides by HPLC. The isotopic coding of BiPS results in characteristic ion signal "doublets" in mass spectra, thereby permitting ready detection of cross-linker-containing peptides. Under MALDI-MS conditions, partial photocleavage of the cross-linker occurs, releasing the cross-linked peptides. This allows differentiation between dead-end, intra-, and interpeptide cross-links based on losses of specific mass fragments. It also allows the use of the isotope doublets as mass spectrometric "signatures." A software program was developed that permits automatic cross-link identification and assignment of the cross-link type. Furthermore photocleavage of BiPS assists in cross-link identification by allowing separate tandem mass spectrometry sequencing of each peptide comprising the original cross-link. By combining the use of BiPS with MS, we have provided the first direct evidence for the docking site of a phosphorylated G-protein-coupled receptor C terminus on the multifunctional adaptor protein beta-arrestin, clearly demonstrating the broad potential and application of this novel cross-linker in structural and cellular biology. PMID:18838738

Petrotchenko, Evgeniy V; Xiao, Kunhong; Cable, Jennifer; Chen, Yiwen; Dokholyan, Nikolay V; Borchers, Christoph H

2009-02-01

73

Isotopically coded cleavable cross-linker for studying protein-protein interaction and protein complexes.  

PubMed

An emerging approach for studying protein-protein interaction in complexes is the combination of chemical cross-linking and mass spectrometric analysis of the cross-linked peptides (cross-links) obtained after proteolysis of the complex. This approach, however, has several challenges and limitations, including the difficulty of detecting the cross-links, the potential interference from non-informative "cross-linked peptides" (dead end and intrapeptide cross-links), and unambiguous identification of the cross-links by mass spectrometry. Thus, we have synthesized an isotopically coded ethylene glycol bis(succinimidylsuccinate) derivate (D12-EGS), which contains 12 deuterium atoms for easy detection of cross-links when applied in a 1:1 mixture with its H12 counterpart and is also cleavable for releasing the cross-linked peptides allowing unambiguous identification by MS sequencing. Moreover, hydrolytic cleavage permits rapid distinguishing between different types of cross-links. Cleavage of a dead end cross-link produces a doublet with peaks 4.03 Da apart, with the lower peak appearing at a molecular mass 162 Da lower than the mass of the H12 form of the original cross-linked peptide. Cleavage of an intrapeptide cross-link leads to a doublet 8.05 Da apart and 62 Da lower than the molecular mass of the H12 form of the original cross-linked peptide. Cleavage of an interpeptide cross-link forms a pair of 4.03-Da doublets, with the lower mass member of each pair each shifted up from its unmodified molecular weight by 82 Da because of the attached portion of the cross-linker. All of this information has been incorporated into a software algorithm allowing automatic screening and detection of cross-links and cross-link types in matrix-assisted laser desorption/ionization mass spectra. In summary, the ease of detection of these species through the use of an isotopically coded cleavable cross-linker and our software algorithm, followed by mass spectrometric sequencing of the cross-linked peptides after cleavage, has been shown to be a powerful tool for studies of multi-component protein complexes. PMID:15901824

Petrotchenko, Evgeniy V; Olkhovik, Vyacheslav K; Borchers, Christoph H

2005-08-01

74

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

PubMed Central

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

2012-01-01

75

Developing new isotope-coded mass spectrometry-cleavable cross-linkers for elucidating protein structures.  

PubMed

Structural characterization of protein complexes is essential for the understanding of their function and regulation. However, it remains challenging due to limitations in existing tools. With recent technological improvements, cross-linking mass spectrometry (XL-MS) has become a powerful strategy to define protein-protein interactions and elucidate structural topologies of protein complexes. To further advance XL-MS studies, we present here the development of new isotope-coded MS-cleavable homobifunctional cross-linkers: d0- and d10-labeled dimethyl disuccinimidyl sulfoxide (DMDSSO). Detailed characterization of DMDSSO cross-linked peptides further demonstrates that sulfoxide-containing MS-cleavable cross-linkers offer robust and predictable MS2 fragmentation of cross-linked peptides, permitting subsequent MS3 analysis for simplified, unambiguous identification. Concurrent usage of these reagents provides a characteristic doublet pattern of DMDSSO cross-linked peptides, thus aiding in the confidence of cross-link identification by MS(n) analysis. More importantly, the unique isotopic profile permits quantitative analysis of cross-linked peptides and therefore expands the capability of XL-MS strategies to analyze both static and dynamic protein interactions. Together, our work has established a new XL-MS workflow for future studies toward the understanding of structural dynamics of protein complexes. PMID:24471733

Yu, Clinton; Kandur, Wynne; Kao, Athit; Rychnovsky, Scott; Huang, Lan

2014-02-18

76

tagging, communities, vocabulary, evolution  

Microsoft Academic Search

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

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

2006-01-01

77

Germ Tag  

NSDL National Science Digital Library

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

Science, American A.

2009-01-01

78

Affinity Chromatography  

NSDL National Science Digital Library

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

Diresta, Dan

2011-05-23

79

Shark Tagging Activities.  

ERIC Educational Resources Information Center

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

Current: The Journal of Marine Education, 1998

1998-01-01

80

Affinity Chromatography  

NSDL National Science Digital Library

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

2011-05-25

81

Ligand affinity of the 67-kD elastin/laminin binding protein is modulated by the protein's lectin domain: visualization of elastin/laminin-receptor complexes with gold-tagged ligands  

PubMed Central

Video-enhanced microscopy was used to examine the interaction of elastin- or laminin-coated gold particles with elastin binding proteins on the surface of live cells. By visualizing the binding events in real time, it was possible to determine the specificity and avidity of ligand binding as well as to analyze the motion of the receptor-ligand complex in the plane of the plasma membrane. Although it was difficult to interpret the rates of binding and release rigorously because of the possibility for multiple interactions between particles and the cell surface, relative changes in binding have revealed important aspects of the regulation of affinity of ligand-receptor interaction in situ. Both elastin and laminin were found to compete for binding to the cell surface and lactose dramatically decreased the affinity of the receptor(s) for both elastin and laminin. These findings were supported by in vitro studies of the detergent-solubilized receptor. Further, immobilization of the ligand-receptor complexes through binding to the cytoskeleton dramatically decreased the ability of bound particles to leave the receptor. The changes in the kinetics of ligand-coated gold binding to living cells suggest that both laminin and elastin binding is inhibited by lactose and that attachment of receptor to the cytoskeleton increases its affinity for the ligand.

1991-01-01

82

Visualizing tags over time  

Microsoft Academic Search

We consider the problem of visualizing the evolution of tags within the Flickr (flickr.com) online image sharing com- munity. Any user of the Flickr service may append a tag to any photo in the system. Over the past year, users have on average added over a million tags each week. Under- standing the evolution of these tags over time is

Micah Dubinko; Ravi Kumar; Joseph Magnani; Jasmine Novak; Prabhakar Raghavan; Andrew Tomkins

2006-01-01

83

Bootstrapping Tagged Islamic Corpora  

Microsoft Academic Search

Among tagged language resources for Arabic there is a high density for Modern Standard Arabic. Nonetheless, the tagged corpora for Classical Arabic are of very low density. Moreover, such corpora are normally developed applying software that are of serious shortcomings. This paper is elaborating on the tagging approach of the Islamic corpora which are being tagged at Noorsoft, Qom, Iran,

Mahmoud Shokrollahi-Far; Behrooz Minaei; Issa Barzegar; Hadi Hossein-Zadeh; Mozhdeh Ghasdi; Salman Hoseini

84

Tagging-Aware Portlets  

Microsoft Academic Search

A corporate portal supports a community of users on cohesively managing a shared set of resources. Such management should\\u000a also include social tagging, i.e. the practice of collaboratively creating and managing tags to annotate and categorize content.\\u000a This task involves to know both what to tag (hence, the rendering of the resource content) and how to tag (i.e. the tagging

Oscar Díaz; Sandy Pérez; Cristóbal Arellano

2009-01-01

85

Myocardial Tagging With SSFP  

PubMed Central

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

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

2007-01-01

86

Donor Tag Game  

MedlinePLUS

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

87

Perspectives of immobilized-metal affinity chromatography  

Microsoft Academic Search

Immobilized Metal-Affinity Chromatography (IMAC) represents a relatively new separation technique that is primarily appropriate for the purification of proteins with natural surface-exposed histidine residues and for recombinant proteins with engineered histidine tags or histidine clusters. Because the method has gained broad popularity in recent years, the main recent developments in the field of new sorbents, techniques and possible applications are

Vladka Gaberc-Porekar; Viktor Menart

2001-01-01

88

EnTag: enhancing social tagging for discovery  

Microsoft Academic Search

The EnTag (Enhanced Tagging for Discovery) project investigated the effect on indexing and retrieval when using only social tagging versus when using social tagging in combination with suggestions from a controlled vocabulary. Two different contexts were explored: tagging by readers of a digital collection and tagging by authors in an institutional repository; also two different controlled vocabularies were examined, Dewey

Koraljka Golub; Jim Moon; Douglas Tudhope; Catherine Jones; Brian Matthews; Bartbomiej Puzod; Marianne Lykke Nielsen

2009-01-01

89

Extracting tag hierarchies.  

PubMed

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

Tibély, Gergely; Pollner, Péter; Vicsek, Tamás; Palla, Gergely

2013-01-01

90

Extracting Tag Hierarchies  

PubMed Central

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.

Tibely, Gergely; Pollner, Peter; Vicsek, Tamas; Palla, Gergely

2013-01-01

91

Mining multi-tag association for image tagging  

Microsoft Academic Search

Automatic media tagging plays a critical role in modern tag-based media retrieval systems. Existing tagging schemes mostly\\u000a perform tag assignment based on community contributed media resources, where the tags are provided by users interactively.\\u000a However, such social resources usually contain dirty and incomplete tags, which severely limit the performance of these tagging\\u000a methods. In this paper, we propose a novel

Yang Yang; Zi Huang; Heng Tao Shen; Xiaofang Zhou

2011-01-01

92

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

PubMed Central

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.

Jing, Chaoran; Cornish, Virginia W.

2013-01-01

93

Density tagging velocimetry  

Microsoft Academic Search

Density tagging velocimetry, a novel optical technique for point-wise measurement of flow velocity is proposed here. This\\u000a new method is based on the detection and subsequent tracking of a local density variation deliberately inserted in the flow.\\u000a The experimental implementation comprising tagging, detection, and velocity evaluation reverts to and combines principles\\u000a of well-known optical measurement techniques. Density tagging velocimetry has

Markus Raffel; Ricardo Hernandez-Rivera; Benjamin Heine; Andreas Schröder; Karen Mulleners

2011-01-01

94

Platform tolerant RFID tag antenna  

Microsoft Academic Search

A passive radio-frequency identification (RFID) tag antenna for UHF applications is proposed. An inductively coupled feed for the tag, employed previously in an earlier design, is modified to realize a new RFID tag antenna. A ground plane is used in the design to make the tag antenna platform-tolerant. The read range and percentage power transfer of the tag antenna are

Y. C; K. W. Leung; R. Mittra; K. V. S. Rao

2007-01-01

95

Method for protein tagging in Schizosaccharomyces pombe.  

PubMed

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

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

2006-01-01

96

Affinity purification of Schistosoma japonicum glutathione-S-transferase and its site-directed mutants with glutathione affinity chromatography and immobilized metal affinity chromatography  

Microsoft Academic Search

A C-terminally polyhistidine-tagged protein of Schistosoma japonicum glutathione-S-transferase, named as SjGST\\/His, and its Cys85?Ser, Cys138?Ser, and Cys178?Ser site-directed mutants were prepared and highly expressed in Escherichia coli. Both immobilized metal affinity chromatography (IMAC) and glutathione (GSH) affinity chromatography were used to purify these four enzymes. All of them were purified with equal efficiency by Ni2+-chelated nitrilotriacetic acid agarose gel, but

Hsiu-Mei Chen; San-Lin Luo; Kai-Ti Chen; Chong-Kuei Lii

1999-01-01

97

Neuron Chain Tag  

NSDL National Science Digital Library

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

Yoshioka, Melissa

2009-01-01

98

Chromatin tandem affinity purification sequencing  

PubMed Central

Chromatin immunoprecipitation coupled with ultra-high-throughput sequencing (ChIP-seq) is a widely used method for mapping the interactions of proteins with DNA. However, the requirements for ChIP-grade antibodies impede wider application of this method, and variations in results can be high owing to differences in affinity and cross-reactivity of antibodies. Therefore, we developed chromatin tandem affinity purification (ChTAP) as an effective alternative to ChIP. Through the use of affinity tags and reagents that are identical for all proteins investigated, ChTAP enables one to directly compare the binding between different transcription factors and to directly assess the background in control experiments. Thus, ChTAP -seq can be used to rapidly map the genome-wide binding of multiple DNA -binding proteins in a wide range of cell types. ChTAP can be completed in 3–4 d, starting from cross-linking of chromatin to purification of ChIP DNA.

Soleimani, Vahab D; Palidwor, Gareth A; Ramachandran, Parameswaran; Perkins, Theodore J; Rudnicki, Michael A

2014-01-01

99

Expanded-bed adsorption immobilized-metal affinity chromatography  

Microsoft Academic Search

The protocol describes a method for capture of secreted hexahistidine-tagged proteins using expanded-bed adsorption immobilized-metal affinity chromatography. The starting material for the procedure is any crude feedstock that contains a histidine (His)-tagged target protein. The protocol is exemplified using unclarified broth from Pichia pastoris fermentation as feedstock. The protocol can be used for laboratory studies or as part of a

Lisa Smith; Richard H J Begent; Kerry A Chester; Berend Tolner

2006-01-01

100

New tags for recombinant protein detection and O-glycosylation reporters.  

PubMed

Monoclonal antibodies (mAbs), because of their unique specificity, are irreplaceable tools for scientific research. Precise mapping of the antigenic determinants allows the development of epitope tagging approaches to be used with recombinant proteins for several purposes. Here we describe a new family of tags derived from the epitope recognized by a single highly specific mAb (anti-roTag mAb), which was obtained from a pool of mAbs reacting with the rotavirus nonstructural protein 5 (NSP5). The variable regions of the anti-roTag mAb were identified and their binding capacity verified upon expression as a single-chain/miniAb. The minimal epitope, termed roTag, was identified as a 10 amino acid sequence (SISSSIFKNE). The affinity of the anti-roTag/roTag interaction was found to be comparable to that of the anti-SV5/SV5 tag interaction. roTag was successfully used for detection of several recombinant cytosolic, secretory and membrane proteins. Two additional variants of roTag of 10 and 13 amino acids containing O-glycosylation susceptible sites (termed OG-tag and roTagO) were constructed and characterised. These tags were useful to detect proteins passing through the Golgi apparatus, the site of O-glycosylation. PMID:24802141

Petris, Gianluca; Bestagno, Marco; Arnoldi, Francesca; Burrone, Oscar R

2014-01-01

101

New Tags for Recombinant Protein Detection and O-Glycosylation Reporters  

PubMed Central

Monoclonal antibodies (mAbs), because of their unique specificity, are irreplaceable tools for scientific research. Precise mapping of the antigenic determinants allows the development of epitope tagging approaches to be used with recombinant proteins for several purposes. Here we describe a new family of tags derived from the epitope recognized by a single highly specific mAb (anti-roTag mAb), which was obtained from a pool of mAbs reacting with the rotavirus nonstructural protein 5 (NSP5). The variable regions of the anti-roTag mAb were identified and their binding capacity verified upon expression as a single-chain/miniAb. The minimal epitope, termed roTag, was identified as a 10 amino acid sequence (SISSSIFKNE). The affinity of the anti-roTag/roTag interaction was found to be comparable to that of the anti-SV5/SV5 tag interaction. roTag was successfully used for detection of several recombinant cytosolic, secretory and membrane proteins. Two additional variants of roTag of 10 and 13 amino acids containing O-glycosylation susceptible sites (termed OG-tag and roTagO) were constructed and characterised. These tags were useful to detect proteins passing through the Golgi apparatus, the site of O-glycosylation.

Arnoldi, Francesca; Burrone, Oscar R.

2014-01-01

102

Purification of proteins containing zinc finger domains using Immobilized Metal Ion Affinity Chromatography  

PubMed Central

Heterologous proteins are frequently purified by Immobilized Metal Ion Affinity Chromatography (IMAC) based on their modification with a hexa-histidine affinity tag (His-tag). The terminal His-tag can, however, alter functional properties of the tagged protein. Numerous strategies for the tag removal have been developed including chemical treatment and insertion of protease target sequences in the protein sequence. Instead of using these approaches, we took an advantage of natural interaction of zinc finger domains with metal ions to purify functionally similar retroviral proteins from two different retroviruses. We found that these proteins exhibited significantly different affinities to the immobilized metal ions, despite that both contain the same type of zinc finger motif (i.e. CCHC). While zinc finger proteins may differ in biochemical properties, the multitude of IMAC platforms should allow relatively simple yet specific method for their isolation in native state.

Vorackova, Irena; Suchanova, Sarka; Ulbrich, Pavel; Diehl, William E.; Ruml, Tomas

2011-01-01

103

Methods for detection of protein-protein and protein-DNA interactions using HaloTag.  

PubMed

HaloTag is a protein fusion tag which was genetically engineered to covalently bind a series of specific synthetic ligands. All ligands carry two groups, the reactive group and the functional/reporter group. The reactive group, the choloroalkane, is the same in all the ligands and is involved in binding to the HaloTag. The functional reporter group is variable and can carry many different moieties including fluorescent dyes, affinity handles like biotin or solid surfaces such as agarose beads. Thus, HaloTag can serve either as a labeling tag or as a protein immobilization tag depending on which ligand is bound to it. Here, we describe a procedure for immobilization of HaloTag fusion proteins and how immobilized proteins can be used to study protein-protein and protein-DNA interactions in vivo and in vitro. PMID:18826056

Urh, Marjeta; Hartzell, Danette; Mendez, Jacqui; Klaubert, Dieter H; Wood, Keith

2008-01-01

104

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

PubMed Central

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.

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

2012-01-01

105

Quantitative Evaluation of Neurotensin Receptor Purification by Immobilized Metal Affinity Chromatography  

Microsoft Academic Search

Immobilized metal affinity chromatography has recently been used for purification of histidine-tagged membrane proteins in the presence of detergents with varying success. Strong binding to the metal resin is essential for purification when expression levels are low. We have investigated the influence of tag length and type of detergent on the purification of a neurotensin receptor fusion protein expressed inEscherichia

Reinhard Grisshammer; Julie Tucker

1997-01-01

106

Discourse Tagging Tool and Discourse-Tagged Multilingual Corpora  

Microsoft Academic Search

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

Chinatsu Aone; Scott W. Bennett

1994-01-01

107

Tag Completion for Image Retrieval.  

PubMed

Many social image search engines are based on keyword/tag matching. This is because tag based image retrieval (TBIR) is not only efficient but also effective. The performance of TBIR is highly dependent on the availability and quality of manual tags. Recent studies have shown that manual tags are often unreliable and inconsistent. In addition, since many users tend to choose general and ambiguous tags in order to minimize their efforts in choosing appropriate words, tags that are specific to the visual content of images tend to be missing or noisy, leading to a limited performance of TBIR. To address this challenge, we study the problem of \\textit{tag completion} where the goal is to automatically fill in the missing tags as well as correct noisy tags for given images. We represent the image-tag relation by a \\textit{tag matrix}, and search for the optimal tag matrix consistent with both the observed tags and the visual similarity. We propose an efficient algorithm for solving the related optimization problem. Extensive empirical studies show that the proposed algorithm is significantly more effective than the state-of-the-art algorithms. Our studies also verify that the proposed algorithm is computationally efficient and scales well to large databases. PMID:22641703

Wu, Lei; Jin, Rong; Jain, Anil K

2012-05-22

108

Tagging mammalian transcription complexity  

Microsoft Academic Search

The nature of the 'transcriptome' is more complex than first realized. Although CAGE, various tagging technol- ogies and tiling arrays show that most of the mammalian genome is transcribed, a large proportion of transcripts do not encode proteins and are either poorly polyade- nylated, involved in sense-antisense pairs or never leave the nucleus. In this article, I review the various

Piero Carninci

2006-01-01

109

Tagging of Arctic Icebergs.  

National Technical Information Service (NTIS)

An air-deployable iceberg tagging system has been developed for use from a C-130 aircraft. The system consists of a steel dart with a trailing buoyant line which can be attached to a floating instrument package. The system allows for considerable melting ...

R. Q. Robe T. S. Ellis

1978-01-01

110

Ontologies and tag-statistics  

NASA Astrophysics Data System (ADS)

Due to the increasing popularity of collaborative tagging systems, the research on tagged networks, hypergraphs, ontologies, folksonomies and other related concepts is becoming an important interdisciplinary area with great potential and relevance for practical applications. In most collaborative tagging systems the tagging by the users is completely ‘flat’, while in some cases they are allowed to define a shallow hierarchy for their own tags. However, usually no overall hierarchical organization of the tags is given, and one of the interesting challenges of this area is to provide an algorithm generating the ontology of the tags from the available data. In contrast, there are also other types of tagged networks available for research, where the tags are already organized into a directed acyclic graph (DAG), encapsulating the ‘is a sub-category of’ type of hierarchy between each other. In this paper, we study how this DAG affects the statistical distribution of tags on the nodes marked by the tags in various real networks. The motivation for this research was the fact that understanding the tagging based on a known hierarchy can help in revealing the hidden hierarchy of tags in collaborative tagging systems. We analyse the relation between the tag-frequency and the position of the tag in the DAG in two large sub-networks of the English Wikipedia and a protein-protein interaction network. We also study the tag co-occurrence statistics by introducing a two-dimensional (2D) tag-distance distribution preserving both the difference in the levels and the absolute distance in the DAG for the co-occurring pairs of tags. Our most interesting finding is that the local relevance of tags in the DAG (i.e. their rank or significance as characterized by, e.g., the length of the branches starting from them) is much more important than their global distance from the root. Furthermore, we also introduce a simple tagging model based on random walks on the DAG, capable of reproducing the main statistical features of tag co-occurrence. This model has high potential for further practical applications, e.g., it can provide the starting point for a benchmark system in ontology retrieval or it may help pinpoint unusual correlations in the co-occurrence of tags.

Tibély, Gergely; Pollner, Péter; Vicsek, Tamás; Palla, Gergely

2012-05-01

111

Extended Affine Weyl Groups  

Microsoft Academic Search

In this paper we study the Weyl groups of reduced extended affine root systems, the root systems of extended affine Lie algebras. We start by describing the extended affine Weyl group as a semidirect product of a finite Weyl group and a Heisenberg-like normal subgroup. This provides a unique expression for the Weyl group elements (in terms of some naturally

Saeid Azam

1999-01-01

112

A novel method of affinity-purifying proteins using a bis-arsenical fluorescein.  

PubMed Central

Genetically-encoded affinity tags constitute an important strategy for purifying proteins. Here, we have designed a novel affinity matrix based on the his-arsenical fluorescein dye FlAsH, which specifically recognizes short alpha-helical peptides containing the sequence CCXXCC (Griffin BA, Adams SR, Tsien RY, 1998, Science 281:269-272). We find that kinesin tagged with this cysteine-containing helix binds specifically to FlAsH resin and can be eluted in a fully active form. This affinity tag has several advantages over polyhistidine, the only small affinity tag in common use. The protein obtained with this single chromatographic step from crude Escherichia coli lysates is purer than that obtained with nickel affinity chromatography of 6xHis tagged kinesin. Moreover, unlike nickel affinity chromatography, which requires high concentrations of imidazole or pH changes for elution, protein bound to the FlAsH column can be completely eluted by dithiothreitol. Because of these mild elution conditions, FlAsH affinity chromatography is ideal for recovering fully active protein and for the purification of intact protein complexes.

Thorn, K. S.; Naber, N.; Matuska, M.; Vale, R. D.; Cooke, R.

2000-01-01

113

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

PubMed Central

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

N Peterson, Scott; Kwon, Keehwan

2012-01-01

114

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

PubMed

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

N Peterson, Scott; Kwon, Keehwan

2012-01-01

115

Anonymous Tracking Using RFID Tags  

Microsoft Academic Search

The increasing use of RFID tags in many applications have brought forth valid concerns of privacy and anonymity among users. One of the primary concerns with RFID tags is their ability to track an individually tagged entity. While this capability is currently thought to be necessary for supporting some features of RFID systems, such practice can lead to potential privacy

Murali S. Kodialam; Thyaga Nandagopal; Wing Cheong Lau

2007-01-01

116

TagMyDoc  

NSDL National Science Digital Library

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

2012-01-01

117

Tagging of MADS domain proteins for chromatin immunoprecipitation  

PubMed Central

Background Most transcription factors fulfill their role in complexes and regulate their target genes upon binding to DNA motifs located in upstream regions or introns. To date, knowledge about transcription factor target genes and their corresponding transcription factor binding sites are still very limited. Two related methods that allow in vivo identification of transcription factor binding sites are chromatin immunoprecipitation (ChIP) and chromatin affinity purification (ChAP). For ChAP, the protein of interest is tagged with a peptide or protein, which can be used for affinity purification of the protein-DNA complex and hence, the identification of the target gene. Results Here, we present the results of experiments aiming at the development of a generic tagging approach for the Arabidopsis MADS domain proteins AGAMOUS, SEPALLATA3, and FRUITFULL. For this, Arabidopsis wild type plants were transformed with constructs containing a MADS-box gene fused to either a double Strep-tag® II-FLAG-tag, a triple HA-tag, or an eGFP-tag, all under the control of the constitutive double 35S Cauliflower Mosaic Virus (CaMV) promoter. Strikingly, in all cases, the number of transformants with loss-of-function phenotypes was much larger than those with an overexpression phenotype. Using endogenous promoters in stead of the 35S CaMV resulted in a dramatic reduction in the frequency of loss-of-function phenotypes. Furthermore, pleiotropic defects occasionally caused by an overexpression strategy can be overcome by using the native promoter of the gene. Finally, a ChAP result is presented using GFP antibody on plants carrying a genomic fragment of a MADS-box gene fused to GFP. Conclusion This study revealed that MADS-box proteins are very sensitive to fusions with small peptide tags and GFP tags. Furthermore, for the expression of chimeric versions of MADS-box genes it is favorable to use the entire genomic region in frame to the tag of choice. Interestingly, though unexpected, it appears that the use of chimeric versions of MADS-box genes under the control of the strong 35S CaMV promoter is a very efficient method to obtain dominant-negative mutants, either caused by cosuppression or by alteration of the activity of the recombinant protein. Finally, we were able to demonstrate AGAMOUS binding to one of its targets by ChAP.

de Folter, Stefan; Urbanus, Susan L; van Zuijlen, Lisette GC; Kaufmann, Kerstin; Angenent, Gerco C

2007-01-01

118

Applications of a peptide ligand for streptavidin: the Strep-tag  

Microsoft Academic Search

The Strep-tag constitutes a nine amino acid-peptide that binds specifically to streptavidin and occupies the same pocket where biotin is normally complexed. Since the Strep-tag participates in a reversible interaction it can be applied for the efficient purification of corresponding fusion proteins on affinity columns with immobilized streptavidin. Elution of the bound recombinant protein can be effected under mild buffer

Arne Skerra; Thomas G. M Schmidt

1999-01-01

119

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

PubMed

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 chromatography-mass 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-, (13)C6-, (15)N2-, or (15)N4-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 Plackett-Burman schemes followed by statistical evaluations. Results confirmed that the most effective approach to avoid chromatographic isotope effect is the use of (15)N or (13)C labeling instead of deuterium labeling, while chromatographic parameters had no general influence. Comparison of the alternate isotope-coded derivatization assay (AIDA) using deuterium versus (15)N 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 (15)N- or (13)C-labeled derivatizing reagent to avoid possible unfavorable consequences of chromatographic isotope effects. PMID:24922593

Szarka, Szabolcs; Prokai-Tatrai, Katalin; Prokai, Laszlo

2014-07-15

120

Implicit human-centered tagging [Social Sciences  

Microsoft Academic Search

Tagging is the annotation of multimedia data with userspecified keywords known as tags, with the aim of facilitating fast and accurate data retrieval based on these tags. In contrast to this process, also referred to as explicit tagging, implicit human-centered tagging (IHCT) refers to exploiting the information on user's nonverbal reactions (e.g., facial expressions like smiles or head gestures like

Maja Pantic; Alessandro Vinciarelli

2009-01-01

121

A dielectric affinity microbiosensor  

NASA Astrophysics Data System (ADS)

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

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

2010-01-01

122

HaloTag: a novel protein labeling technology for cell imaging and protein analysis.  

PubMed

We have designed a modular protein tagging system that allows different functionalities to be linked onto a single genetic fusion, either in solution, in living cells, or in chemically fixed cells. The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands). The synthetic ligands comprise a chloroalkane linker attached to a variety of useful molecules, such as fluorescent dyes, affinity handles, or solid surfaces. Covalent bond formation between the protein tag and the chloroalkane linker is highly specific, occurs rapidly under physiological conditions, and is essentially irreversible. We demonstrate the utility of this system for cellular imaging and protein immobilization by analyzing multiple molecular processes associated with NF-kappaB-mediated cellular physiology, including imaging of subcellular protein translocation and capture of protein--protein and protein--DNA complexes. PMID:18533659

Los, Georgyi V; Encell, Lance P; McDougall, Mark G; Hartzell, Danette D; Karassina, Natasha; Zimprich, Chad; Wood, Monika G; Learish, Randy; Ohana, Rachel Friedman; Urh, Marjeta; Simpson, Dan; Mendez, Jacqui; Zimmerman, Kris; Otto, Paul; Vidugiris, Gediminas; Zhu, Ji; Darzins, Aldis; Klaubert, Dieter H; Bulleit, Robert F; Wood, Keith V

2008-06-20

123

Presence of imidazole in loading buffer prevents formation of free radical in immobilized metal affinity chromatography and dramatically improves the recovery of herpes simplex virus type 1 gene therapy vectors  

Microsoft Academic Search

We have recently shown that immobilized metal affinity chromatography (IMAC) is an effective technique for purification of herpes simplex virus type 1 (HSV-1) gene vector engineered to display cobalt affinity tag on the envelope. However, the tagged HSV-1 viruses were severely inactivated by oxidative hydroxyl free radicals when crude HSV-1 supernatant was applied on an immobilized cobalt column and eluted

Canping Jiang; Joseph C Glorioso; Mohammad Ataai

2006-01-01

124

Semiotic dynamics and collaborative tagging.  

PubMed

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 cooccurrence. 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. PMID:17244704

Cattuto, Ciro; Loreto, Vittorio; Pietronero, Luciano

2007-01-30

125

Semiotic dynamics and collaborative tagging  

PubMed Central

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 cooccurrence. 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.

Cattuto, Ciro; Loreto, Vittorio; Pietronero, Luciano

2007-01-01

126

HaloTag-based purification of functional human kinases from mammalian cells.  

PubMed

Although cultured mammalian cells are preferred for producing functional mammalian proteins with appropriate post-translational modifications, purification of recombinant proteins is frequently hampered by low expression. We have addressed this by creating a new method configured specifically for mammalian cell culture that provides rapid detection and efficient purification. This approach is based on HaloTag, a protein fusion tag designed to bind rapidly, selectively and covalently to a series of synthetic ligands that can carry a variety of functional groups, including fluorescent dyes for detection or solid supports for purification. Since the binding of HaloTag to the HaloLink resin is essentially irreversible, it overcomes the equilibrium-based binding limitations associated with affinity tags and enables efficient capture and purification of target protein, even at low expression levels. The target protein is released from the HaloLink resin by specific cleavage using a TEV protease fused to HaloTag (HaloTEV), leaving both HaloTag and HaloTEV permanently attached to the resin and highly pure, tag-free protein in solution. HaloTag fluorescent ligands enable fluorescent labeling of HaloTag fusion proteins, providing a convenient way to monitor expression, and thus facilitate the identification of optimal transient transfection conditions as well as the selection of high expression stable cell lines. The capabilities of this method have been demonstrated by the efficient purification of five functional human kinases from HEK293T cells. In addition, when purifications using FLAG, 3xFLAG, His(6)Tag and HaloTag were performed in parallel, HaloTag was shown to provide significantly higher yields, purity and overall recovery of the expressed proteins. PMID:21129486

Ohana, Rachel Friedman; Hurst, Robin; Vidugiriene, Jolanta; Slater, Michael R; Wood, Keith V; Urh, Marjeta

2011-04-01

127

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

PubMed Central

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.

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

2013-01-01

128

Surface histidine mutations for the metal affinity purification of a ?-carbonic anhydrase.  

PubMed

Metal affinity chromatography using polyhistidine tags is a standard laboratory technique for the general purification of proteins from cellular systems, but there have been no attempts to explore whether the surface character of a protein may be engineered to similar affinity. We present the Arg160His mutation of Haemophilus influenzae carbonic anhydrase (HICA), which mimics the endogenous metal affinity of Escherichia coli carbonic anhydrase (ECCA). The purity and activity of the mutant are reported, and the purification is discussed. This is the first step toward developing a general method to engineer surface metal affinity for use in purification and metal labeling techniques. PMID:24792153

Hoffmann, Katherine M; Wood, Kaitlin M; Labrum, Alysha D; Lee, Dave K; Bolinger, Ingmar M; Konis, Mary E; Blount, Adam G; Prussia, Gregory A; Schroll, Monica M; Watson, Jeffrey M

2014-08-01

129

Immobilized metal affinity chromatography in the presence of arginine  

Microsoft Academic Search

Arginine hydrochloride (ArgHCl) is a versatile solvent additive, as it suppresses protein aggregation. ArgHCl has been used for protein refolding and to solubilize proteins from loose inclusion bodies. Immobilized metal affinity chromatography (IMAC) is one of the most commonly used technologies for purification of recombinant proteins. Here we have evaluated compatibility of ArgHCl with IMAC purification for his-tag proteins. ArgHCl

Ryota Abe; Motonori Kudou; Yoshikazu Tanaka; Tsutomu Arakawa; Kouhei Tsumoto

2009-01-01

130

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

Microsoft Academic Search

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

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

131

Chapter 27 Immobilized-Metal Affinity Chromatography (IMAC)  

Microsoft Academic Search

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

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

2009-01-01

132

D-TAG: erasing the tag of gang membership.  

PubMed

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

Gurke, B; Armstrong, M L

1997-04-01

133

Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands.  

PubMed

Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenable to modular synthetic design. Taken together, the design features and molecular evolution of HT7 have resulted in a superior alternative to common tags for the overexpression, detection, and isolation of target proteins. PMID:23248739

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

2012-01-01

134

Behavioral tagging of extinction learning  

PubMed Central

Extinction of contextual fear in rats is enhanced by exposure to a novel environment at 1–2 h before or 1 h after extinction training. This effect is antagonized by administration of protein synthesis inhibitors anisomycin and rapamycin into the hippocampus, but not into the amygdala, immediately after either novelty or extinction training, as well as by the gene expression blocker 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole administered after novelty training, but not after extinction training. Thus, this effect can be attributed to a mechanism similar to synaptic tagging, through which long-term potentiation can be enhanced by other long-term potentiations or by exposure to a novel environment in a protein synthesis-dependent fashion. Extinction learning produces a tag at the appropriate synapses, whereas novelty learning causes the synthesis of plasticity-related proteins that are captured by the tag, strengthening the synapses that generated this tag.

de Carvalho Myskiw, Jociane; Benetti, Fernando; Izquierdo, Ivan

2013-01-01

135

Sensitivity of tag-recovery mortality estimates to inaccuracies in tag shedding, handling mortality, and tag reporting  

Microsoft Academic Search

We used Monte Carlo simulations to evaluate the sensitivity of tag-recovery mortality estimates to inaccuracies in tag shedding, handling mortality, and tag reporting. The data-generating model used in the simulations assumed that tagging was conducted annually for 4 years with tag recoveries occurring over a 4-year period. Several different combinations of instantaneous fishing (F) and natural (M) mortality were evaluated

Travis O. Brenden; Michael L. Jones; Mark P. Ebener

2010-01-01

136

A repertoire of Peptide tags for controlled drug release from injectable noncovalent hydrogel.  

PubMed

A repertoire of conjugable tags for controlling the release of drugs from biomaterials is highly interesting for the development of combinatorial drug administration techniques. This paper describes such a system of 11 peptide tags derived from our previous work on a physical hydrogel system cross-linked through peptide-heparin interactions. The release kinetics of the tags correlate well with their affinity to heparin and obey Fick's second law of diffusion, with the exception of the ATIII peptide, which displays a stable release profile close to a zero-order reaction. A system for release experiments over seven months was built, using the hydrogel matrix as a barrier between the reservoirs of tagged compounds and supernatant. The gel matrix can be injected without affecting the releasing properties. A tagged cyclosporin A derivative was also tested, and its release was monitored by measuring its biological activity. This work represents a design of biomaterials with an integral system of drug delivery, where both the assembly process of the matrix and affinity capture/release of tagged compounds are based on the noncovalent interaction of heparin with one class of peptides. PMID:24825401

Wieduwild, Robert; Lin, Weilin; Boden, Annett; Kretschmer, Karsten; Zhang, Yixin

2014-06-01

137

Usage patterns of collaborative tagging systems  

Microsoft Academic Search

Collaborative tagging describes the process by which many users add metadata in the form of keywords to shared content. Recently, collaborative tagging has grown in popularity on the web, on sites that allow users to tag bookmarks, photographs and other content. In this paper we analyze the structure of collaborative tagging systems as well as their dynamic aspects. Specifically, we

Scott A. Golder; Bernardo A. Huberman

2006-01-01

138

The Structure of Collaborative Tagging Systems  

Microsoft Academic Search

Collaborative tagging describes the process by which many users add metadata in the form of keywords to shared content. Recently, collaborative tagging has grown in popularity on the web, on sites that allow users to tag bookmarks, photographs and other content. In this paper we analyze the structure of collaborative tagging systems as well as their dynamical aspects. Specifically, we

Scott A. Golder; Bernardo A. Huberman

2005-01-01

139

Identifying RFID tag categories in linear time  

Microsoft Academic Search

Given a large set of RFID tags, we are interested in determining the categories of tags that are present in the shortest time possible. Since there can be more than one tag present in a particular category, pure randomized strategies that rely on resolving individual tags are very inefficient. Instead, we rely on a pseudo-random strategy that utilizes a uniform

Murali Kodialam; Wing Cheong Lau; Thyaga Nandagopal

2009-01-01

140

The affine matched filter  

Microsoft Academic Search

The hyperspectral matched filter (MF) is a popular tool in remote sensing problems for locating objects that extend over several pixels. However, it is ideally suited only for the detection of sub-pixel targets with known mean signature in radiance space. Here we develop an alternative affine matched filter (AMF) that is more appropriate for detecting extended targets, and which accommodates

A. Schaum; Richard Priest

2009-01-01

141

Affine field theories  

SciTech Connect

The author constructs a non-Abelian field theory by gauging a Kac-Moody algebra, obtaining an infinite tower of interacting vector fields and associated ghosts, that obey slightly modified Feynman rules. She discusses the spontaneous symmetry breaking of such theory via the Higgs mechanism. If the Higgs particle lies in the Cartan subalgebra of the Kac-Moody algebra, the previously massless vectors acquire a mass spectrum that is linear in the Kac-Moody index and has additional fine structure depending on the associated Lie algebra. She proceeds to show that there is no obstacle in implementing the affine extension of supersymmetric Yang-Mills theories. The result is valid in four, six and ten space-time dimensions. Then the affine extension of supergravity is investigated. She discusses only the loop algebra since the affine extension of the super-Poincare algebra appears inconsistent. The construction of the affine supergravity theory is carried out by the group manifold method and leads to an action describing infinite towers of spin 2 and spin 3/2 fields that interact subject to the symmetries of the loop algebra. The equations of motion satisfy the usual consistency check. Finally, she postulates a theory in which both the vector and scalar fields lie in the loop algebra of SO(3). This theory has an expanded soliton sector, and corresponding to the original 't Hooft-Polyakov solitonic solutions she now finds an infinite family of exact, special solutions of the new equations. She also proposes a perturbation method for obtaining an arbitrary solution of those equations for each level of the affine index.

Cadavid, A.C.

1989-01-01

142

Noisy Tags: A Pretty Good Key Exchange Protocol for RFID Tags  

Microsoft Academic Search

We propose a protocol that can be used between an RFID tag and a reader to exchange a secret without performing any expensive computation. Similarly to the famous blocker tag suggested by Juels, Rivest, and Szydlo, our scheme makes use of special tags that we call noisy tags. Noisy tags are owned by the reader's manager and set out within

Claude Castelluccia; Gildas Avoine

2006-01-01

143

A System for Recommending Tags of Images Using Co-Occurrence of Tags and Similar Images  

Microsoft Academic Search

In this paper, we propose a system for recommending tags of images. The proposed method presents to its user various tags with high precision by taking into account both the co-occurrence of tags and tags of similar images. Additional search with the user feedback enables us to find some new tags relevant to the image of interest. In order to

Naoto Sezaki; Koichi Kise

2009-01-01

144

Radio Tag Retention and Tag-Related Mortality among Adult Sockeye Salmon  

Microsoft Academic Search

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

Kristina M. Ramstad; Carol Ann Woody

2003-01-01

145

Learning to Tag Multilingual Texts Through Observation  

Microsoft Academic Search

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

Scott W. Bennett; Chinatsu Aone; Craig Lovell

1997-01-01

146

Iminodiacetic acid functionalized porous hydroxyapatite nanoparticles for capturing histidine-tagged proteins.  

PubMed

A simple strategy has been developed to synthesize hydroxyapatite (HAP) nanoparticles (NPs) in a simulated body fluid (SBF). The HAP NPs have an average diameter of 50nm and present porous structure. By taking advantage of surface hydroxyl groups, the HAP NPs are further modified with iminodiacetic acid (IDA), followed by chelating Ni(2+) ions. The HAP/IDA-Ni(2+) NPs as novel adsorbent can capture directly histidine-tagged (His-tagged) proteins from the mixture of lysed cells without sample pretreatment. Results indicated that the HAP/IDA-Ni(2+) NPs present negligible nonspecific adsorption and high protein binding ability, and their specificity and affinity toward His-tagged proteins can remain after 5 times of recycling. The HAP/IDA-Ni(2+) NPs are especially suitable for purification of His-tagged proteins with low molecule weight. PMID:24863189

Yao, Shasha; Huang, Yanqin; Zhao, Yanbao; Zhang, Yu; Zou, Xueyan; Song, Chunpeng

2014-06-01

147

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

NASA Astrophysics Data System (ADS)

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.

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

2012-04-01

148

Antibodies with infinite affinity  

Microsoft Academic Search

Here we report an approach to the design and production of antibody\\/ligand pairs, to achieve functional affinity far greater than avidin\\/biotin. Using fundamental chemical principles, we have developed antibody\\/ligand pairs that retain the binding specificity of the antibody, but do not dissociate. Choosing a structurally characterized antibody\\/ligand pair as an example, we engineered complementary reactive groups in the antibody binding

Albert J. Chmura; Molly S. Orton; Claude F. Meares

2001-01-01

149

Phase-sensitive cardiac tagging--REALTAG.  

PubMed

Fully inverting spins, instead of merely saturating them, provides superior contrast for tagging procedures. The resulting improvement in tag contrast-to-noise ratio (CNR) yields higher-precision tag detection. Also, thinner slices and hence reduced tag separations can be employed, providing displacement and strain measurements with better spatial resolution. Alternatively, the improved tag contrast can be used to obtain cine images covering a greater portion of the cardiac cycle. The use of standard magnitude reconstruction for images of these inversion tags causes rectification of the negative-valued signals from the tags, confounding the image interpretation. Therefore, a phase-sensitive reconstruction scheme of the inverted tags must be employed. Here we demonstrate the implementation of inverted tags with phase-sensitive reconstruction in a ramped-flip-angle, steady-state free precession (SSFP) sequence. PMID:17659612

Derbyshire, J Andrew; Sampath, Smita; McVeigh, Elliot R

2007-07-01

150

Antibodies with infinite affinity.  

PubMed

Here we report an approach to the design and production of antibody/ligand pairs, to achieve functional affinity far greater than avidin/biotin. Using fundamental chemical principles, we have developed antibody/ligand pairs that retain the binding specificity of the antibody, but do not dissociate. Choosing a structurally characterized antibody/ligand pair as an example, we engineered complementary reactive groups in the antibody binding pocket and the ligand, so that they would be in close proximity in the antibody/ligand complex. Cross-reactions with other molecules in the medium are averted because of the low reactivity of these groups; however, in the antibody/ligand complex the effective local concentrations of the complementary reactive groups are very large, allowing a covalent reaction to link the two together. By eliminating the dissociation of the ligand from the antibody, we have made the affinity functionally infinite. This chemical manipulation of affinity is applicable to other biological binding pairs. PMID:11447282

Chmura, A J; Orton, M S; Meares, C F

2001-07-17

151

SparTag.us: a low cost tagging system for foraging of web content  

Microsoft Academic Search

Tagging systems such as del.icio.us and Diigo have become important ways for users to organize information gathered from the Web. However, despite their popularity among early adopters, tagging still incurs a relatively high interaction cost for the general users. We introduce a new tagging system called SparTag.us, which uses an intuitive Click2Tag technique to provide in situ, low cost tagging

Lichan Hong; Ed H. Chi; Raluca Budiu; Peter Pirolli; Les Nelson

2008-01-01

152

Synaptic Tagging During Memory Allocation  

PubMed Central

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.

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

2014-01-01

153

WebTag: Web Browsing into Sensor Tags over NFC  

PubMed Central

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

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

2012-01-01

154

WebTag: Web browsing into sensor tags over NFC.  

PubMed

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

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

2012-01-01

155

An alternative tandem affinity purification strategy applied to Arabidopsis protein complex isolation  

Microsoft Academic Search

Summary Tandem affinity purification (TAP) strategies constitute an efficient approach for protein complex purification from many different organisms. However, the application of such strategies for purifying endogenous Arabidopsis multi-protein complexes has not yet been reported. Here, we describe an alternative TAP (TAPa) system that successfully allows protein complex purification from Arabidopsis. In our newly generated TAPa tag we have replaced

Vicente Rubio; Yunping Shen; Yusuke Saijo; Yule Liu; Giuliana Gusmaroli; Savithramma P. Dinesh-Kumar; Xing Wang Deng

2005-01-01

156

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

NASA Astrophysics Data System (ADS)

-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

Sekine-Aizawa, Yoko; Huganir, Richard L.

2004-12-01

157

Preparation of hollow nickel silicate nanospheres for separation of His-tagged proteins.  

PubMed

Hollow nickel silicate nanospheres (NiSiO3 NSs) with hierarchical shells were hydrothermally synthesized by using silica spheres as a template. The NiSiO3 NSs have an average diameter of 250 nm with a shell thickness of 50 nm, and the hierarchical shell consists of a large number of sheets. By taking advantage of the high affinity of Ni(2+) toward histidine-tagged (His-tagged) proteins, hollow NiSiO3 NSs can be used to enrich and separate His-tagged proteins directly from a mixture of lysed cells. Results indicated that the hollow NiSiO3 NSs presented negligible nonspecific protein adsorption and a high protein binding ability with a high binding capacity of 13.2 mmol g(-1). Their specificity and affinity toward His-tagged proteins remained after recycling 5 times. The hollow NiSiO3 NSs are especially suitable for rapid purification of His-tagged proteins. PMID:24149676

Wu, Yonghui; Chang, Guanxiao; Zhao, Yanbao; Zhang, Yu

2014-01-14

158

The structure of the SBP-Tag-streptavidin complex reveals a novel helical scaffold bridging binding pockets on separate subunits  

PubMed Central

The 38-residue SBP-Tag binds to streptavidin more tightly (K d ? 2.5–4.9?nM) than most if not all other known peptide sequences. Crystallographic analysis at 1.75?Å 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 12–14) and a C-­terminal HPQ sequence (residues 31–33) 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 17–28) 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 1–10 and 35–38 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 11–34 (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-Tag–streptavidin complex and establish a minimal bivalent streptavidin-binding tag from which further rational design and optimization can proceed.

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

2013-01-01

159

Harmonic scattering from passive UHF RFID tags  

Microsoft Academic Search

This paper discusses harmonic scattering from passive UHF RFID tags. We describe the problem and the basic theory; explain our measurement setup, and present experimental results for three different commercial Gen2 tags.

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

2009-01-01

160

Freedom System Text and Graphics System (TAGS)  

NASA Technical Reports Server (NTRS)

The Text and Graphics System (TAGS) is a high-resolution facsimile system that scans text or graphics material and converts the analog SCAN data into serial digital data. This video shows the TAGS in operation.

1989-01-01

161

Buddy Tag's motion sensing and analysis subsystem  

SciTech Connect

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

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

1991-01-01

162

High affinity tamoxifen derivatives  

US Patent & Trademark Office Database

The synthesis of tamoxifen derivatives, most particularly halo, halo alkyl, hydroxy, and amino tamoxifen derivatives is disclosed. The native tamoxifen molecule includes a substituted chemical group positioned on the aliphatic chain of the tamoxifen molecule. Particular tamoxifen derivatives of the invention include chloro, bromo, iodo, fluoro, amino and DTPA tamoxifen derivatives, and corresponding lower alkyl halogenated forms. The halogenated tamoxifen derivatives possess superior binding affinities for estrogen receptor rich tissues, such as uterine tissue and breast tissue, relative to unsubstituted native tamoxifen. Radiolabeled forms of the tamoxifen derivatives may be used as highly specific imaging agents for estrogen receptor rich tissues. The fluoro and bromo tamoxifen derivatives are particularly useful for imaging estrogen receptors by PET whereas the iodinated tamoxifens are particularly useful in imaging estrogen receptors by SPECT. Rapid and efficient methods of preparing the tamoxifen derivatives having high specific activity (>6 Ci/.mu.mol) are also disclosed. Aliphatic chain substituted tamoxifen derivatives are shown to possess greater estrogen receptor binding affinity and more potent tumor cell inhibition than tamoxifen or tamoxifen derivatives substituted at other locations on the molecule (i.e., non-aliphatic chain substituted tamoxifen). The tanioxifen derivatives of the present invention may advantageously be used as anti-cancer therapeutic agents to halt estrogen-receptor positive tumors, such as those of breast and uterine tissue. The present invention also provides a hydrophilic DTPA-tamnoxifen analogue, and uses thereof in imaging estrogen receptor positive ER+ lesions.

2000-08-01

163

Tag Gardening for Folksonomy Enrichment and Maintenance  

Microsoft Academic Search

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

Isabella Peters; Katrin Weller

2008-01-01

164

Requesting Pervasive Services by Touching RFID Tags  

Microsoft Academic Search

We suggest a general framework for requesting pervasive services by touching RFID tags. The tags conne ct the physical and digital environments. Visual symbols c ommunicate to users the objects that can be touched and the services that can be activated. When a user touches such a symbol with a mobile phone, the data stored in the tag and other

Jukka Riekki; Timo Salminen; Ismo Alakärppä

2006-01-01

165

Efficient Object Identification with Passive RFID Tags  

Microsoft Academic Search

Radio frequency identification systems with passive tags are power- ful tools for object identification. However, if multiple tags are to be identified simultaneously, messages from the tags can collide and cancel each other out. Therefore, multiple read cycles have to be performed in order to achieve a high recognition rate. For a typical stochastic anti-collision scheme, we show how to

Harald Vogt

2002-01-01

166

Multiple object identification with passive RFID tags  

Microsoft Academic Search

We investigate the applicability of passive RFID systems to the task of identifying multiple tagged objects simultaneously, assuming that the number of tags is not known in advance. We present a combinatorial model of the communication mechanism between the reader device and the tags, and use this model to derive the optimal parameter setting for the reading process, based on

Harald Vogt

2002-01-01

167

Method for designing gas tag compositions  

DOEpatents

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.

Gross, K.C.

1995-04-11

168

Method for designing gas tag compositions  

DOEpatents

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.

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

1995-01-01

169

Use of META Tags for Internet Documents  

Microsoft Academic Search

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

Elaine A. Nowick

2002-01-01

170

Using Blog Tags To Identify Topic Authorities  

Microsoft Academic Search

The Web has experienced an exponential growth in the use of weblogs or blogs. Blog entries are generally organised using tags, informally defined labels which are increasingly being proposed as a 'grassroots' answer to Semantic Web standards. Despite this, tags have been shown to be weak at partition- ing blog data. In this paper, we demonstrate how tags pro- vide

Conor Hayes ITC-IRST; Paolo Avesani ITC-IRST

171

Tagging as a Social Literacy Practice  

ERIC Educational Resources Information Center

Tagging is not simply an act of vandalism or violence; it is a social practice with its own rules and codes--a literacy practice imbued with intent and meaning. Three aspects of tagging reflect its nature as a literate practice: (1) The purpose of tagging to achieve particular social goals and group affiliations; (2) The role of talent to be…

MacGillivray, Laurie; Curwen, Margaret Sauceda

2007-01-01

172

Low cost silver ink RFID tag antennas  

Microsoft Academic Search

We discuss silver ink as a low cost option for manufacturing RFID tags at ultra high frequencies (UHF). An analysis of two different RFID tag antennas, made from silver ink and from copper, is presented at UHF. The influence of each material on tag performance is discussed along with simulation results and measurement data which are in good agreement. It

P. V. Nikitin; S. Lam; K. V. S. Rao

2005-01-01

173

Active tag emulation for pedestrian localization applications  

Microsoft Academic Search

Active tags are currently used for various tasks in transport and distribution industries, for factory automation or asset tracking. Pedestrian localization is another application of active tags, for which we develop a technique and a practical system. Following preliminary real-world experiments with a system prototype, we continued to develop our active tag based pedestrian localization technique by an emulation approach

Razvan Beuran; Junya Nakata; Yoshihiro Suzuki; Tetsuya Kawakami; Ken-ichi Chinen; Yasuo Tan; Yoichi Shinoda

2008-01-01

174

Infrared tag and track technique  

SciTech Connect

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.

Partin, Judy K. (Idaho Falls, ID); Stone, Mark L. (Idaho Falls, ID); Slater, John (Albuquerque, NM); Davidson, James R. (Idaho Falls, ID)

2007-12-04

175

Electronic Tag and Position Sensor  

SciTech Connect

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.

Not Available

1992-01-20

176

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

PubMed Central

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

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

2012-01-01

177

Click chemistry facilitates formation of reporter ions and simplified synthesis of amine-reactive multiplexed isobaric tags for protein quantification.  

PubMed

We report the development of novel reagents for cell-level protein quantification, referred to as Caltech isobaric tags (CITs), which offer several advantages in comparison with other isobaric tags (e.g., iTRAQ and TMT). Click chemistry, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), is applied to generate a gas-phase cleavable linker suitable for the formation of reporter ions. Upon collisional activation, the 1,2,3-triazole ring constructed by CuAAC participates in a nucleophilic displacement reaction forming a six-membered ring and releasing a stable cationic reporter ion. To investigate its utility in peptide mass spectrometry, the energetics of the observed fragmentation pathway are examined by density functional theory. When this functional group is covalently attached to a target peptide, it is found that the nucleophilic displacement occurs in competition with formation of b- and y-type backbone fragment ions regardless of the amino acid side chains present in the parent bioconjugate, confirming that calculated reaction energetics of reporter ion formation are similar to those of backbone fragmentations. Based on these results, we apply this selective fragmentation pathway for the development of CIT reagents. For demonstration purposes, duplex CIT reagent is prepared using a single isotope-coded precursor, allyl-d(5)-bromide, with reporter ions appearing at m/z 164 and 169. Isotope-coded allyl azides for the construction of the reporter ion group can be prepared from halogenated alkyl groups which are also employed for the mass balance group via N-alkylation, reducing the cost and effort for synthesis of isobaric pairs. Owing to their modular designs, an unlimited number of isobaric combinations of CIT reagents are, in principle, possible. The reporter ion mass can be easily tuned to avoid overlapping with common peptide MS/MS fragments as well as the low mass cutoff problems inherent in ion trap mass spectrometers. The applicability of the CIT reagent is tested with several model systems involving protein mixtures and cellular systems. PMID:22225568

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

2012-02-01

178

His-tag protein monitoring by a fast mix-and-measure immunoassay.  

PubMed

Here, we present a fast mix-and-measure immunoassay for the specific semiquantitative detection of His-tagged proteins, for example in E. coli cell lysate. The assay is based on Förster resonance energy transfer (FRET) between a lanthanide dye-labeled low-affinity His-peptide and an acceptor-labeled anti-His-tag antibody. The targeted His-tag protein in the sample displaces the donor-labeled peptide and leads to a concentration-dependent time-resolved fluorescence signal. The assay has a total assay time of less than two minutes including sample preparation. The assay recognizes both, N- and C-terminally tagged proteins. The detection limit is comparable to those obtained in SDS-PAGE or Western Blot, which are used as standard methods for the characterization of His-tag protein expression. Additionally, we demonstrate a full compatibility of the developed assay to cell lysate, and a correlation to detectable bands in a western blot application. In conclusion, this fast, sensitive, specific and affordable mix-and-measure assay provides a timesaving and user-friendly way to quantify recombinant protein expression. It substantially reduces the workload for recombinant protein detection, especially when His-tag-protein-containing fractions in manual chromatographic purifications have to be identified. PMID:25000910

Kreisig, Thomas; Prasse, Agneta A; Zscharnack, Kristin; Volke, Daniela; Zuchner, Thole

2014-01-01

179

His-tag protein monitoring by a fast mix-and-measure immunoassay  

PubMed Central

Here, we present a fast mix-and-measure immunoassay for the specific semiquantitative detection of His-tagged proteins, for example in E. coli cell lysate. The assay is based on Förster resonance energy transfer (FRET) between a lanthanide dye-labeled low-affinity His-peptide and an acceptor-labeled anti-His-tag antibody. The targeted His-tag protein in the sample displaces the donor-labeled peptide and leads to a concentration-dependent time-resolved fluorescence signal. The assay has a total assay time of less than two minutes including sample preparation. The assay recognizes both, N- and C-terminally tagged proteins. The detection limit is comparable to those obtained in SDS-PAGE or Western Blot, which are used as standard methods for the characterization of His-tag protein expression. Additionally, we demonstrate a full compatibility of the developed assay to cell lysate, and a correlation to detectable bands in a western blot application. In conclusion, this fast, sensitive, specific and affordable mix-and-measure assay provides a timesaving and user-friendly way to quantify recombinant protein expression. It substantially reduces the workload for recombinant protein detection, especially when His-tag-protein-containing fractions in manual chromatographic purifications have to be identified.

Kreisig, Thomas; Prasse, Agneta A.; Zscharnack, Kristin; Volke, Daniela; Zuchner, Thole

2014-01-01

180

Adaptive binary splitting for efficient RFID tag anti-collision  

Microsoft Academic Search

Tag collision arbitration for passive RFID tags is a significant issue for fast tag identification. This letter presents a novel tag anti-collision scheme called adaptive binary splitting (ABS). For reducing collisions, ABS assigns distinct timeslots to tags by using information obtained from the last identification process. Our performance evaluation shows that ABS outperforms other tree based tag anti-collision protocols.

Jihoon Myung; Wonjun Lee; J. Srivastava

2006-01-01

181

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

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.

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

2009-01-01

182

Identification of RNA aptamers against recombinant proteins with a hexa-histidine tag.  

PubMed

Artificial riboswitches that respond to the concentrations of intracellular proteins are a promising tool with a variety of applications. They can be designed and engineered using existing RNA aptamers that target proteins. Aptamers are generated via an iterative selection-amplification process, known as systematic evolution of ligands by exponential enrichment (SELEX). This chapter describes a SELEX procedure for the identification of RNA aptamers against hexa-histidine-tagged proteins. For the efficient enrichment of higher affinity aptamers, the selection stringency should be gradually increased. Undesired RNA species that bind to affinity resins can be eliminated from the pool by using a negative selection step and alternating different types of resins. PMID:24549611

Ohuchi, Shoji

2014-01-01

183

Functional Differentiation of Bundle Sheath and Mesophyll Maize Chloroplasts Determined by Comparative Proteomics  

Microsoft Academic Search

Chloroplasts of maize (Zea mays) leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C4 photosynthesis. Consequences for other plastid functions are not well understood but are addressed here through a quantitative comparative proteome analysis of purified M and BS chloroplast stroma. Three independent techniques were used, including cleavable stable isotope coded affinity tags. Enzymes involved

Wojciech Majeran; Yang Cai; Qi Sun; Klaas J. van Wijka

2005-01-01

184

Directional Radio-Frequency Identification Tag Reader  

NASA Technical Reports Server (NTRS)

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).

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

2004-01-01

185

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

Microsoft Academic Search

Affinity tagging has been used in many global stu- dies towards protein function. We describe a highly efficient system for in vivo biotinylation of transcrip- tion factors in the yeast Saccharomyces cerevisiae, which is based on the bacterial BirA biotin ligase. The strength of the biotin-streptavidin interaction was exploited to improve detection of in vivo protein-DNA complexes in chromatin immunopre-

Folkert J. van Werven; Marc Timmers

2006-01-01

186

Electron Affinity Calculations for Thioethers  

NASA Technical Reports Server (NTRS)

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.

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

1997-01-01

187

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

PubMed Central

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

Strohmaier, Markus; Korner, Christian; Kern, Roman

2012-01-01

188

Securing RFID Systems by Detecting Tag Cloning  

Microsoft Academic Search

Cloning of RFID tags can lead to financil losses in many commercial RFID applications. There are two general strategies to\\u000a provide security: prevention and detection. The security community and the RFID chip manufacturers are currently focused on\\u000a the former by making tags hard to clone. This paper focuses on the latter by investigating a method to pinpoint tags with\\u000a the

Mikko Lehtonen; Daniel Ostojic; Alexander Ilic; Florian Michahelles

2009-01-01

189

Emulation system for active tag applications  

Microsoft Academic Search

In this paper we present an active tag emulation system that is used to carry out live experiments with active tag applications. Its two main components are the wireless communication emulator QOMET, and an active tag processor emulator. Experiments are performed using the experiment-support software RUNE on StarBED, the large-scale network testbed at the Hokuriku Research Center of the National

Razvan Beuran; Junya Nakata; Takashi Okada; Tetsuya Kawakami; Ken-ichi Chinen; Yasuo Tan; Yoichi Shinoda

2008-01-01

190

Minimal affinizations as projective objects  

NASA Astrophysics Data System (ADS)

We prove that the specialization to q=1 of a Kirillov-Reshetikhin module for an untwisted quantum affine algebra of classical type is projective in a suitable category. This yields a uniform character formula for the Kirillov-Reshetikhin modules. We conjecture that these results hold for specializations of minimal affinization with some restriction on the corresponding highest weight. We discuss the connection with the conjecture of Nakai and Nakanishi on q-characters of minimal affinizations. We establish this conjecture in some special cases. This also leads us to conjecture an alternating sum formula for Jacobi-Trudi determinants.

Chari, Vyjayanthi; Greenstein, Jacob

2011-03-01

191

Tandem Affinity Purification Combined with Mass Spectrometry to Identify Components of Protein Complexes  

PubMed Central

Most biological processes are governed by multiprotein complexes rather than individual proteins. Identification of protein complexes therefore is becoming increasingly important to gain a molecular understanding of cells and organisms. Mass spectrometry–based proteomics combined with affinity-tag-based protein purification is one of the most effective strategies to isolate and identify protein complexes. The development of tandem-affinity purification approaches has revolutionized proteomics experiments. These two-step affinity purification strategies allow rapid, effective purification of protein complexes and, at the same time, minimize background. Identification of even very low-abundant protein complexes with modern sensitive mass spectrometers has become routine. Here, we describe two general strategies for tandem-affinity purification followed by mass spectrometric identification of protein complexes.

Kaiser, Peter; Meierhofer, David; Wang, Xiaorong; Huang, Lan

2011-01-01

192

Identification of secreted bacterial proteins by noncanonical amino acid tagging  

PubMed Central

Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy.

Mahdavi, Alborz; Szychowski, Janek; Ngo, John T.; Sweredoski, Michael J.; Graham, Robert L. J.; Hess, Sonja; Schneewind, Olaf; Mazmanian, Sarkis K.; Tirrell, David A.

2014-01-01

193

Identification of secreted bacterial proteins by noncanonical amino acid tagging.  

PubMed

Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy. PMID:24347637

Mahdavi, Alborz; Szychowski, Janek; Ngo, John T; Sweredoski, Michael J; Graham, Robert L J; Hess, Sonja; Schneewind, Olaf; Mazmanian, Sarkis K; Tirrell, David A

2014-01-01

194

Minimizing adsorption of histidine-tagged proteins for the study of protein-deoxyribonucleic acid interactions by kinetic capillary electrophoresis.  

PubMed

Affinity interactions between DNA and proteins play a crucial role in many cellular processes. Kinetic Capillary Electrophoresis is a highly efficient tool for kinetic and equilibrium studies of protein-DNA interactions. Recombinant proteins, which are typically used for in vitro studies of protein-DNA interactions, are often expressed with a His tag to aid in their purification. In this work, we study how His tags affect Kinetic Capillary Electrophoresis analysis of protein-DNA interactions. We found that the addition of a His tag can increase or decrease protein adsorption to a bare-silica capillary wall, dependent on the protein. For Kinetic Capillary Electrophoresis measurements, it is essential to have as little protein adsorption as possible. We screened a number of capillary coatings to reduce adsorption of the His-tagged DNA mismatch repair protein MutS to the capillary wall and found that UltraTrol LN was the most effective coating. The effectiveness of the coating was confirmed with the prevention of adsorption of His-tagged fat mass and obesity-associated protein. Under typical conditions, the coating reduced protein adsorption to a level at which accurate Kinetic Capillary Electrophoresis analysis of protein-DNA interactions was possible. We further used Kinetic Capillary Electrophoresis to study how the His tag affected Kd of protein-DNA interactions for the MutS protein. Using UltraTrol LN, we found that the effect of the His tag was insignificant. PMID:24275486

Liyanage, Ruchi; Krylova, Svetlana M; Krylov, Sergey N

2013-12-27

195

Survival and Tag Retention of Pacific Lamprey Larvae and Macrophthalmia Marked with Coded Wire Tags  

Microsoft Academic Search

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%

Michael H. Meeuwig; Amy L. Puls; Jennifer M. Bayer

2007-01-01

196

Impedance Characterization of RFID Tag Antennas and Application in Tag Co-Design  

Microsoft Academic Search

In this paper, an experimental methodology for the characterization of the impedance of balanced RF identification (RFID) tag antennas is presented, and the application of the proposed method in RFID tag co-design is demonstrated. The balanced tag antenna is considered as a two-port network and the impedance of the antenna is characterized using network parameters. In the measurement, the antenna

Xianming Qing; Chean Khan Goh; Zhi Ning Chen

2009-01-01

197

Beyond tag relevance: integrating visual attention model and multi-instance learning for tag saliency ranking  

Microsoft Academic Search

Tag ranking has emerged as an important research topic recently due to its potential application on web image search. Conventional tag ranking approaches mainly rank the tags according to their relevance levels with respect to a given image. Nonetheless, such algorithms heavily rely on the large-scale image dataset and the proper similarity measurement to retrieve semantic relevant images with multi-labels.

Songhe Feng; Congyan Lang; De Xu

2010-01-01

198

Towards a Noise-Tagging Auditory BCI-Paradigm  

Microsoft Academic Search

Stimulus tagging is an important technique for investigating the functional operation of the brain. In this paper we propose the novel noise tagging method as an alternative to the more commonly used frequency tagging technique. Noise tagging is based on spread-spectrum signal processing techniques and has a number of theoretical advantages over frequency tagging in terms of noise robustness and

J. Farquhar; J. Blankespoor; R. Vlek; P. Desain

199

Evaluation of visible implant elastomer tags in zebrafish (Danio rerio).  

PubMed

The use of the visible implant elastomer (VIE) tagging system in zebrafish (Danio rerio) was examined. Two tag orientations (horizontal and vertical) at the dorsal fin base were tested for tag retention, tag fragmentation and whether VIE tags affected growth and survival of juvenile zebrafish (1-4 month post hatch). Six tag locations (abdomen, anal fin base, caudal peduncle, dorsal fin base, pectoral fin base, isthmus) and 5 tag colors (yellow, red, pink, orange, blue) were evaluated for ease of VIE tag application and tag visibility in adult zebrafish. Long-term retention (1 year) and multiple tagging sites (right and left of dorsal fin and pectoral fin base) were examined in adult zebrafish. Lastly, survival of recombination activation gene 1(-/-) (rag1(-/-)) zebrafish was evaluated after VIE tagging. The best tag location was the dorsal fin base, and the most visible tag color was pink. Growth rate of juvenile zebrafish was not affected by VIE tagging. Horizontal tagging is recommended in early stages of fish growth (1-2 months post hatch). VIE tags were retained for 1 year and tagging did not interfere with long-term growth and survival. There was no mortality associated with VIE tagging in rag1(-/-) zebrafish. The VIE tagging system is highly suitable for small-sized zebrafish. When familiar with the procedure, 120 adult zebrafish can be tagged in one hour. It does not increase mortality in adult zebrafish or interfere with growth in juvenile or adult zebrafish. PMID:24285706

Hohn, Claudia; Petrie-Hanson, Lora

2013-01-01

200

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

SciTech Connect

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.

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

201

Production of a His-tagged canecystatin in transgenic sugarcane.  

PubMed

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

Henrique-Silva, Flavio; Soares-Costa, Andrea

2012-01-01

202

Photodissociation of Charge Tagged Peptides  

NASA Astrophysics Data System (ADS)

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.

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

2012-07-01

203

Transposon tagging in diploid strawberry.  

PubMed

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

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

204

Identification of Signaling Protein Complexes by Parallel Affinity Precipitation Coupled with Mass Spectrometry  

PubMed Central

Protein–protein interactions play a pivotal role in both inter- and intra-cellular signaling. Identification of signaling protein complexes can thus shed important new insights into cell communications. We developed a parallel affinity precipitation protocol to overcome the disadvantages of the tandem affinity purification procedure, such as the potential disruption of target protein conformation, subcellular localization or function by epitope tags, the potential need of large amounts of cell culture or generation of stable cell lines, and relatively long duration the two-step precipitation takes. This new simplified assay of protein interaction is quick, economic and specific. This paper describes the details in the design and method of the assay.

Lu, Heng; Lin, Qishan; Zhao, Jihe

2014-01-01

205

Environmental effects on RFID tag antennas  

Microsoft Academic Search

We have studied the effects of nearby objects on the read range of several types of RFID tags, and the impedance, pattern, and radiative efficiency of antennas that closely emulate the tag structures, using measurements and simulations. We find that the main reason for the decrease in read range at perpendicular incidence in close proximity to metals or dielectrics (such

Daniel M. Dobkin; Steven M. Weigand

2005-01-01

206

Revisting Tag Collision Problem in RFID Systems  

Microsoft Academic Search

In RFID systems, the reader is unable to discriminate concurrently reported IDs of tags from the overlapped signals, and a collision happens. Many algorithms for anticollision are proposed to improve the throughput and reduce the latency for tag identification. Existing anti-collision algorithms mainly employ CRC based collision detection functions for determining whether the collision happens. Generating CRC codes, however, requires

Lei Yang; Jinsong Han; Yong Qi; Cheng Wang; Yunhao Liu; Ying Cheng; Xiao Zhong

2010-01-01

207

Tagging English Text with a Probabilistic Model  

Microsoft Academic Search

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

Bernard Merialdo

1994-01-01

208

Harmonic radar identification tag for insect tracking  

Microsoft Academic Search

Design considerations and simulations are presented for an identification tag to be used in tracking insects through the use of harmonic radar. Radar is used instead of a radio transmitter in order to minimize the loading of the insect which for the Colorado potato beetle under study will require a tag of less than 5 mg mass. Frequency selection, antenna

B. Colpitts; D. Luke; G. Boiteau; M. Doyle

1999-01-01

209

Five Approaches to Collecting Tags for Music  

Microsoft Academic Search

We compare five approaches to collecting tags for music: conducting a survey, harvesting social tags, deploying anno- tation games, mining web documents, and autotagging audio content. The comparison includes a discussion of both scala- bility (financial cost, human involvement, and computational resources) and quality (the cold start problem & popularity bias, strong vs. weak labeling, vocabulary structure & size, and

Douglas Turnbull; Luke Barrington; Gert R. G. Lanckriet

2008-01-01

210

Multimedia Content Adaptation through Tag Libraries  

Microsoft Academic Search

This paper presents Alembik, a framework for the transcoding of multimedia content. Alembik has been designed to adapt and transform image, audio and video files in a mobile Web environment. This objective is achieved through a collection of tags defined by the framework. Developers exploit the collection of tags to integrate and adapt multimedia information to the applications needs, as

Davide Bellinzona; Patrick Vitali

2008-01-01

211

Harnessing Collective Knowledge Inherent in Tag Clouds  

ERIC Educational Resources Information Center

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).…

Cress, U.; Held, C.

2013-01-01

212

Tagging text with a probabilistic model  

Microsoft Academic Search

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

B. Merialdo

1991-01-01

213

Method and apparatus for manufacturing gas tags  

DOEpatents

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.

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

1996-12-17

214

Method and apparatus for manufacturing gas tags  

DOEpatents

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.

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

1996-01-01

215

50 CFR 20.36 - Tagging requirement.  

Code of Federal Regulations, 2013 CFR

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

2013-10-01

216

Analogue Fingerprinting for Passive RFID Tags  

Microsoft Academic Search

Traditionally, modern RFID technology has always been assumed to be reliable, but as technology has advanced, so has the ability to replicate the signals that an RFID tag emits upon being interrogated. This paper presents a method of identifying the authenticity of an RFID tag by means of creating an intentional fingerprint. To achieve this we propose an addition to

Gregory Stuart Smith; Marijke Coetzee

2008-01-01

217

Dialog Act Tagging Using Graphical Models  

Microsoft Academic Search

Detecting discourse patterns such as dialog acts (DAs) is an important factor for processing spoken conversations and meetings. Different techniques have been used to tag dialog acts in the past such as hidden Markov models and neural networks. In this work, a full analysis of dialog act tagging using different generative and conditional dynamic Bayesian networks (DBNs) is performed, where

Gang Ji; Jeff Bilmes

2005-01-01

218

A Radio Tag for Big Whales  

ERIC Educational Resources Information Center

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

Watkins, William A.

1978-01-01

219

Verification of Cryptographic Protocols: Tagging Enforces Termination  

Microsoft Academic Search

In experiments with a resolution-based verification method for cryptographic protocols, we could enforce its termination by tagging, a syntactic transformation of messages that leaves attack-free executions invariant. In this paper, we generalize the experimental evidence: we prove that the verification method always terminates for tagged protocols.

Bruno Blanchet; Andreas Podelski

2003-01-01

220

Notes on SAW Tag Interrogation Techniques  

NASA Technical Reports Server (NTRS)

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.

Barton, Richard J.

2010-01-01

221

Self-organization in social tagging systems.  

PubMed

Individuals often imitate each other to fall into the typical group, leading to a self-organized state of typical behaviors in a community. In this paper, we model self-organization in social tagging systems and illustrate the underlying interaction and dynamics. Specifically, we introduce a model in which individuals adjust their own tagging tendency to imitate the average tagging tendency. We found that when users are of low confidence, they tend to imitate others and lead to a self-organized state with active tagging. On the other hand, when users are of high confidence and are stubborn to change, tagging becomes inactive. We observe a phase transition at a critical level of user confidence when the system changes from one regime to the other. The distributions of post length obtained from the model are compared to real data, which show good agreement. PMID:21797438

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

2011-06-01

222

Intrinsic-surface-tag image authentication  

SciTech Connect

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

Palm, R.G.; DeVolpi, A.

1991-12-01

223

Automated grammatical tagging of child language samples.  

PubMed

Recent studies of the automated grammatical categorization ("tagging") of words using probabilistic methods have reported substantial levels of accuracy-over 95% agreement with manual tagging for words from a variety of texts. However, the texts with which this method has been tested were written by adults and edited by publishers. The present study examined the accuracy with which such methods could tag transcribed conversational language samples from 30 normally developing children. On a word-by-word basis, automated accuracy levels ranged from 92.9% to 97.4%, averaging 95.1%. Accuracy at correctly tagging whole utterances was lower, ranging from 60.5% to 90.3%, with an average of 77.7%. Probabilistic methods of coding language samples hold potential as a viable tool for child language research. Further study and improvement of automated grammatical tagging is warranted and necessary before widespread use can be made of this technology. PMID:10391635

Channell, R W; Johnson, B W

1999-06-01

224

Tag Location and Retention in Black Rockfish: Feasibility of Using PIT Tags in a Wild Marine Species  

Microsoft Academic Search

Tag and recovery programs can provide valuable information on population size and exploitation rates in fishes. Passive integrated transponder (PIT) tags are ideal for use in such programs because they provide identification of individual fish and are invisible to anglers, circumventing problems with nonreporting of tags. Our objective was to determine whether PIT tags could be used successfully to tag

Steven J. Parker; Polly S. Rankin

2003-01-01

225

Visible Implant Elastomer Color Determination, Tag Visibility, and Tag Loss: Potential Sources of Error for Mark–Recapture Studies  

Microsoft Academic Search

Errors in visible implant elastomer (VIE) color determination may exert stronger influences on mark–recapture data quality than poor tag visibility and tag loss. I applied individual VIE tags to 567 wild long-snouted seahorses Hippocampus guttulatus using four fluorescent colors (red, orange, green, and yellow). Given VIE tag data were compared with tag data recorded by observers as they released recently

Janelle M. R. Curtis

2006-01-01

226

False positive RNA binding activities after Ni-affinity purification from Escherichia coli  

PubMed Central

A His-tag is often added by means of recombinant DNA technology to a heterologous protein of interest, which is then over-produced in Escherchia coli and purified by one-step immobilized metal-affinity chromatography (IMAC). Owing to the presence of 24 histidines at the C-termini of the hexameric E. coli RNA chaperone Hfq, the protein co-purifies with His-tagged proteins of interest. As Hfq can bind to distinct RNA substrates with high affinity, its presence can obscure studies performed with (putative) RNA binding activities purified by IMAC. Here, we present results for a seemingly positive RNA-binding activity, exemplifying that false-positive results can be avoided if the protein of interest is either subjected to further purification step(s) or produced in an E. coli hfq- strain.

Milojevic, Tetyana; Sonnleitner, Elisabeth; Romeo, Alessandra; Djinovic-Carugo, Kristina; Blasi, Udo

2013-01-01

227

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

PubMed

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

Furuyama, Takehito; Henikoff, Steven

2006-06-01

228

Extended-Range Passive RFID and Sensor Tags.  

National Technical Information Service (NTIS)

Extended-range passive radio-frequency identification (RFID) tags and related sensor tags are undergoing development. A tag of this type incorporates a retroreflective antenna array, so that it reflects significantly more signal power back toward an inter...

G. Y. Lin P. W. Fink R. Barton T. F. Kennedy

2012-01-01

229

48 CFR 908.7101-7 - Government license tags.  

Code of Federal Regulations, 2013 CFR

...System DEPARTMENT OF ENERGY COMPETITION ACQUISITION...tags. Assignment of new tag numbers will be...via the UNICOR online vehicle license tag ordering...Transportation, Motor Vehicles Services Branch, District...Columbia, for all motor vehicles (except vehicles...

2013-10-01

230

Proteomic Analysis of Rat Striatal Synaptosomes during Acrylamide Intoxication at a Low Dose Rate  

Microsoft Academic Search

We have hypothesized that acrylamide (ACR) intoxication causes cumulative nerve terminal damage by forming adducts with nucleophilic cysteine sulfhydryl groups on critical pre- synaptic proteins. To determine the cumulative effects of ACR on the cysteine-containing proteome of nerve terminal, we employed cleavable isotope-coded affinity tagging (ICAT) and liquid chromatography-tandem mass spectrometry. ICAT analysis uses a sulfhydryl-specific tag to identify and

David S. Barber; Stanley Stevens; Richard M. LoPachin

2007-01-01

231

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

Microsoft Academic Search

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

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

2006-01-01

232

Protein Purification with Polymeric Affinity Membranes Containing Functionalized Poly(acid) Brushes  

PubMed Central

Porous nylon membranes modified with poly(acid) brushes and their derivatives can rapidly purify proteins via ion-exchange and metal-ion affinity interactions. Membranes containing poly(2-(methacryloyloxy)ethyl succinate) (poly(MES)) brushes bind 118 ± 8 mg of lysozyme per cm3 of membrane and facilitate purification of lysozyme from chicken egg white. Moreover, functionalization of the poly(MES) brushes with nitrilotriacetate (NTA)-Ni2+ complexes yields membranes that bind poly(histidine)-tagged (His-tagged) ubiquitin with a capacity of 85 ± 2 mg of protein per cm3 of membrane. Most importantly, the membranes modified with poly(MES)-NTA-Ni2+ allow isolation of His-tagged cellular retinaldehyde-binding protein directly from a cell extract in less than 10 min, and the protein purity is comparable to that achieved with commercial affinity columns. Thus, porous nylon membranes containing functionalized poly(MES) brushes are attractive candidates for rapid, high-capacity purification of His-tagged proteins from cell extracts.

Jain, Parul; Vyas, Mukesh Kumar; Geiger, James H.; Baker, Gregory L.; Bruening, Merlin L.

2010-01-01

233

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

SciTech Connect

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

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

2007-07-01

234

DICOM involving XML path-tag  

NASA Astrophysics Data System (ADS)

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

Zeng, Qiang; Yao, Zhihong; Liu, Lei

2011-03-01

235

Enhanced UHF RFID tags for drug tracing.  

PubMed

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

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

2012-12-01

236

Applying thiouracil tagging to mouse transcriptome analysis.  

PubMed

Transcriptional profiling is a powerful approach for studying mouse development, physiology and disease models. Here we describe a protocol for mouse thiouracil tagging (TU tagging), a transcriptome analysis technology that includes in vivo covalent labeling, purification and analysis of cell type-specific RNA. TU tagging enables the isolation of RNA from a given cell population of a complex tissue, avoiding transcriptional changes induced by cell isolation trauma, as well as the identification of actively transcribed RNAs and not preexisting transcripts. Therefore, in contrast to other cell-specific transcriptional profiling methods based on the purification of tagged ribosomes or nuclei, TU tagging provides a direct examination of transcriptional regulation. We describe how to (i) deliver 4-thiouracil to transgenic mice to thio-label cell lineage-specific transcripts, (ii) purify TU-tagged RNA and prepare libraries for Illumina sequencing and (iii) follow a straightforward bioinformatics workflow to identify cell type-enriched or differentially expressed genes. Tissue containing TU-tagged RNA can be obtained in 1 d, RNA-seq libraries can be generated within 2 d and, after sequencing, an initial bioinformatics analysis can be completed in 1 additional day. PMID:24457332

Gay, Leslie; Karfilis, Kate V; Miller, Michael R; Doe, Chris Q; Stankunas, Kryn

2014-02-01

237

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

DOEpatents

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.

Burghard, Brion J. (W. Richland, WA) [W. Richland, WA; Skorpik, James R. (Kennewick, WA) [Kennewick, WA

2008-04-22

238

Affinity chromatography for antibody purification.  

PubMed

The availability of purified antibodies is prerequisite for many applications and the appropriate choice(s) of antibody-purification steps is crucial. Numerous methods have been developed for the purification of antibodies; however, affinity chromatography-based methods are the most extensively utilized. These methods are based on highly specific and reversible biological interactions between two molecules (e.g., between receptor and ligand or antibody and antigen). Affinity chromatography offers very high selectivity, involving minimal steps, providing simplicity of approach and rapidity. Implementing an effective protocol often requires meticulous planning and testing in order to achieve high purity and yields of desired antibody types/subtypes. This chapter describes the basic techniques for purification of monoclonal, polyclonal, and recombinant antibodies employing affinity chromatography. PMID:24648096

Arora, Sushrut; Ayyar, B Vijayalakshmi; O'Kennedy, Richard

2014-01-01

239

Time-Tag Generation Script  

NASA Technical Reports Server (NTRS)

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.

Jackson, Dan E.

2010-01-01

240

MUBs inequivalence and affine planes  

NASA Astrophysics Data System (ADS)

There are fairly large families of unitarily inequivalent complete sets of N + 1 mutually unbiased bases (MUBs) known in CN for various prime powers N. The number of such sets is not bounded above by any polynomial as a function of N. While it is standard that there is a superficial similarity between complete sets of MUBs and finite affine planes, there is an intimate relationship between these large families and affine planes. This note briefly summarizes ``old'' results that do not appear to be well known concerning known families of complete sets of MUBs and their associated planes.

Kantor, W. M.

2012-03-01

241

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

PubMed Central

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.

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

2013-01-01

242

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

PubMed

In molecular biology and protein detection the immobilized metal ion clusters using a NTA-chelator is a powerful technique in identification and isolation of histidine-tagged fusion proteins. The Oligo-histidine tag should serve as a high affinity binding sequence for the purification of any fusion protein via metal chelating adsorbents. We described the preparation and characterization of bioinorganic conjugates made with highly luminescent semiconductor CdTe-CdS core-shell quantum dots (QDs) for biological labeling. A biocompatible surface-functionalized nanoparticle was designed to sense histidine-tagged fusion proteins. This study demonstrates the synthesis of Ni-NTA conjugated QD nanoparticles and the successful application of these nanoparticles to the detection of histidine-tagged fusion proteins. It is believed that this approach will provide a more convenient methodology for the intracellular localization of histidine-tagged protein, as compared with current methods. These Ni-NTA-QD clusters were shown to target the 6x histidine region of tagged proteins specially. PMID:19027151

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

2009-02-01

243

An AIL/IL-based liquid/liquid extraction system for the purification of His-tagged proteins.  

PubMed

A sorbent based on affinity ionic liquid (AIL), triazacyclononane-ionic liquid, was synthesized, characterized, and applied to the extraction of histidine (His)-tagged proteins from aqueous buffer to ionic liquid (IL) phase. The adsorbed His-tagged proteins could be back-extracted from the IL phase to the aqueous buffer with an imidazole solution. The specific binding of His-tagged proteins with AIL/IL could be affected by a few factors including the ionic strength and coordinated metal ions. In the case of His-tagged enhanced green fluorescent protein (EGFP), the maximum binding capacity of Cu(2+)-AIL/IL reached 2.58 ?g/?mol under the optimized adsorption conditions. The eluted His-tagged EGFP kept fluorescent and remained active through the purification process. Moreover, a tandem extraction process successively using Cu(2+)-AIL/IL and Zn(2+)-AIL/IL systems was developed, which was proven very efficient to obtain the ultimate protein with a purity of about 90 %. An effective reclamation method for the AIL/IL extraction system was further established. The sorbent could be easily regenerated by removing metal ions with EDTA and the followed reimmobilization of metal ions. Easy handling of the presented M(2+)-AIL/IL system and highly specific ability to absorb His-tagged proteins make it attractive and potentially applicable in biomolecular separation. PMID:24743984

Xu, Weiyuan; Cao, Huazhen; Ren, Guangwei; Xie, Hujun; Huang, Jianying; Li, Shijun

2014-06-01

244

Intrinsic-surface-tag image authentication  

SciTech Connect

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

Palm, R.G.; DeVolpi, A.

1991-12-01

245

50 CFR 20.81 - Tagging requirement.  

Code of Federal Regulations, 2013 CFR

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

2013-10-01

246

Jet Fuel Production from TAG and FAME.  

National Technical Information Service (NTIS)

The Energy & Environmental Research Center (EERC) and its partners have developed thermocatalytic technologies to produce a 100% renewable fuel from crop oil-derived triglyceride (TAG) feedstock that meets the critical military specification requirements ...

B. G. Oster

2010-01-01

247

Unseeded molecular flow tagging in cold and hot flows using ozone and hydroxyl tagging velocimetry  

Microsoft Academic Search

Two complementary unseeded molecular flow tagging techniques for gas-flow velocity field measurement at low and high temperature are demonstrated. Ozone tagging velocimetry (OTV) is applicable to low-temperature air flows whereas hydroxyl tagging velocimetry (HTV) is amenable to use in high-temperature reacting flows containing water vapour. In OTV, a grid of ozone lines is created by photodissociation of O2 by a

Robert W Pitz; Joseph A Wehrmeyer; Lubomir A Ribarov; Douglas A Oguss; Farrokh Batliwala; Peter A DeBarber; Stefan Deusch; Paul E Dimotakis

2000-01-01

248

Estimation of an affine motion  

Microsoft Academic Search

This paper discusses the 3D affine motion estimation problem using two cameras via observations of a single feature point. The unknown parameters to be estimated include the nine rotational parameters, the three translational parameters, and the 3D position. One camera assumes a parabolic projection. The other camera is the conventional camera that has a planar projection surface. The parabolic camera

Lili Ma; Chengyu Cao; Naira Hovakimyan; Craig Woolsey; Guoqiang Hu

2009-01-01

249

Automorphisms of Affine Surfaces. Ii  

Microsoft Academic Search

Affine surfaces X completed by an irreducible rational curve C are studied. The integer m = (C2) is an invariant of X. It is shown that the set of all such surfaces with fixed invariant m is described in terms of orbits of a group action on the space of \\

M. H. Gizatullin; V. I. Danilov

1977-01-01

250

Programmable reflectors for SAW-ID-tags  

Microsoft Academic Search

Surface acoustic wave devices for identification systems (SAW-ID-tags or SAW wireless labels) have a large potential for future applications. We concentrate in this paper on reflective SAW-ID-tags with amplitude modulation. We use splitfinger interdigital transducers as reflecting structures. If the transducers are short circuited or capacitively loaded the reflection disappears almost entirely. On the other hand, if an open circuit

L. Reindl; W. Ruile

1993-01-01

251

Lock-Out / Tag-Out  

NSDL National Science Digital Library

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

2010-05-14

252

MRI of myocardial infarction with tissue tagging  

Microsoft Academic Search

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

Daniel A. Herzka; Elliot R. McVeigh

2009-01-01

253

b-tagging at D0  

SciTech Connect

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.

Hanagaki, K.; /Fermilab

2005-07-01

254

A generic protocol for the expression and purification of recombinant proteins in Escherichia coli using a combinatorial His6-maltose binding protein fusion tag  

Microsoft Academic Search

We describe a generic protocol for the overproduction and purification of recombinant proteins in Escherichia coli. The strategy utilizes a dual His6-maltose binding protein (HisMBP) affinity tag that can be removed from the target protein by digestion of the fusion protein at a designed site by tobacco etch virus protease. The MBP moiety serves to enhance the solubility and promote

Sreedevi Nallamsetty; David S Waugh

2007-01-01

255

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

USGS Publications Warehouse

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.

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

2007-01-01

256

SNAP-tagging the retrograde route.  

PubMed

We have developed a chemical biology strategy to identify proteins that follow the retrograde transport route from the plasma membrane to the Golgi apparatus, via endosomes. The general principle is the following: plasma membrane proteins are covalently tagged with a first probe. Only the ones that are then transported to trans-Golgi/TGN membranes are covalently bound to a capture reagent that has been engineered into this compartment. Specifically, the first probe is benzylguanine (BG) that is conjugated onto primary amino groups of plasma-membrane proteins. The capture reagent includes an O(6)-alkylguanine-DNA alkyltransferase-derived fragment, the SNAP-tag, which forms a covalent linkage with BG. The SNAP-tag is fused to the GFP-tagged Golgi membrane anchor from galactosyl transferase for proper targeting to trans-Golgi/TGN membranes. Cell-surface BG-tagged proteins that are transported to trans-Golgi/TGN membranes (i.e., that are retrograde cargoes) are thereby covalently captured by the SNAP-tag fusion protein. For identification, the latter is immunopurified using GFP-Trap, and associated retrograde cargo proteins are identified by mass spectrometry. We here provide a step-by-step protocol of this method. PMID:24295305

Johannes, Ludger; Shafaq-Zadah, Massiullah

2013-01-01

257

Nanomechanics of HaloTag tethers.  

PubMed

The active site of the Haloalkane Dehydrogenase (HaloTag) enzyme can be covalently attached to a chloroalkane ligand providing a mechanically strong tether, resistant to large pulling forces. Here we demonstrate the covalent tethering of protein L and I27 polyproteins between an atomic force microscopy (AFM) cantilever and a glass surface using HaloTag anchoring at one end and thiol chemistry at the other end. Covalent tethering is unambiguously confirmed by the observation of full length polyprotein unfolding, combined with high detachment forces that range up to ?2000 pN. We use these covalently anchored polyproteins to study the remarkable mechanical properties of HaloTag proteins. We show that the force that triggers unfolding of the HaloTag protein exhibits a 4-fold increase, from 131 to 491 pN, when the direction of the applied force is changed from the C-terminus to the N-terminus. Force-clamp experiments reveal that unfolding of the HaloTag protein is twice as sensitive to pulling force compared to protein L and refolds at a slower rate. We show how these properties allow for the long-term observation of protein folding-unfolding cycles at high forces, without interference from the HaloTag tether. PMID:23909704

Popa, Ionel; Berkovich, Ronen; Alegre-Cebollada, Jorge; Badilla, Carmen L; Rivas-Pardo, Jaime Andrés; Taniguchi, Yukinori; Kawakami, Masaru; Fernandez, Julio M

2013-08-28

258

Harvesting Intelligence in Multimedia Social Tagging Systems  

NASA Astrophysics Data System (ADS)

As more people adopt tagging practices, social tagging systems tend to form rich knowledge repositories that enable the extraction of patterns reflecting the way content semantics is perceived by the web users. This is of particular importance, especially in the case of multimedia content, since the availability of such content in the web is very high and its efficient retrieval using textual annotations or content-based automatically extracted metadata still remains a challenge. It is argued that complementing multimedia analysis techniques with knowledge drawn from web social annotations may facilitate multimedia content management. This chapter focuses on analyzing tagging patterns and combining them with content feature extraction methods, generating, thus, intelligence from multimedia social tagging systems. Emphasis is placed on using all available "tracks" of knowledge, that is tag co-occurrence together with semantic relations among tags and low-level features of the content. Towards this direction, a survey on the theoretical background and the adopted practices for analysis of multimedia social content are presented. A case study from Flickr illustrates the efficiency of the proposed approach.

Giannakidou, Eirini; Kaklidou, Foteini; Chatzilari, Elisavet; Kompatsiaris, Ioannis; Vakali, Athena

259

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

PubMed

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

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

2012-01-01

260

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

PubMed Central

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

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

2006-01-01

261

Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry  

Microsoft Academic Search

To investigate protein-protein interaction sites in the DNA mismatch repair system we developed a crosslinking\\/mass spectrometry technique employ- ing a commercially available trifunctional cross- linker with a thiol-specific methanethiosulfonate group, a photoactivatable benzophenone moiety and a biotin affinity tag. The XACM approach combines photocrosslinking (X), in-solution digestion of the crosslinked mixtures, affinity purification via the biotin handle (A), chemical coding

Robert Ahrends; Jan Kosinski; Dieter Kirsch; Laura Manelyte; Luis Giron-Monzon; Lars Hummerich; Oliver Schulz; Bernhard Spengler; Peter Friedhoff

2006-01-01

262

Lightweight Authentication Protocols for Low-Cost RFID Tags  

Microsoft Academic Search

Providing security in low-cost RFID tags is a challenging task because tags are highly resource con- strained and cannot support strong cryptography. Special lightweight algorithms and protocols need to be designed that take into account the limitations of the tags. In this paper, we propose a set of extremely lightweight tag authentication protocols. We also provide an analysis of the

Istvan Vajda

2003-01-01

263

Theory and measurement of backscattering from RFID tags  

Microsoft Academic Search

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

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

2006-01-01

264

Analysis and Development of Urdu POS Tagged Corpus  

Microsoft Academic Search

In this paper, two corpora of Urdu (with 110K and 120K words) tagged with different POS tagsets are used to train TnT and Tree taggers. Error analysis of both taggers is done to identi- fy frequent confusions in tagging. Based on the analysis of tagging, and syntactic structure of Urdu, a more refined tagset is derived. The existing tagged corpora

Ahmed Muaz; Aasim Ali; Sarmad Hussain

2009-01-01

265

Rule Based Part of Speech Tagging of Sindhi Language  

Microsoft Academic Search

Part of speech (POS) tagging is a process of assigning correct syntactic categories to each word in the text. Tag set and word disambiguation rules are fundamental parts of any POS tagger. No work has hitherto been published of tag set in Sindhi language. The Sindhi lexicon for computational processing is also not available. In this study, the tag set

Javed Ahmed Mahar; Ghulam Qadir Memon

2010-01-01

266

SEARCHING THE LONG TAIL: HIDDEN STRUCTURE IN SOCIAL TAGGING  

Microsoft Academic Search

In this paper we explore a method of decomposition of compound tags found in social tagging systems and outline several results, including improvement of search indexes, extraction of semantic information, and benefits to usability. Analysis of tagging habits demonstrates that social tagging systems such as del.icio.us and flickr include both formal metadata, such as geotags, and informally created metadata, such

Emma Tonkin

2006-01-01

267

Quantifying tag representativeness of visual content of social images  

Microsoft Academic Search

Social tags describe images from many aspects including the visual content observable from the images, the context and usage of images, user opinions and others. Not all tags are therefore useful for image search and are appropriate for tag recommendation with respect to visual content of images. However, the relationship between a given tag and the visual content of its

Aixin Sun; Sourav S. Bhowmick

2010-01-01

268

Emulation of an Active Tag Location Tracking System  

Microsoft Academic Search

In this paper we present the emulation of a location tracking system that uses active tags to identify the position of the active tag wearer. Using emulation we were able to carry out live experiments with such active tag applications. Emulation is done using QOMET, a wireless communication emulator, and the active tag processor emulator. Experiments are performed using the

Razvan Beuran; Junya Nakata; Takashi Okada; Tetsuya Kawakami; Ken-ichi Chinen; Yasuo Tan; Yoichi Shinoda

2008-01-01

269

Earth Processes: Reading the Isotopic Code  

NASA Astrophysics Data System (ADS)

Publication of this monograph will coincide, to a precision of a few per mil, with the centenary of Henri Becquerel's discovery of "radiations actives" (C. R. Acad. Sci., Feb. 24, 1896). In 1896 the Earth was only 40 million years old according to Lord Kelvin. Eleven years later, Boltwood had pushed the Earth's age past 2000 million years, based on the first U/Pb chemical dating results. In exciting progression came discovery of isotopes by J. J. Thomson in 1912, invention of the mass spectrometer by Dempster (1918) and Aston (1919), the first measurement of the isotopic composition of Pb (Aston, 1927) and the final approach, using Pb-Pb isotopic dating, to the correct age of the Earth: close—2.9 Ga (Gerling, 1942), closer—3.0 Ga (Holmes, 1949) and closest—4.50 Ga (Patterson, Tilton and Inghram, 1953).

Basu, Asish; Hart, Stan

270

Histidine-tag-directed chromophores for tracer analyses in the analytical ultracentrifuge  

PubMed Central

Many recombinant proteins carry an oligohistidine (HisX)-tag that allows their purification by immobilized metal affinity chromatography (IMAC). This tag can be exploited for the site-specific attachment of chromophores and fluorophores, using the same metal ion–nitrilotriacetic acid (NTA) coordination chemistry that forms the basis of popular versions of IMAC. Labeling proteins in this way can allow their detection at wavelengths outside of the absorption envelopes of un-modified proteins and nucleic acids. Here we describe use of this technology in tracer sedimentation experiments that can be performed in a standard analytical ultracentrifuge equipped with absorbance or fluorescence optics. Examples include sedimentation velocity in the presence of low molecular weight chromophoric solutes, sedimentation equilibrium in the presence of high concentrations of background protein and selective labeling to simplify the assignment of species in a complex interacting mixture.

Hellman, Lance M.; Zhao, Chunxia; Melikishvili, Manana; Tao, Xiaorong; Hopper, James E.; Whiteheart, Sidney W.; Fried, Michael G.

2011-01-01

271

Examining the complexity of human RNA polymerase complexes using HaloTag technology coupled to label free quantitative proteomics.  

PubMed

Efficient determination of protein interactions and cellular localization remains a challenge in higher order eukaryotes and creates a need for robust technologies for functional proteomics studies. To address this, the HaloTag technology was developed for highly efficient and rapid isolation of intracellular complexes and correlative in vivo cellular imaging. Here we demonstrate the strength of this technology by simultaneous capture of human eukaryotic RNA polymerases (RNAP) I, II, and III using a shared subunit, POLR2H, fused to the HaloTag. Affinity purifications showed successful isolation, as determined using quantitative proteomics, of all RNAP core subunits, even at expression levels near endogenous. Transient known RNAP II interacting partners were identified as well as three previously uncharacterized interactors. These interactions were validated and further functionally characterized using cellular imaging. The multiple capabilities of the HaloTag technology demonstrate the ability to efficiently isolate highly challenging multiprotein complexes, discover new interactions, and characterize cellular localization. PMID:22149079

Daniels, Danette L; Méndez, Jacqui; Mosley, Amber L; Ramisetty, Sreenivasa R; Murphy, Nancy; Benink, Hélène; Wood, Keith V; Urh, Marjeta; Washburn, Michael P

2012-02-01

272

Neural net controlled tag gas sampling system for nuclear reactors  

DOEpatents

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.

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

1997-02-11

273

Neural net controlled tag gas sampling system for nuclear reactors  

US Patent & Trademark Office Database

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.

1997-02-11

274

Neural net controlled tag gas sampling system for nuclear reactors  

DOEpatents

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

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

1997-01-01

275

Associated Particle Tagging (APT) in Magnetic Spectrometers  

SciTech Connect

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

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

2012-10-16

276

Stille couplings in supercritical CO 2 catalyzed with perfluoro-tagged and un-tagged Pd complexes  

Microsoft Academic Search

Stille CC-couplings in supercritical CO2 (scCO2) were performed with perfluoro-tagged and un-tagged Pd complexes in high yields. With fluoro-tagged complexes yields were generally slightly higher. A recycling of the perfluoro-tagged catalyst was also achieved.

Thomas Osswald; Siegfried Schneider; Shaoning Wang; Willi Bannwarth

2001-01-01

277

A brief examination of optical tagging technologies.  

SciTech Connect

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

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

2003-07-01

278

Influence of material properties upon immobilization of histidine-tagged protein on Ni-Co coated chip.  

PubMed

In protein research, protein microarray facilitates high-throughput study of protein abundance and function. An appropriate microarray surface that can be used to immobilize protein samples is a prerequisite for the investigation of molecular interactions. Ni-Co alloy coated protein microarray chip has been found to adsorb histidine-tagged proteins effectively based on the method of immobilized metal affinity chromatography. Due to the ingredient of bi-metallic elements, different electroplating conditions resulted in distinct binding affinities. Therefore, the influence of Ni-Co material properties on the immobilization of histidine-tagged protein was systematically investigated in this study. In the experiments, the contact angle measurement suggested that no strong relationship can be established between the wettability of chip surface and its corresponding protein immobilization. ESCA test demonstrated that the major ingredients of the Ni-Co alloy coated protein microarray chip were Ni and Co. In addition, the XRD test concluded that a Ni-Co protein chip that consists mostly of hcp lattice has better binding capability. SEM micrographs provide direct image evidence. These material tests summarize that the Ni-Co alloy coated protein microarray chip adsorbs His-tagged proteins through its surface morphology. Therefore, it can provide specific binding due to the affinity adsorption between the intermediate metals and the protein. PMID:24582262

Chang, Yaw-Jen; Ho, Ching-Yuan; Chang, Cheng-Hao

2014-04-01

279

Tagging of functional ribosomes in living cells by HaloTag® technology.  

PubMed

Ribosomal proteins and ribosomal associated proteins are complicated subjects to target and study because of their high conservation through evolution which led to highly structured and regulated proteins. Tagging of ribosomal proteins may allow following of protein synthesis in vivo and isolating translated mRNAs. HaloTag® is a new technology which allows detection in living cells, biochemical purification, and localization studies. In the present work, we tested HaloTag®-based ribosomal tagging. We focused on eIF6 (eukaryotic Initiation Factor 6 free 60S ribosomal marker), RACK1 (Receptor for Activated C Kinase 1; 40S and polysomes, not nuclear), and rpS9 (40S ribosomes, both in the nucleus and in the cytoplasm). Experiments performed on HEK293 cells included ribosomal profiles and Western blot on the fractions, purification of HaloTag® proteins, and fluorescence with time-lapse microscopy. We show that tagged proteins can be incorporated on ribosomes and followed by time-lapse microscopy. eIF6 properly accumulates in the nucleolus, and it is redistributed upon actinomycin D treatment. RACK1 shows a specific cytoplasmic localization, whereas rpS9 is both nucleolar and cytoplasmic. However, efficiency of purification varies due to steric hindrances. In addition, the level of overexpression and degradation may vary upon different constructs. In summary, HaloTag® technology is highly suitable to ribosome tagging, but requires prior characterization for each construct. PMID:21082278

Gallo, Simone; Beugnet, Anne; Biffo, Stefano

2011-02-01

280

Tagmantic: a social recommender service based on semantic tag graphs and tag clusters  

Microsoft Academic Search

Tagmantic.com is a web based social recommendation and search service which exploits the rich information within folksonomies. By developing multiple layers of organizational structure on top of folksonomies the relations between tags, users and resources can be utilized in order to create advanced recommender engines. The first layer of organizational structure is a tag graph which relates different entities within

János Moldvay; Ingo Bax; Alexander Frerichs; Mirko Schuh

2010-01-01

281

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

Microsoft Academic Search

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

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

2008-01-01

282

GST-His purification: a two-step affinity purification protocol yielding full-length purified proteins.  

PubMed

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST) and a C-terminal 10xHis tag, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest. PMID:24193370

Maity, Ranjan; Pauty, Joris; Krietsch, Jana; Buisson, Rémi; Genois, Marie-Michelle; Masson, Jean-Yves

2013-01-01

283

Automorphisms of Affine Surfaces. I  

Microsoft Academic Search

We study the group of automorphisms Aut(X) of an affine surface X which can be made complete by adding a zigzag. This study is based on the computation of the action of Aut(X) on a certain tree DeltaX associated with the surface X. Our results are used to give a description of forms of the surface X and of algebraic

M. H. Gizatullin; V. I. Danilov

1975-01-01

284

Affine Buildings and Tropical Convexity  

Microsoft Academic Search

The notion of convexity in tropical geometry is closely related to notions of convexity in the theory of affine buildings. We explore this relationship from a combinatorial and computational perspective. Our results include a convex hull algorithm for the Bruhat--Tits building of SL$_d(K)$ and techniques for computing with apartments and membranes. While the original inspiration was the work of Dress

Michael Joswig; Bernd Sturmfels; Josephine Yu

2007-01-01

285

Affinity Hemodialysis for Antiviral Therapy  

Microsoft Academic Search

Background: HIV-1 gp120 may play a role in the progression from HIV infection to AIDS. Aims: We investigated affinity hemodialysis for removing gp120 from cell culture and whole blood. Methods: Anti-gp120 antibodies covalently coupled to agarose beads were packed into columns or hollow-fiber hemodialysis cartridges. Supernatants from HIV-infected HL2\\/3 cells or gp120 containing whole blood were pumped over the columns

Richard H. Tullis; R. Paul Duffin; Marvin Zech; Julian L. Ambrus

2003-01-01

286

Lectin affinity chromatography of glycolipids  

SciTech Connect

Since glycolipids (GLs) are either insoluble or form mixed micelles in water, lectin affinity chromatography in aqueous systems has not been applied to their separation. They have overcome this problem by using tetrahydrofuran (THF) in the mobile phase during chromatography. Affinity columns prepared with the GalNAc-specific Helix pomatia agglutinin (HPA) and equilibrated in THF specifically bind the (/sup 3/H)oligosaccharide derived from Forssman GL indicating that the immobilized HPA retained its carbohydrate-binding specificity in this solvent. Intact Forssman GL was bound by the HPA-column equilibrated in THF and was specifically eluted with 0.1 mg/ml GalNAc in THF. Purification of the Forssman GL was achieved when a crude lipid extract of sheep erythrocyte membranes was applied to the HPA-column in THF. Non-specifically bound GLs were eluted from the column using a step gradient of aqueous buffer in THF, while the addition of GalNAc was required to elute the specifically bound GLs. Using this procedure the A-active GLs were purified from a crude lipid extract of type A human erythrocytes in a single chromatographic step. The use of solvents that maintain carbohydrate-binding specificity and lipid solubility will permit the application of affinity chromatography on immobilized carbohydrate-binding proteins to intact GLs.

Torres, B.V.; Smith, D.F.

1987-05-01

287

Sequence tagging reveals unexpected modifications in toxicoproteomics  

PubMed Central

Toxicoproteomic samples are rich in posttranslational modifications (PTMs) of proteins. Identifying these modifications via standard database searching can incur significant performance penalties. Here we describe the latest developments in TagRecon, an algorithm that leverages inferred sequence tags to identify modified peptides in toxicoproteomic data sets. TagRecon identifies known modifications more effectively than the MyriMatch database search engine. TagRecon outperformed state of the art software in recognizing unanticipated modifications from LTQ, Orbitrap, and QTOF data sets. We developed user-friendly software for detecting persistent mass shifts from samples. We follow a three-step strategy for detecting unanticipated PTMs in samples. First, we identify the proteins present in the sample with a standard database search. Next, identified proteins are interrogated for unexpected PTMs with a sequence tag-based search. Finally, additional evidence is gathered for the detected mass shifts with a refinement search. Application of this technology on toxicoproteomic data sets revealed unintended cross-reactions between proteins and sample processing reagents. Twenty five proteins in rat liver showed signs of oxidative stress when exposed to potentially toxic drugs. These results demonstrate the value of mining toxicoproteomic data sets for modifications.

Dasari, Surendra; Chambers, Matthew C.; Codreanu, Simona G.; Liebler, Daniel C.; Collins, Ben C.; Pennington, Stephen R.; Gallagher, William M.; Tabb, David L.

2010-01-01

288

Social Tagging for Personalized Web Search  

NASA Astrophysics Data System (ADS)

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

Biancalana, Claudio

289

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

SciTech Connect

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

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

2012-02-07

290

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3).  

PubMed

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for isolation of protein complexes under physiological conditions. Fusion proteins containing two copies of Strep-tag II, designated twin-Strep-tag or SIII-tag, have the advantage of higher affinity for Strep-Tactin compared to those containing only a single Strep-tag, thus allowing more efficient protein purification. However, this advantage is offset by the fact that elution of twin-Strep-tagged proteins with biotin may be incomplete, leading to low protein recovery. The recovery can be dramatically improved by using denaturing elution with sodium dodecyl sulfate (SDS), but this leads to sample contamination with Strep-Tactin released from the resin, making the assay incompatible with downstream proteomic analysis. To overcome this limitation, we have developed a method whereby resin-coupled tetramer of Strep-Tactin is first stabilized by covalent cross-linking with Bis(sulfosuccinimidyl) suberate (BS3) and the resulting cross-linked resin is then used to purify target protein complexes in a single batch purification step. Efficient elution with SDS ensures good protein recovery, while the absence of contaminating Strep-Tactin allows downstream protein analysis by mass spectrometry. As a proof of concept, we describe here a protocol for purification of SIII-tagged viral protein VPg-Pro from nuclei of virus-infected N. benthamiana plants using the Strep-Tactin polymethacrylate resin cross-linked with BS3. The same protocol can be used to purify any twin-Strep-tagged protein of interest and characterize its physiological binding partners. PMID:24796313

Ivanov, Konstantin I; Baši?, Marta; Varjosalo, Markku; Mäkinen, Kristiina

2014-01-01

291

Expression and affinity purification of recombinant proteins from plants  

NASA Technical Reports Server (NTRS)

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).

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

2002-01-01

292

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

PubMed

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

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

2012-10-15

293

B mixing and flavor tagging at CDF  

SciTech Connect

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

Russ, James S.; /Carnegie Mellon U.

2004-12-01

294

Tags to Track Illicit Uranium and Plutonium  

SciTech Connect

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.

Haire, Marvin Jonathan [ORNL

2007-01-01

295

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

NASA Astrophysics Data System (ADS)

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

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

2003-06-01

296

Small-molecule hydrophobic tagging-induced degradation of HaloTag fusion proteins.  

PubMed

The ability to regulate any protein of interest in living systems with small molecules remains a challenge. We hypothesized that appending a hydrophobic moiety to the surface of a protein would mimic the partially denatured state of the protein, thus engaging the cellular quality control machinery to induce its proteasomal degradation. We designed and synthesized bifunctional small molecules to bind a bacterial dehalogenase (the HaloTag protein) and present a hydrophobic group on its surface. Hydrophobic tagging of the HaloTag protein with an adamantyl moiety induced the degradation of cytosolic, isoprenylated and transmembrane HaloTag fusion proteins in cell culture. We demonstrated the in vivo utility of hydrophobic tagging by degrading proteins expressed in zebrafish embryos and by inhibiting Hras1(G12V)-driven tumor progression in mice. Therefore, hydrophobic tagging of HaloTag fusion proteins affords small-molecule control over any protein of interest, making it an ideal system for validating potential drug targets in disease models. PMID:21725302

Neklesa, Taavi K; Tae, Hyun Seop; Schneekloth, Ashley R; Stulberg, Michael J; Corson, Timothy W; Sundberg, Thomas B; Raina, Kanak; Holley, Scott A; Crews, Craig M

2011-08-01

297

The accuracy and value of machine-generated image tags: design and user evaluation of an end-to-end image tagging system  

Microsoft Academic Search

Automated image tagging is a problem of great interest, due to the proliferation of photo sharing services. Researchers have achieved considerable advances in understanding motivations and usage of tags, recognizing relevant tags from image content, and leveraging community input to recommend more tags. In this work we address several important issues in building an end-to-end image tagging application, including tagging

Lexing Xie; Apostol Natsev; Matthew Hill; John R. Smith; Alex Phillips

2010-01-01

298

The Dual Use of RNA Aptamer Sequences for Affinity Purification and Localization Studies of RNAs and RNA-Protein Complexes  

PubMed Central

RNA affinity tags (aptamers) have emerged as useful tools for the isolation of RNAs and ribonucleoprotein complexes from cell extracts. The streptavidin binding RNA aptamer binds with high affinity and is quickly and cleanly eluted with biotin under mild conditions that retain intact complexes. We describe the use of the streptavidin binding aptamer as a tool for purification and discuss strategies towards the design and production of tagged RNAs with a focus on structured target RNAs. The aptamer site can be further exploited as a unique region for the hybridization of oligonucleotide probes and localization by fluorescent in situ hybridization (FISH). The aptamer insertion will allow the localization of a population of RNA species (such as mutants) to be viewed specifically, while in the presence of the wild type RNA. We describe the production of labeled oligonucleotide probes and the preparation of yeast cells for the localization of RNAs by FISH.

Walker, Scott C.; Good, Paul D.; Gipson, Theresa A.; Engelke, David R.

2013-01-01

299

Untraceable RFID tags via insubvertible encryption  

Microsoft Academic Search

We introduce a new cryptographic primitive, called insubvertible encryption, that produces ciphertexts which can be randomized without the need of any key material. Unlike plain universal re-encryption schemes, insubvertible encryption prevents against adversarial exploitation of hidden channels, by including certificates proving that the ciphertext can only be decrypted by authorized parties.The scheme can be applied to RFID tags, providing strong

Giuseppe Ateniese; Jan Camenisch; Breno de Medeiros

2005-01-01

300

RFID-Tags for Anti-counterfeiting  

Microsoft Academic Search

RFID-tags are becoming very popular tools for identiflcation of products. As they have a small microchip on board, they ofier func- tionality that can be used for security purposes. This chip functionality makes it possible to verify the authenticity of a product and hence to detect and prevent counterfeiting. In order to be successful for these secu- rity purposes too,

Pim Tuyls; Lejla Batina

2006-01-01

301

The Tagalog Stemming Algorithm (TagSA)  

Microsoft Academic Search

TagSA, a Tagalog Stemming Algorithm, was developed for all forms of Tagalog words. It can be used specifically for morphological analysis to derive root words. In addition, it can also be applied to information retrieval (IR) to conflate different word forms to a common canonical form. It uses the principle of iterative affix removal and is context sensitive. The system

Don Erick; J. Bonus

302

Harmonic radar tag measurement and characterization  

Microsoft Academic Search

Conventional radar systems transmit and receive at a single, fundamental frequency. It is sometimes challenging to identify a small object against a large background with these radars due to the relatively large return from the background. In effect, the background represents a clutter environment. Harmonic radar tags can be attached to an item, such as insects, and then used to

Gregory L. Charvat; Edward J. Rothwell; Leo C. Kempel

2003-01-01

303

Automated tag reading system for material tracking  

NASA Astrophysics Data System (ADS)

An automated product tag reading system based on CCD cameras and computer image processing has been developed by West Virginia University, and demonstrated at the Weirton Steel Corporation. The system was developed to read painted steel identification tags which are fastened to the ends of steel slabs. The prototype was mounted on a slab hauler and tested in a steel mill environment. It demonstrated the ability to survey a wide-angle image of a scene, locate the target tags in the image, pan and zoom on each one in turn, and read the contents of the tag -- both bar code and alphanumeric code. The system could communicate via radio modem to a stationary computer which could be interfaced to the plant's inventory management database. We are aware of no other system which can both point and read a bar code scanner. The pointing function is important, in that it allows operation in bright sunlight or at long distances: two conditions where humans encounter difficulties in aiming. Our system can read 50-mil bar codes at distances of 30 feet without the use of retro reflective targets. This paper provides an overview of the technical approach used, and the results of stem testing.

Banta, Larry E.; Rosenberry Friend, Kimberly A.; Pertl, Franz A.

1997-09-01

304

Improving SAGE di-tag processing  

Microsoft Academic Search

BACKGROUND: SAGE is a genome-wide method for obtaining gene expression profiles. It generates tags of 10 nucleotides in length, which are assumed to determine the corresponding gene transcript. In practice however, this is not always sufficient for uniquely identifying a gene. RESULTS: We propose an improved processing of SAGE sequences that allows us to obtain one extra base for reasonably

Jacques Colinge; Georg Feger

2001-01-01

305

Novel and efficient tag SNPs selection algorithms.  

PubMed

SNPs are the most abundant forms of genetic variations amongst species; the association studies between complex diseases and SNPs or haplotypes have received great attention. However, these studies are restricted by the cost of genotyping all SNPs; thus, it is necessary to find smaller subsets, or tag SNPs, representing the rest of the SNPs. In fact, the existing tag SNP selection algorithms are notoriously time-consuming. An efficient algorithm for tag SNP selection was presented, which was applied to analyze the HapMap YRI data. The experimental results show that the proposed algorithm can achieve better performance than the existing tag SNP selection algorithms; in most cases, this proposed algorithm is at least ten times faster than the existing methods. In many cases, when the redundant ratio of the block is high, the proposed algorithm can even be thousands times faster than the previously known methods. Tools and web services for haplotype block analysis integrated by hadoop MapReduce framework are also developed using the proposed algorithm as computation kernels. PMID:24212035

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

2014-01-01

306

Closeup of Solar Tadpoles without time tags  

NSDL National Science Digital Library

Here is a close-up view of dark tentacles or tadpoles moving towards the solar surface in this solar flare of April 21, 2002 seen by TRACE. One theory proposed in this press release is that they are due to voids created by magnetic reconnection in the flare. This version of the visualization does not display the instrument clock time tags.

Bridgman, Tom; Deluca, Edward; Mckenzie, David; Cooper, Fenwick; Nakariakov, Valery; Seaton, Daniel

2003-04-11

307

Closeup of Solar Tadpoles with time tags  

NSDL National Science Digital Library

Here is a close-up view of dark tentacles or tadpoles moving towards the solar surface in this solar flare of April 21, 2002 seen by TRACE. One theory proposed in this press release is that they are due to voids created by magnetic reconnection in the flare. This version of the visualization displays the instrument clock time tags.

Bridgman, Tom; Deluca, Edward; Mckenzie, David; Cooper, Fenwick; Nakariakov, Valery; Seaton, Daniel

2003-04-11

308

Tag SNP selection via a genetic algorithm  

Microsoft Academic Search

Single Nucleotide Polymorphisms (SNPs) provide valuable information on human evolutionary history and may lead us to identify genetic variants responsible for human complex diseases. Unfortunately, molecular haplotyping methods are costly, laborious, and time consuming; therefore, algorithms for constructing full haplotype patterns from small available data through computational methods, Tag SNP selection problem, are convenient and attractive. This problem is proved

Ghasem Mahdevar; Javad Zahiri; Mehdi Sadeghi; Abbas Nowzari-Dalini; Hayedeh Ahrabian

2010-01-01

309

Tagging, Tracking and Locating without GPS.  

National Technical Information Service (NTIS)

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

J. Shuler J. V. Cordaro

2012-01-01

310

TAGGING, TRACKING AND LOCATING WITHOUT GPS  

Microsoft Academic Search

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

J. Cordaro; T. Coleman; D. Shull

2012-01-01

311

Semantic Enhancement of Social Tagging Systems  

NASA Astrophysics Data System (ADS)

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.

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

312

Myocardial tissue tagging with cardiovascular magnetic resonance  

Microsoft Academic Search

Cardiovascular magnetic resonance (CMR) is currently the gold standard for assessing both global and regional myocardial function. New tools for quantifying regional function have been recently developed to characterize early myocardial dysfunction in order to improve the identification and management of individuals at risk for heart failure. Of particular interest is CMR myocardial tagging, a non-invasive technique for assessing regional

Monda L Shehata; Susan Cheng; Nael F Osman; David A Bluemke; João AC Lima

2009-01-01

313

Pulse Shaping for Localized Magnetic Resonance Tagging.  

National Technical Information Service (NTIS)

Magnetic resonance tagging is usually achieved by means of a train of non-selective radio-frequency pulses separated by gradient pulses. Thus, the modulation of the M(sub z) magnetization component expands all over the imaging plane. It has been proposed ...

V. N. Ikonomidou G. D. Sergiadis

2001-01-01

314

RFID tag antenna mountable on metallic plates  

Microsoft Academic Search

RFID tag antenna which could be mountable on metallic plate is designed and measured for 900MHz band. The proposed antenna consists of ground plane, substrate (?r = 4.7), feed line with shorted circuit and radiating patch with L-shaped slit. The feed line with shorted circuit is designed for the direct impedance matching between the antenna and the RFID microchip. Especially,

Sung-Joo Kim; Byongkil Yu; Ho-Jun Lee; Myun-Joo Park; Frances J. Harackiewicz; Byungje Lee

2005-01-01

315

RFID tag antenna based temperature sensing  

Microsoft Academic Search

Temperature monitoring is important in a number of fields, particularly cold supply chain applications. Most commercial wireless temperature sensors consist of transceivers, memory and batteries to maintain a temperature time history but this is expensive and allows for limited sensor deployment. In this paper, we propose a low cost temperature sensor based on the paradigm of passive RFID tag antenna

Rahul Bhattacharyya; Christian Floerkemeier; Sanjay Sarma

2010-01-01

316

Techniques for Tagging and Tracking Deepwater Rockfishes  

Microsoft Academic Search

Using scuba in August and September 1997 and 1998, we surgically implanted acoustic transmitters (Vemco V16 series) in 6 greenspotted rockfish Sebastes chlorostictus and 16 bocaccio S. paucispinis on the flank of Soquel Canyon in Monterey Bay, California. In 1997 we used longline gear to capture and tag greenspotted rockfish, and in 1998 we worked with a commercial fisherman who

Richard M. Starr; John N. Heine; Korie A. Johnson

2000-01-01

317

Active tag based pedestrian localization emulation system  

Microsoft Academic Search

We develop an emulation system for performing experiments related to active tag based pedestrian localization. We use emulation as an integral part of our development approach so as to be able to carry out large-scale experiments with ease, and in a repeatable manner. Our demonstration will show how to perform live emulation experiments on a remote network testbed located in

R. Beuran; Junya Nakata; T. Okada; K. Chinen; Y. Tan; Y. Shinoda; Y. Suzuki; T. Kawakami

2008-01-01

318

TANGO: Vocabulary Learning Environment Using RFID Tags  

Microsoft Academic Search

Ubiquitous computing will help in the organization and mediation of social interactions wherever and whenever these situations might occur. With those technologies, learning environment can be embedded in daily real life. Especially, RFID (Radio Frequency Identification) tags are very useful and important technology to realize ubiquitous computing, because they enable to bridge real objects and information in a virtual world.

Hiroaki OGATA; Ryo Akamatsu; Yoneo YANO

319

Multi-Exemplar Affinity Propagation.  

PubMed

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 multi-subclasses 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 determines automatically 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 super-exemplars. 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:23358283

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

2013-01-24

320

Indian craniometric variability and affinities.  

PubMed

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

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

2013-01-01

321

Bioseparation of recombinant cellulose-binding module-proteins by affinity adsorption on an ultra-high-capacity cellulosic adsorbent  

Microsoft Academic Search

Low-cost protein purification methods are in high demand for mass production of low-selling price enzymes that play an important role in the upcoming bioeconomy. A simple protein purification method was developed based on affinity adsorption of a cellulose-binding module-tagged protein on regenerated amorphous cellulose (RAC) followed by modest desorption. The biodegradable cellulosic adsorbent RAC had a very high protein-binding capacity

Jiong Hong; Xinhao Ye; Yiran Wang; Y.-H. Percival Zhang

2008-01-01

322

Virginia Game Fish Tagging Program: 2007 Annual Report.  

National Technical Information Service (NTIS)

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

J. A. Lucy L. Gillingham

2008-01-01

323

Identification of hydrophobic tags for the degradation of stabilized proteins.  

PubMed

New HyTs are a knockout: we previously reported that labeling HaloTag proteins with low molecular weight hydrophobic tags (HyTs) leads to targeted degradation of HaloTag fusion proteins. In this report, we employed a chemical approach to extend this hydrophobic tagging methodology to highly stabilized proteins by synthesizing and evaluating a library of HyTs, which led to the identification of HyT36. PMID:22271667

Tae, Hyun Seop; Sundberg, Thomas B; Neklesa, Taavi K; Noblin, Devin J; Gustafson, Jeffrey L; Roth, Anke G; Raina, Kanak; Crews, Craig M

2012-03-01

324

Nickel nanoparticle decorated graphene for highly selective isolation of polyhistidine-tagged proteins  

NASA Astrophysics Data System (ADS)

Nickel nanoparticle decorated graphene (GP-Ni) is prepared by one-pot hydrothermal reduction of graphene oxide and nickel cations by hydrazine hydrate in the presence of poly(sodium-p-styrenesulfonate) (PSS). The GP-Ni hybrid is characterized by XRD, TEM, SEM, XPS, Raman and FT-IR spectra, demonstrating the formation of poly-dispersed nickel nanoparticles with an average size of 83 nm attached on the surface of graphene sheets. The GP-Ni hybrid exhibits ferromagnetic behavior with a magnetization saturation of 31.1 emu g-1 at 10?000 Oersted (Oe). The GP-Ni also possesses favorable stability in aqueous medium and rapid magnetic response to an external magnetic field. These make it a novel magnetic adsorbent for the separation/isolation of His6-tagged recombinant proteins from a complex sample matrix (cell lysate). The targeted protein species is captured onto the surface of the GP-Ni hybrid via specific metal affinity force between polyhistidine groups and nickel nanoparticles. The SDS-PAGE assay indicates highly selective separation of His6-tagged Smt A from cell lysate. The GP-Ni hybrid displays favorable performance on the separation/isolation of His6-tagged recombinant proteins with respect to the commercial NTA-Ni2+ column.

Liu, Jia-Wei; Yang, Ting; Ma, Lin-Yu; Chen, Xu-Wei; Wang, Jian-Hua

2013-12-01

325

A new TAG-72 cancer marker peptide identified by phage display  

PubMed Central

Radiolabeled peptides as markers of cancer targets have demonstrated their value in diagnostic imaging and radiotherapy. The 16 mer f88-4/Cys6 phage display library was applied to affinity purified TAG-72 and three consensus peptides were identified: VHHSCTKLTHCCQNWH (A2–13), GGVSCMQTSPVCENNL (A2–6) and TKRDCSAQNYGCQKAI (A2–11). A2–13 and A2–6 phages showed the highest percent binding to LS-174T cells by flow cytometry that was 3-fold higher than a control phage. Fluorescence microscopy showed that both A2–6 and A2–13 phages bound to the LS-174T cell membrane. However, only the A2–6 phage demonstrated specificity by low binding to the TAG-72 negative cell HT-29. Furthermore, the synthesized A2–6 peptide demonstrated specific binding to LS-174T cells by flow cytometry and by immunohistochemical staining of xenograph tumor compared to normal colon. These data indicate that the A2–6 peptide is specific and selective for the TAG-72 cancer target.

Chen, Ling; Dou, Shuping; Liu, Xinrong; Liu, Guozheng; Wang, Yi; Hnatowich, Donald J.; Rusckowski, Mary

2008-01-01

326

Nickel nanoparticle decorated graphene for highly selective isolation of polyhistidine-tagged proteins.  

PubMed

Nickel nanoparticle decorated graphene (GP-Ni) is prepared by one-pot hydrothermal reduction of graphene oxide and nickel cations by hydrazine hydrate in the presence of poly(sodium-p-styrenesulfonate) (PSS). The GP-Ni hybrid is characterized by XRD, TEM, SEM, XPS, Raman and FT-IR spectra, demonstrating the formation of poly-dispersed nickel nanoparticles with an average size of 83 nm attached on the surface of graphene sheets. The GP-Ni hybrid exhibits ferromagnetic behavior with a magnetization saturation of 31.1 emu g(-1) at 10,000 Oersted (Oe). The GP-Ni also possesses favorable stability in aqueous medium and rapid magnetic response to an external magnetic field. These make it a novel magnetic adsorbent for the separation/isolation of His6-tagged recombinant proteins from a complex sample matrix (cell lysate). The targeted protein species is captured onto the surface of the GP-Ni hybrid via specific metal affinity force between polyhistidine groups and nickel nanoparticles. The SDS-PAGE assay indicates highly selective separation of His6-tagged Smt A from cell lysate. The GP-Ni hybrid displays favorable performance on the separation/isolation of His6-tagged recombinant proteins with respect to the commercial NTA-Ni(2+) column. PMID:24270901

Liu, Jia-Wei; Yang, Ting; Ma, Lin-Yu; Chen, Xu-Wei; Wang, Jian-Hua

2013-12-20

327

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

PubMed Central

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

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

2008-01-01

328

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

NASA Astrophysics Data System (ADS)

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

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

2011-10-01

329

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

ERIC Educational Resources Information Center

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

Papper, Marc; Kempter, Richard; Leibold, Christian

2011-01-01

330

A global SAW ID tag with large data capacity  

Microsoft Academic Search

The Global SAW Tag uses a recently-invented digital modulation based on simultaneous time position and phase shifting. A unique feature of this tag is that it satisfies global RFID requirements using the international 2.44 GHz ISM band. Precision amplitude and phase weighting of reflectors and accurate control of parasitic effects is critical to implementing this device. This tag has significantly

Clinton S. Hartmann

2002-01-01

331

The Searching Effectiveness of Social Tagging in Museum Websites  

ERIC Educational Resources Information Center

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

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

2012-01-01

332

Inline SAW RFID tag using time position and phase encoding  

Microsoft Academic Search

Surface acoustic wave (SAW) radio-frequency identification (RFID) tags are encoded according to partial reflections of an interrogation signal by short metal reflectors. The standard encryption method involves time position encoding that uses time delays of response signals. However, the data capacity of a SAW RFID tag can be significantly enhanced by extracting additional phase information from the tag responses. In

Sanna Harma; Wesley G. Arthur; Clinton S. Hartmann; Roman G. Maev; Victor P. Plessky

2008-01-01

333

A model-based approach to selection of tag SNPs  

Microsoft Academic Search

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

Pierre Nicolas; Fengzhu Sun; Lei M. Li

2006-01-01

334

Evaluation of RFID tag antenna performance using radar cross sections  

Microsoft Academic Search

Evaluation of a RFID tag antenna performance using RCS is shown to be very effective compared with that based on a detection distance. A tag antenna of which impedance is 35Q at 911MHz is designed and its performances are evaluated by RCS'. The procedures to obtain RCS' by EM simulation are described in detail. How to interpret the tag RCS'

Hongil Kwon; Bomson Lee

2005-01-01

335

AN ACCURATE METHOD FOR IMPEDANCE MEASUREMENT OF RFID TAG ANTENNA  

Microsoft Academic Search

This paper presents a method of antenna impedance measurement for RFID tag antenna based on a differential probe. The importance of accurate impedance measurement in optimal design of tag antenna, especially for the metal tags, is first addressed. Afterwards, an overview of the existing methods based on the single- ended probe and the balun probe is presented. The proposed method

Shih-Kang Kuo; Sung-Lin Chen; Chang-Tsun Lin

2008-01-01

336

High gain Yagi-Uda UHF RFID tag antennas  

Microsoft Academic Search

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

Kyounghwan Lee; You Chung Chung

2007-01-01

337

Novel RFID tag antenna with stability to material  

Microsoft Academic Search

In this paper, we suggested a novel RFID tag antenna in UHF band based on a new concept that the tag should always attain good readability irrespective of material characteristics of object. To optimize antenna structure we used Friss transmission equation considered return loss, radiation efficiency and directivity corresponding material of objects. The optimal tag was fabricated and measured the

J. Choo; J. Ryoo; J. Hong

2008-01-01

338

Design of Novel RFID Tag Antennas for Metallic Objects  

Microsoft Academic Search

In this paper, a novel tag antenna is proposed; it has a very simple structure that does not require a ground plane or shorting pins. The proposed tag antenna has significant advantages over other types of tag antennas for metallic objects, such as low cost, light weight, and ease of fabrication. The body of the antenna is printed as a

Chihyun Cho; Hosung Choo

2006-01-01

339

Developing a Better Method of Tag Attachment for Cetaceans.  

National Technical Information Service (NTIS)

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

J. T. Harvey

2009-01-01

340

The maximal affinity of ligands  

PubMed Central

We explore the question of what are the best ligands for macromolecular targets. A survey of experimental data on a large number of the strongest-binding ligands indicates that the free energy of binding increases with the number of nonhydrogen atoms with an initial slope of ??1.5 kcal/mol (1 cal = 4.18 J) per atom. For ligands that contain more than 15 nonhydrogen atoms, the free energy of binding increases very little with relative molecular mass. This nonlinearity is largely ascribed to nonthermodynamic factors. An analysis of the dominant interactions suggests that van der Waals interactions and hydrophobic effects provide a reasonable basis for understanding binding affinities across the entire set of ligands. Interesting outliers that bind unusually strongly on a per atom basis include metal ions, covalently attached ligands, and a few well known complexes such as biotin–avidin.

Kuntz, I. D.; Chen, K.; Sharp, K. A.; Kollman, P. A.

1999-01-01

341

A method for affine invariant curve smoothing  

NASA Astrophysics Data System (ADS)

This paper proposes a new curve smoothing method invariant to affine transformation. Curve smoothing is one of the important challenges in computer vision as a procedure for noise suppression in shape analysis such as Curvature Scale Space (CSS). Currently, Gaussian filtering is widely used among a lot of smoothing methods. However Gaussian filtering is not affine invariant. This paper proposes a new method for curve smoothing that is invariant under affine transformation such that area of any region in the image does not change. Specifically, we introduce an affine invariant evaluate function with a metric tensor. The original curve is smoothed by minimizing the evaluation function. We mathematically prove that this method is affine invariant. Further, experimental results show that the proposed method is almost never affected by affine transformation different from usual Gaussian filtering. In the proposed method, processing results are expected to be not affected much by variation of the viewpoint.

Nishida, Takahiro; Toriu, Takashi

2014-01-01

342

Robust Speaker Clustering Using Affinity Propagation  

NASA Astrophysics Data System (ADS)

In this letter, a recently proposed clustering algorithm named affinity propagation is introduced for the task of speaker clustering. This novel algorithm exhibits fast execution speed and finds clusters with low error. However, experiments show that the speaker purity of affinity propagation is not satisfying. Thus, we propose a hybrid approach that combines affinity propagation with agglomerative hierarchical clustering to improve the clustering performance. Experiments show that compared with traditional agglomerative hierarchical clustering, the hybrid method achieves better performance on the test corpora.

Zhang, Xiang; Lu, Ping; Suo, Hongbin; Zhao, Qingwei; Yan, Yonghong

343

Affinity purification of DNA-binding proteins.  

PubMed

The focus of this review is on DNA affinity chromatography, which is the most powerful tool for purification of DNA binding proteins. The use of nonspecific-, sequence specific- and single stranded-DNA affinity columns in purification of various DNA binding proteins is discussed. The purification strategies for transcription factors, restriction enzymes, telomerases, DNA and RNA polymerase and DNA binding antibodies are described. Different applications of DNA affinity chromatography are presented. PMID:11694305

Gadgil, H; Oak, S A; Jarrett, H W

2001-10-30

344

Identification of protein complexes from filamentous fungi with tandem affinity purification.  

PubMed

Fungal molecular biology has benefited from the enormous advances in understanding protein-protein interactions in prokaryotic or eukaryotic organisms of the past decade. Tandem affinity purification (TAP) allows the enrichment of native protein complexes from cell extracts under mild conditions. We codon-optimized tags and established TAP, previously not applicable to filamentous fungi, for the model organism Aspergillus nidulans. We could identify by this method the trimeric Velvet complex VelB/VeA/LaeA or the eight subunit COP9 signalosome. Here, we describe an optimized protocol for A. nidulans which can also be adapted to other filamentous fungi. PMID:23065618

Bayram, Ozgür; Bayram, Ozlem Sarikaya; Valerius, Oliver; Jöhnk, Bastian; Braus, Gerhard H

2012-01-01

345

Non-affine deformations in polymer hydrogels  

PubMed Central

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

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

2012-01-01

346

Analysis of STIS time-tag data  

NASA Technical Reports Server (NTRS)

Very high time resolution data can be obtained from the Space Telescope Imaging Spectrograph (STIS) Multi-Anode Microchannel Array (MAMA) detectors using the time-tag observing mode. In this mode, the photon events are not accumulated onboard the spacecraft. Instead, each event is recorded internally and transmitted to the ground as an X and Y location with an event time. Event times are recorded in units of 125 microseconds. Analysis of STIS Crab Pulsar data demonstrates that a time resolution of approaching 125 microseconds can be achieved. Furthermore, the time-tag observing mode has been demonstrated to be a very powerful diagnostic tool and can be used to increase the resolution of both imaging and spectral data.

Lindler, Don J.; Gull, Theodore R.; Kraemer, Steven B.; Hulbert, Stephen J.

1997-01-01

347

Chemoenzymatic Fc Glycosylation via Engineered Aldehyde Tags  

PubMed Central

Glycoproteins with chemically defined glycosylation sites and structures are important biopharmaceutical targets and critical tools for glycobiology. One approach toward constructing such molecules involves chemical glycosylation of aldehyde-tagged proteins. Here, we report the installation of a genetically encoded aldehyde tag at the internal glycosylation site of the crystallizable fragment (Fc) of IgG1. We replaced the natural Fc N-glycosylation sequon with a five amino-acid sequence that was efficiently converted by recombinant formylglycine generating enzyme in vitro, thereby introducing aldehyde groups for subsequent chemical elaboration. Oxime-linked glycoconjugates were synthesized by conjugating aminooxy N-acetylglucosamine to the modified Fc followed by enzymatic transfer of complex N-glycans from corresponding glycan oxazolines by an EndoS-derived glycosynthase. In this manner we generated specific Fc glycoforms without relying on natural protein glycosylation machineries.

2014-01-01

348

The tagged RIBs facility of LNS  

SciTech Connect

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

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

2007-11-30

349

A fractal circular polarized RFID tag antenna  

NASA Astrophysics Data System (ADS)

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

Chaouki, Guesmi; Ferchichi, Abdelhak; Gharsallah, Ali

2013-09-01

350

Physics with tagged forward protons at RHIC  

SciTech Connect

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

Yip,K.

2009-08-30

351

RTCGD: retroviral tagged cancer gene database  

Microsoft Academic Search

Retroviral insertional mutagenesis in mouse hematopoietic tumors provides a potent cancer gene discovery tool in the post-genome-sequence era. To manage multiple high-throughput insertional mutagenesis screening projects, we developed the Retroviral Tagged Cancer Gene Database (RTCGD; http:\\/\\/RTCGD.ncifcrf.gov). A sequence analysis pipeline determines the genomic position of each retroviral integration site cloned from a mouse tumor, the distance between it and the

Keiko Akagi; Takeshi Suzuki; Robert M. Stephens; Nancy A. Jenkins; Neal G. Copeland

2004-01-01

352

Automatic Sense Tagging Using Parallel Corpora  

Microsoft Academic Search

This article reports the results of an analysis of translation equivalents in six languages from different language families, extracted from an on-line parallel corpus of George Orwell's Nineteen Eighty-Four. The goal is to determine sense distinctions that can be used to automatically sense-tag the data. Our results show that sense distinctions derived from cross- lingual information correspond to those made

Nancy Ide; Tomaz Erjavec; Dan Tufis

2001-01-01

353

Morphosyntactic Tagging of Slovene Using Progol  

Microsoft Academic Search

We consider the task of tagging Slovene words with morpho- syntactic descriptions (MSDs). MSDs contain not only part-of-speech information but also attributes such as gender and case. In the case of Slovene there are 2,083 possible MSDs. P-Progol was used to learn mor- phosyntactic disambiguation rules from annotated data (consisting of 161,314 examples) produced by the MULTEXT-East project. P-Progol produced

James Cussens; Saso Dzeroski; Tomaz Erjavec

1999-01-01

354

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

PubMed Central

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

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

2005-01-01

355

An approach to sequence DNA without tagging  

NASA Astrophysics Data System (ADS)

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

Niu, Sanjun; Saraf, Ravi F.

2002-10-01

356

Single tag for total carbohydrate analysis.  

PubMed

Anthranilic acid (2-aminobenzoic acid, 2-AA) has the remarkable property of reacting rapidly with every type of reducing carbohydrate. Reactivity of 2-AA with carbohydrates in aqueous solutions surpasses all other tags reported to date. This unique capability is attributed to the strategically located -COOH which accelerates Schiff base formation. Monosaccharides, oligosaccharides (N-, O-, and lipid linked and glycans in secretory fluids), glycosaminoglycans, and polysaccharides can be easily labeled with 2-AA. With 2-AA, labeling is simple in aqueous solutions containing proteins, peptides, buffer salts, and other ingredients (e.g., PNGase F, glycosidase, and transferase reaction mixtures). In contrast, other tags require relatively pure glycans for labeling in anhydrous dimethyl sulfoxide-acetic acid medium. Acidic conditions are known to cause desialylation, thus requiring a great deal of attention to sample preparation. Simpler labeling is achieved with 2-AA within 30-60 min in mild acetate-borate buffered solution. 2-AA provides the highest sensitivity and resolution in chromatographic methods for carbohydrate analysis in a simple manner. Additionally, 2-AA is uniquely qualified for quantitative analysis by mass spectrometry in the negative mode. Analyses of 2-AA-labeled carbohydrates by electrophoresis and other techniques have been reported. Examples cited here demonstrate that 2-AA is the universal tag for total carbohydrate analysis. PMID:24769375

Anumula, Kalyan Rao

2014-07-15

357

High-affinity VEGF antagonists by oligomerization of a minimal sequence VEGF-binding domain.  

PubMed

Vascular endothelial growth factor (VEGF) neutralizing antagonists including antibodies or receptor extracellular domain Fc fusions have been applied clinically to control angiogenesis in cancer, wet age-related macular degeneration, and edema. We report here the generation of high-affinity VEGF-binding domains by chemical linkage of the second domain of the VEGF receptor Flt-1 (D2) in several configurations. Recombinant D2 was expressed with a 13 a.a. C-terminal tag, including a C-terminal cysteine to enable its dimerization by disulfide bond formation or by attachment to divalent PEGs and oligomerization by coupling to multivalent PEGs. Disulfide-linked dimers produced by Cu(2+) oxidation of the free-thiol form of the protein demonstrated picomolar affinity for VEGF in solution, comparable to that of a D2-Fc fusion (sFLT01) and ~50-fold higher than monomeric D2, suggesting the 26 a.a. tag length between the two D2 domains permits simultaneous interaction of both faces of the VEGF homodimer. Extending the separation between the D2 domains by short PEG spacers from 0.35 kD to 5 kD produced a modest ~2-fold increase in affinity over the disulfide, thus defining the optimal distance between the two D2 domains for maximum affinity. By surface plasmon resonance (SPR), a larger (~5-fold) increase in affinity was observed by conjugation of the D2 monomer to the termini of 4-arm PEG, and yielding a product with a larger hydrodynamic radius than sFLT01. The higher affinity displayed by these D2 PEG tetramers than either D2 dimer or sFLT01 was largely a consequence of a slower rate of dissociation, suggesting the simultaneous binding by these tetramers to neighboring surface-bound VEGF. Finally, disulfide-linked D2 dimers showed a greater resistance to autocatalytic fragmentation than sFLT01 under elevated temperature stress, indicating such minimum-sequence constructs may be better suited for sustained-release formulations. Therefore, these constructs represent novel Fc-independent VEGF antagonists with ultrahigh affinity, high stability, and a range of hydrodynamic radii for application to multiple therapeutic targets. PMID:23176598

Stefano, James E; Bird, Julie; Kyazike, Josephine; Cheng, Anthony Wai-Ming; Boudanova, Ekaterina; Dwyer, Markryan; Hou, Lihui; Qiu, Huawei; Matthews, Gloria; O'Callaghan, Michael; Pan, Clark Q

2012-12-19

358

Using TAGs to speed up the ATLAS analysis process  

NASA Astrophysics Data System (ADS)

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.

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

359

Subcellular localization and function of an epitope-tagged p7 viroporin in hepatitis C virus-producing cells.  

PubMed

The hepatitis C virus (HCV) viroporin p7 is crucial for production of infectious viral progeny. However, its role in the viral replication cycle remains incompletely understood, in part due to the poor availability of p7-specific antibodies. To circumvent this obstacle, we inserted two consecutive hemagglutinin (HA) epitope tags at its N terminus. HA-tagged p7 reduced peak virus titers ca. 10-fold and decreased kinetics of virus production compared to the wild-type virus. However, HA-tagged p7 rescued virus production of a mutant virus lacking p7, thus providing formal proof that the tag does not disrupt p7 function. In HCV-producing cells, p7 displayed a reticular staining pattern which colocalized with the HCV envelope glycoprotein 2 (E2) but also partially with viral nonstructural proteins 2, 3, and 5A. Using coimmunoprecipitation, we confirmed a specific interaction between p7 and NS2, whereas we did not detect a stable interaction with core, E2, or NS5A. Moreover, we did not observe p7 incorporation into affinity-purified virus particles. Consistently, there was no evidence supporting a role of p7 in viral entry, as an anti-HA antibody was not able to neutralize Jc1 virus produced from an HA-p7-tagged genome. Collectively, these findings highlight a stable interaction between p7 and NS2 which is likely crucial for production of infectious HCV particles. Use of this functional epitope-tagged p7 variant should facilitate the analysis of the final steps of the HCV replication cycle. PMID:23175364

Vieyres, Gabrielle; Brohm, Christiane; Friesland, Martina; Gentzsch, Juliane; Wölk, Benno; Roingeard, Philippe; Steinmann, Eike; Pietschmann, Thomas

2013-02-01

360

Subcellular Localization and Function of an Epitope-Tagged p7 Viroporin in Hepatitis C Virus-Producing Cells  

PubMed Central

The hepatitis C virus (HCV) viroporin p7 is crucial for production of infectious viral progeny. However, its role in the viral replication cycle remains incompletely understood, in part due to the poor availability of p7-specific antibodies. To circumvent this obstacle, we inserted two consecutive hemagglutinin (HA) epitope tags at its N terminus. HA-tagged p7 reduced peak virus titers ca. 10-fold and decreased kinetics of virus production compared to the wild-type virus. However, HA-tagged p7 rescued virus production of a mutant virus lacking p7, thus providing formal proof that the tag does not disrupt p7 function. In HCV-producing cells, p7 displayed a reticular staining pattern which colocalized with the HCV envelope glycoprotein 2 (E2) but also partially with viral nonstructural proteins 2, 3, and 5A. Using coimmunoprecipitation, we confirmed a specific interaction between p7 and NS2, whereas we did not detect a stable interaction with core, E2, or NS5A. Moreover, we did not observe p7 incorporation into affinity-purified virus particles. Consistently, there was no evidence supporting a role of p7 in viral entry, as an anti-HA antibody was not able to neutralize Jc1 virus produced from an HA-p7-tagged genome. Collectively, these findings highlight a stable interaction between p7 and NS2 which is likely crucial for production of infectious HCV particles. Use of this functional epitope-tagged p7 variant should facilitate the analysis of the final steps of the HCV replication cycle.

Vieyres, Gabrielle; Brohm, Christiane; Friesland, Martina; Gentzsch, Juliane; Wolk, Benno; Roingeard, Philippe; Steinmann, Eike

2013-01-01

361

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

PubMed Central

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

McKercher, Ginette; Beaulieu, Pierre L.; Lamarre, Daniel; LaPlante, Steven; Lefebvre, Sylvain; Pellerin, Charles; Thauvette, Louise; Kukolj, George

2004-01-01

362

The use of tags in monitoring limits on mobile missiles  

SciTech Connect

Three tagging systems were considered in this paper: as a supplement to on-site inspection (OSI), as a supplement to national technical means (NTM), and as a supplement to site surveillance systems. Each system would require a different type of tag, perhaps ranging from microchip tags with infrared transponders to navigation receivers. Use of tags as a supplement to OSIs may be the simplest system to implement because it places the least demands on technology. Tags may make OSI more acceptable by replacing humans with remote sensors, thereby decreasing the perceived potential for espionage. Using tags as a supplement to NTM decreases the necessity for human OSI even further, but places higher demands on technology and may affect the normal operation of deployment areas. Site surveillance systems using tags have the potential for excellent missile verification, but they may be excessively intrusive and expensive, and could have a large effect on the normal operation of declared facilities.

Fetter, S.

1987-03-01

363

TagRecon: High-Throughput Mutation Identification through Sequence Tagging  

PubMed Central

Shotgun proteomics produces collections of tandem mass spectra that contain all the data needed to identify mutated peptides from clinical samples. Identifying these sequence variations, however, has not been feasible with conventional database search strategies, which require exact matches between observed and expected sequences. Searching for mutations as mass shifts on specified residues through database search can incur significant performance penalties and generate substantial false positive rates. Here we describe TagRecon, an algorithm that leverages inferred sequence tags to identify unanticipated mutations in clinical proteomic data sets. TagRecon identifies unmodified peptides as sensitively as the related MyriMatch database search engine. In both LTQ and Orbitrap data sets, TagRecon outperformed state of the art software in recognizing sequence mismatches from data sets with known variants. We developed guidelines for filtering putative mutations from clinical samples, and we applied them in an analysis of cancer cell lines and an examination of colon tissue. Mutations were found in up to 6% of identified peptides, and only a small fraction corresponded to dbSNP entries. The RKO cell line, which is DNA mismatch repair deficient, yielded more mutant peptides than the mismatch repair proficient SW480 line. Analysis of colon cancer tumor and adjacent tissue revealed hydroxyproline modifications associated with extracellular matrix degradation. These results demonstrate the value of using sequence tagging algorithms to fully interrogate clinical proteomic data sets.

Dasari, Surendra; Chambers, Matthew C.; Slebos, Robbert J.; Zimmerman, Lisa J.; Ham, Amy-Joan L.; Tabb, David L.

2010-01-01

364

Scaling analysis of affinity propagation  

NASA Astrophysics Data System (ADS)

We analyze and exploit some scaling properties of the affinity propagation (AP) clustering algorithm proposed by Frey and Dueck [Science 315, 972 (2007)]. Following a divide and conquer strategy we setup an exact renormalization-based approach to address the question of clustering consistency, in particular, how many cluster are present in a given data set. We first observe that the divide and conquer strategy, used on a large data set hierarchically reduces the complexity O(N2) to O(N(h+2)/(h+1)) , for a data set of size N and a depth h of the hierarchical strategy. For a data set embedded in a d -dimensional space, we show that this is obtained without notably damaging the precision except in dimension d=2 . In fact, for d larger than 2 the relative loss in precision scales such as N(2-d)/(h+1)d . Finally, under some conditions we observe that there is a value s? of the penalty coefficient, a free parameter used to fix the number of clusters, which separates a fragmentation phase (for ss? ) of the underlying hidden cluster structure. At this precise point holds a self-similarity property which can be exploited by the hierarchical strategy to actually locate its position, as a result of an exact decimation procedure. From this observation, a strategy based on AP can be defined to find out how many clusters are present in a given data set.

Furtlehner, Cyril; Sebag, Michèle; Zhang, Xiangliang

2010-06-01

365

Scaling analysis of affinity propagation.  

PubMed

We analyze and exploit some scaling properties of the affinity propagation (AP) clustering algorithm proposed by Frey and Dueck [Science 315, 972 (2007)]. Following a divide and conquer strategy we setup an exact renormalization-based approach to address the question of clustering consistency, in particular, how many cluster are present in a given data set. We first observe that the divide and conquer strategy, used on a large data set hierarchically reduces the complexity O(N2) to O(N((h+2)/(h+1))) , for a data set of size N and a depth h of the hierarchical strategy. For a data set embedded in a d -dimensional space, we show that this is obtained without notably damaging the precision except in dimension d=2 . In fact, for d larger than 2 the relative loss in precision scales such as N((2-d)/(h+1)d). Finally, under some conditions we observe that there is a value s* of the penalty coefficient, a free parameter used to fix the number of clusters, which separates a fragmentation phase (for ss*) of the underlying hidden cluster structure. At this precise point holds a self-similarity property which can be exploited by the hierarchical strategy to actually locate its position, as a result of an exact decimation procedure. From this observation, a strategy based on AP can be defined to find out how many clusters are present in a given data set. PMID:20866473

Furtlehner, Cyril; Sebag, Michèle; Zhang, Xiangliang

2010-06-01

366

Photovoltaic Voltage Regulation by Affine Parameterization  

Microsoft Academic Search

Photovoltaic power systems work under uncertain, nonlinear, and time-variant environments, making the controller design of solar converters a challenging problem. This paper proposes an application of the affine parameterization design (APD) to photovoltaic voltage regulation for DC\\/DC converters. The affine parameterization approach greatly simplifies the control design by directly choosing parameters from desired closed loop characteristics. The tradeoff between robustness

Weidong Xiao; Peng Zhang

2012-01-01

367

Lorentzian Affine Hypersurfaces with Parallel Cubic Form  

Microsoft Academic Search

We study Lorentzian affine hypersurfaces of $${\\\\mathbb{R}^{n+1}}$$ having parallel cubic form with respect to the Levi-Civita connection of the affine Berwald-Blaschke metric. As main result,\\u000a we obtain a complete classification of these hypersurfaces.

Zejun Hu; Cece Li; Haizhong Li; Luc Vrancken

2011-01-01

368

Credit risk modeling with affine processes  

Microsoft Academic Search

This article combines an orientation to credit risk modeling with an introduction to affine Markov processes, which are particularly useful for financial modeling. We emphasize corporate credit risk and the pricing of credit derivatives. Applications of affine processes that are mentioned include survival analysis, dynamic term-structure models, and option pricing with stochastic volatility and jumps. The default-risk applications include default

Darrell Duffie

2005-01-01

369

Affine\\/ Photometric Invariants for Planar Intensity Patterns  

Microsoft Academic Search

The paper contributes to the viewpoint invariant recognition of planar patterns, especially labels and signs under affine deformations. By their nature, the information of such eye-catchers is not contained in the outline or frame — they often are affinely equivalent like parallelograms and ellipses — but in the intensity content within. Moment invariants are well suited for their recognition. They

Luc J. Van Gool; Theo Moons; Dorin Ungureanu

1996-01-01

370

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

USGS Publications Warehouse

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.

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

1999-01-01

371

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

PubMed

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

Mills, Brittney J; Mu, Qingxin; Krause, Mary E; Laurence, Jennifer S

2014-06-18

372

Improving image segmentation by learning region affinities  

SciTech Connect

We utilize the context information of other regions in hierarchical image segmentation to learn new regions affinities. It is well known that a single choice of quantization of an image space is highly unlikely to be a common optimal quantization level for all categories. Each level of quantization has its own benefits. Therefore, we utilize the hierarchical information among different quantizations as well as spatial proximity of their regions. The proposed affinity learning takes into account higher order relations among image regions, both local and long range relations, making it robust to instabilities and errors of the original, pairwise region affinities. Once the learnt affinities are obtained, we use a standard image segmentation algorithm to get the final segmentation. Moreover, the learnt affinities can be naturally unutilized in interactive segmentation. Experimental results on Berkeley Segmentation Dataset and MSRC Object Recognition Dataset are comparable and in some aspects better than the state-of-art methods.

Prasad, Lakshman [Los Alamos National Laboratory; Yang, Xingwei [TEMPLE UNIV.; Latecki, Longin J [TEMPLE UNIV.

2010-11-03

373

Identification of okadaic acid-induced phosphorylation events by a mass spectrometry approach  

Microsoft Academic Search

Okadaic acid (OA) is a widely used small-molecule phosphatase inhibitor that is thought to selectively inhibit protein phosphatase 2A (PP2A). Multiple studies have demonstrated that PP2A activity is compromised in the brains of Alzheimer’s disease patients. Thus, we set out to determine changes in phosphorylation that occur upon OA treatment of neuronal cells. Utilizing isotope-coded affinity tags and mass spectrometry

Jennifer J.. Hill; Deborah A. Callaghan; Wen Ding; John F. Kelly; Balu R. Chakravarthy

2006-01-01

374

Dynamic changes in transcription factor complexes during erythroid differentiation revealed by quantitative proteomics  

Microsoft Academic Search

During erythroid differentiation, ?-globin gene expression is regulated by the locus control region (LCR). The transcription factor NF-E2p18\\/MafK binds within this region and is essential for ?-globin expression in murine erythroleukemia (MEL) cells. Here we use the isotope-coded affinity tag (ICAT) technique of quantitative mass spectrometry to compare proteins interacting with NF-E2p18\\/MafK during differentiation. Our results define MafK as a

Marjorie Brand; Jeffrey A Ranish; Nicolas T Kummer; Joan Hamilton; Kazuhiko Igarashi; Claire Francastel; Tian H Chi; Gerald R Crabtree; Ruedi Aebersold; Mark Groudine

2003-01-01

375

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

DOEpatents

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.

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

2006-09-12

376

A novel approach to investigating protein/protein interactions and their functions by TAP-tagged yeast strains and its application to examine yeast transcription machinery.  

PubMed

Tandem affinity purification (TAP) method combined with LC-MS/MS is the most accurate and reliable way to study the interaction of proteins or proteomics in a genome-wide scale. For the first time, we used a TAP-tag as a mutagenic tool to disrupt protein interactions at the specific site. Although lots of commonly used mutational tools exist to study functions of a gene, such as deletional mutations and site-directed mutagenesis, each method has its own demerit. To test the usefulness of a TAP-tag as a mutagenic tool, we applied a TAP-tag to RNA polymerase II, which is the key enzyme of gene expression and is controlled by hundreds of transcription factors even to transcribe a gene. Our experiment is based on the hypothesis that there will be interrupted interactions between Pol II and transcription factors owing to the TAP-tag attached at the C-terminus of each subunit of Pol II, and the abnormality caused by interrupted protein interactions can be observed by measuring a cell-cycle of each yeast strain. From ten different TAP-tagged strains, Rpb7- and Rpb12-TAP-tagged strains show severe defects in growth rate and morphology. Without a heterodimer of Rpb4/Rpb7, only the ten subunits Pol II can conduct transcription normally, and there is no previously known function of Rpb7. The observed defect of the Rpb7-TAP-tagged strain shows that Rpb7 forms a complex with other proteins or compounds and the interruption of the interaction can interfere with the normal cell cycle and morphology of the cell and nucleus. This is a novel attempt to use a TAP-tag as a proteomic tool to study protein interactions. PMID:18467854

Jung, Junho; Ahn, Yeh-Jin; Kang, Lin-Woo

2008-04-01

377

Integrated Management and Visualization of Electronic Tag Data with Tagbase  

PubMed Central

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.

Lam, Chi Hin; Tsontos, Vardis M.

2011-01-01

378

Identification of new tag sequences with differential and selective recognition properties for the anti-FLAG monoclonal antibodies M1, M2 and M5  

Microsoft Academic Search

Summary The FLAG® peptides DYKDDDDK and MDYKDDDDK are widely used affinity tags. Here we describe new variants of the FLAG peptides which, in direct ELISA, showed selective and differential binding to the commercially available anti-FLAG monoclonal antibodies M1, M2 and M5. Variants of the FLAG peptides were synthesized on polymer-grafted plastic pins, and in an ELISA incubated with mAbs M1,

J. W. Slootstra; D. Kuperus; A. Plückthun; R. H. Meloen

1997-01-01

379

His-tag truncated butyrylcholinesterase as a useful construct for in vitro characterization of wild-type and variant butyrylcholinesterases  

PubMed Central

Human butyrylcholinesterase (BChE) can scavenge and thereby provide protection against various toxic esters, including organophosphate-based chemical warfare agents and the recreational drug cocaine. It is currently being used in molecular evolution studies to generate novel enzymes with improved ability to hydrolyze toxic ester compounds. Currently, the most commonly used purification strategies for recombinant BChE enzymes involve using affinity resins based on small molecule interactions with the enzyme’s substrate binding site. However, as BChE variants are discovered and developed, a generic purification protocol that is insensitive to amino acid substitutions is necessary. In the current manuscript, an expression vector encoding a C-terminal truncation and His6-tag was designed for BChE and used to express recombinant “wild-type” enzyme and two variants (i.e., G117H BChE and G117H/E197Q BChE). All three His6-tagged enzymes were successfully purified via metal-affinity columns using similar procedures with good recovery. Steady-state kinetic parameters were determined for each enzyme, and values were compared to those obtained with the corresponding non-truncated non-His6-tagged enzymes. Rates of inhibition by echothiophate, a model compound for organophosphate-based pesticides, and rates of oxime-mediated reactivation after inhibition with a nerve agent model compound were also determined for selected enzymes. Rates of spontaneous reactivation from ETP inhibition were determined for G117H variants. In all instances examined, truncation of the C-terminus of BChE and introduction of a His6-tag had no significant effects on the observed kinetic parameters, making this a highly useful construct for in vitro characterization of wild-type and variant BChEs.

Ralph, Erik C.; Xiang, Longkuan; Cashman, John R.; Zhang, Jun

2011-01-01

380

Affinity-based Protein Surface Pattern Formation by Ligand Self-Selection from Mixed Protein Solutions.  

PubMed

Photolithographically prepared surface patterns of two affinity ligands (biotin and chloroalkane) specific for two proteins (streptavidin and HaloTag®, respectively) are used to spontaneously form high-fidelity surface patterns of the two proteins from their mixed solution. High affinity protein-surface self-selection onto patterned ligands on surfaces exhibiting low non-specific adsorption rapidly yields the patterned protein surfaces. Fluorescence images after protein immobilization show high specificity of the target proteins to their respective surface patterned ligands. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging further supports the chemical specificity of streptavidin and HaloTag® for their surface patterned ligands from mixed protein solutions. However, ToF-SIMS did detect some non-specific adsorption of bovine serum albumin, a masking protein present in excess in the adsorbing solutions, on the patterned surfaces. Protein amino acid composition, surface coverage, density and orientation are important parameters that determine the relative ToF-SIMS fragmentation pattern yields. ToF-SIMS amino acid-derived ion fragment yields summed to produce surface images can reliably determine which patterned surface regions contain bound proteins, but do not readily discriminate between different co-planar protein regions. Principal component analysis (PCA) of these ToF-SIMS data, however, improves discrimination of ions specific to each protein, facilitating surface pattern discrimination and contrast. PMID:23504611

Grainger, David W; Castner, David G; Dubey, Manish; Emoto, Kazunori; Takahashi, Hironobu

2009-10-01

381

Development of Translating Ribosome Affinity Purification for Zebrafish  

PubMed Central

Summary The regulation of transcription and translation by specific cell types is essential to generate the cellular diversity that typifies complex multicellular organisms. Tagging and purification of ribosomal proteins has been shown to be an innovative and effective means of characterizing the ribosome bound transcriptome of highly specific cell populations in vivo. To test the feasibility of using translating ribosome affinity purification (TRAP) in zebrafish, we have generated both a ubiquitous TRAP line and a melanocyte-specific TRAP line using the native zebrafish rpl10a ribosomal protein. We have demonstrated the capacity to capture mRNA transcripts bound to ribosomes, and confirmed the expected enrichment of melanocyte specific genes and depletion of non-melanocyte genes when expressing the TRAP construct with a cell specific promoter. We have also generated a generic EGFP-rpl10a Tol2 plasmid construct (Tol2-zTRAP) that can be readily modified to target any additional cell populations with characterized promoters in zebrafish.

Tryon, Robert C.; Pisat, Nilambari; Johnson, Stephen L.; Dougherty, Joseph D.

2013-01-01

382

High-throughput analysis of protein-DNA binding affinity.  

PubMed

Sequence-specific protein-DNA interactions mediate most regulatory processes underlying gene expression, such as transcriptional regulation by transcription factors (TFs) or chromatin organization. Current knowledge about DNA-binding specificities of TFs is based mostly on low- to medium-throughput methodologies that are time-consuming and often fail to identify DNA motifs recognized by a TF with lower affinity but retaining biological relevance. The use of protein-binding microarrays (PBMs) offers a high-throughput alternative for the identification of protein-DNA specificities. PBM consists in an array of pseudorandomized DNA sequences that are optimized to include all the possible 10- or 11-mer DNA sequences, allowing the determination of binding specificities of most eukaryotic TFs. PBMs that can be synthesized by several manufacturing companies as single-stranded DNA are converted into double-stranded in a simple primer extension reaction. The protein of interest fused to an epitope tag is then incubated onto the PBM, and specific DNA-protein complexes are revealed in a series of immunological reactions coupled to a fluorophore. After scanning and quantifying PBMs, specific DNA motifs recognized by the protein are identified with ready-to-use scripts, generating comprehensive but accessible information about the DNA-binding specificity of the protein. This chapter describes detailed procedures for preparation of double-stranded PBMs, incubation with recombinant protein, and detection of protein-DNA complexes. Finally, we outline some cues for evaluating the biological role of DNA motifs obtained in vitro. PMID:24057393

Franco-Zorrilla, José M; Solano, Roberto

2014-01-01

383

Affinity Purification Strategies for Proteomic Analysis of Transcription Factor Complexes  

PubMed Central

Affinity purification (AP) coupled to mass spectrometry (MS) has been successful in elucidating protein molecular networks of mammalian cells. These approaches have dramatically increased the knowledge of the interconnectivity present among proteins and highlighted biological functions within different protein complexes. Despite significant technical improvements reached in the past years, it is still challenging to identify the interaction networks and the subsequent associated functions of nuclear proteins such as transcription factors (TFs). A straightforward and robust methodology is therefore required to obtain unbiased and reproducible interaction data. Here we present a new approach for TF AP-MS, exemplified with the CCAAT/enhancer binding protein alpha (C/EBPalpha). Utilizing the advantages of a double tag and three different MS strategies, we conducted a total of six independent AP-MS strategies to analyze the protein–protein interactions of C/EBPalpha. The resultant data were combined to produce a cohesive C/EBPalpha interactome. Our study describes a new methodology that robustly identifies specific molecular complexes associated with transcription factors. Moreover, it emphasizes the existence of TFs as protein complexes essential for cellular biological functions and not as single, static entities.

2013-01-01

384

Reprint of: Minimal affinizations as projective objects  

NASA Astrophysics Data System (ADS)

We prove that the specialization to q=1 of a Kirillov-Reshetikhin module for an untwisted quantum affine algebra of classical type is projective in a suitable category. This yields a uniform character formula for the Kirillov-Reshetikhin modules. We conjecture that these results hold for specializations of minimal affinization with some restriction on the corresponding highest weight. We discuss the connection with the conjecture of Nakai and Nakanishi on q-characters of minimal affinizations. We establish this conjecture in some special cases. This also leads us to conjecture an alternating sum formula for Jacobi-Trudi determinants.

Chari, Vyjayanthi; Greenstein, Jacob

2011-09-01

385

Harvesting weakly tagged images for computer vision tasks  

NASA Astrophysics Data System (ADS)

To crawl large amounts of weakly-tagged images for computer vision tasks such as object detection and scene recognition, it is very important to develop new techniques for tag cleansing and word sense disambiguation (i.e., removing irrelevant images from the crawled results). Based on this observation, a topic network is first generated to characterize both the semantic similarity contexts and the visual similarity contexts between the image topics more sufficiently. The topic network is used to represent the classes of objects and scenes of interest. Second, both the visual similarity contexts between the images and the semantic similarity contexts between their tags are integrated for tag cleansing and word sense disambiguation. By addressing the issues of polysemes and synonyms more effectively, our word sense disambiguation algorithm can determine the relevance between the images and the associated tags more precisely, and thus it can allow us to crawl large-scale weakly-tagged images for computer vision tasks.

Shen, Yi; Yang, Chunlei; Gao, Yuli; Fan, Jianping

2010-02-01

386

Method for nonlinear optimization for gas tagging and other systems  

DOEpatents

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.

Chen, T.; Gross, K.C.; Wegerich, S.

1998-01-06

387

Tagging module for lesion localization in capsule endoscopy.  

PubMed

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

Chandrappan, Jayakrishnan; Ruiqi, Lim; Su, Nandar; Qiang, Tanq Shao; Vaidyanathan, Kripesh

2010-01-01

388

Method for nonlinear optimization for gas tagging and other systems  

DOEpatents

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.

Chen, Ting (Chicago, IL); Gross, Kenny C. (Bolingbrook, IL); Wegerich, Stephan (Glendale Heights, IL)

1998-01-01

389

Triosephosphate isomerase is a common crystallization contaminant of soluble His-tagged proteins produced in Escherichia coli  

PubMed Central

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?Å resolution, which is a significant improvement over the previous structure.

Kozlov, Guennadi; Vinaik, Roohi; Gehring, Kalle

2013-01-01

390

Some Fundamental Limits on SAW RFID Tag Information Capacity and Collision Resolution  

NASA Technical Reports Server (NTRS)

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

Barton, Richard J.

2013-01-01

391

Minimalist Cryptography for Low-Cost RFID Tags  

Microsoft Academic Search

A radio-frequency identification (RFID) tag is a small, inexpensive microchip that emits an identifier in response to a query from a nearby reader. The price of these tags promises to drop to the range of $0.05 per unit in the next several years, oering a viable and powerful replacement for barcodes. The challenge in providing security for low-cost RFID tags

Ari Juels

2004-01-01

392

HTML Tags as Extraction Cues for Web Page Description Construction  

Microsoft Academic Search

Using four previously identified samples of Web pages containing meta-tagged descriptions, the value of meta-tagged keywords, the first 200 characters of the body, and text marked with common HTML tags as extracts helpful for writing summaries was estimated by applying two measures: density of de- scription words and density of two-word description phrases. Generally, titles and keywords showed the highest

Timothy C. Craven

2003-01-01

393

Tag-based approaches for deep transcriptome analysis in plants  

Microsoft Academic Search

Serial analysis of gene expression (SAGE) has pioneered the use of short-tag sequences derived from the 3?-ends of cDNAs for transcriptome research. Many new tag-based technologies, capitalizing on the success of SAGE, have been developed in the last decade greatly improving the tag length, cloning efficacy and the depth of transcriptome analysis in targeted genomes. Moreover, the recent introduction of

Miguel E. Vega-Sánchez; Malali Gowda; Guo-Liang Wang

2007-01-01

394

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

PubMed

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

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

2013-10-10

395

Current test results for the Athena radar responsive tag  

NASA Astrophysics Data System (ADS)

Sandia National Laboratories has teamed with General Atomics and Sierra Monolithics to develop the Athena tag for the Army's Radar Tag Engagement (RaTE) program. The radar-responsive Athena tag can be used for Blue Force tracking and Combat Identification (CID) as well as data collection, identification, and geolocation applications. The Athena tag is small (~4.5" x 2.4" x 4.2"), battery-powered, and has an integral antenna. Once remotely activated by a Synthetic Aperture Radar (SAR) or Moving Target Indicator (MTI) radar, the tag transponds modulated pulses to the radar at a low transmit power. The Athena tag can operate Ku-band and X-band airborne SAR and MTI radars. This paper presents results from current tag development testing activities. Topics covered include recent field tests results from the AN/APY-8 Lynx, F16/APG-66, and F15E/APG-63 V(1) radars and other Fire Control radars. Results show that the Athena tag successfully works with multiple radar platforms, in multiple radar modes, and for multiple applications. Radar-responsive tags such as Athena have numerous applications in military and government arenas. Military applications include battlefield situational awareness, combat identification, targeting, personnel recovery, and unattended ground sensors. Government applications exist in nonproliferation, counter-drug, search-and-rescue, and land-mapping activities.

Ormesher, Richard C.; Martinez, Ana; Plummer, Kenneth W.; Erlandson, David; Delaware, Sheri; Clark, David R.

2006-06-01

396

Remote object authentication using distortion-invariant ID tags  

NASA Astrophysics Data System (ADS)

A number of applications in security or inventory control may benefit from an authentication system able to identify a remote object viewed from different perspectives or distances. Object identification can be accomplished by using optical ID tags, which include relevant information of the target and are located on a visible part of the object under surveillance. Encryption of the information codified in the ID tag allows increasing security and deters from unauthorized usage of optical tags. The identification process encompasses several steps such as detection, information decoding and verification which are all detailed in this work. Design of distortion-invariant ID tags has to be taken into account to achieve a correct object authentication even if the ID tag is detected and captured at different distances (i.e. different scales) or from different views (i.e. rotated versions of the original ID tag). Description of diverse distortion-invariant ID tags and authentication results using the proposed ID tags are provided. We show that distortion-tolerance is achieved by the described identification system. Information encrypted on the tested ID tags is correctly decoded and verified even if variations in scale and rotations are considered. The effects of environmental degradation are taken into account in the recognition process.

Perez-Cabre, Elisabet; Millan, Maria S.; Javidi, Bahram

2005-09-01

397

PIT tags increase effectiveness of freshwater mussel recaptures  

USGS Publications Warehouse

Translocations are used increasingly to conserve populations of rare freshwater mussels. Recovery of translocated mussels is essential to accurate assessment of translocation success. We designed an experiment to evaluate the use of passive integrated transponder (PIT) tags to mark and track individual freshwater mussels. We used eastern lampmussels (Lampsilis radiata radiata) as a surrogate for 2 rare mussel species. We assessed internal and external PIT-tag retention in the laboratory and field. Internal tag retention was high (75-100%), and tag rejection occurred primarily during the first 3 wk after tagging. A thin layer of nacre coated internal tags 3 to 4 mo after insertion, suggesting that long-term retention is likely. We released mussels with external PIT tags at 3 field study sites and recaptured them with a PIT pack (mobile interrogation unit) 8 to 10 mo and 21 to 23 mo after release. Numbers of recaptured mussels differed among study sites; however, we found more tagged mussels with the PIT-pack searches with visual confirmation (72-80%) than with visual searches alone (30-47%) at all sites. PIT tags offer improved recapture of translocated mussels and increased accuracy of posttranslocation monitoring. ?? 2007 by The North American Benthological Society.

Kurth, J.; Loftin, C.; Zydlewski, J.; Rhymer, J.

2007-01-01

398

Fluorinated NAD as an affinity surfactant.  

PubMed

Nicatinamide adenine dinucleotide (NAD) with an attached perfluoropolyether tail acts as an affinity surfactant in the extraction of the enzyme horse liver alcohol dehydrogenase (HLADH) from an aqueous medium into a fluorous solvent. PMID:12123052

Panza, Janice L; Russell, Alan J; Beckman, Eric J

2002-05-01

399

Compounds with High Monoamine Transporter Affinity.  

National Technical Information Service (NTIS)

Featured compounds have high monoamine transport affinity and are characterized by one of the following two general formulas set out above. The compounds bind selectively or non-selectively to monoamine transporters. The compounds are useful to treat vari...

B. K. Madras P. Blundell P. Wang P. C. Meltzer

2005-01-01

400

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...

401

Electron Affinities of Atoms, Molecules, and Radicals.  

National Technical Information Service (NTIS)

We review briefly but comprehensively the theoretical, semiempirical and experimental methods employed to determine electron affinities (EAs) of atoms, molecules and radicals, and summarize the EA data obtained by these methods. The detailed processes und...

A. A. Christodoulides, D. L. McCorkle, L. G. Christophorou

1982-01-01

402

Laplacian Discriminant Projection Based on Affinity Propagation  

NASA Astrophysics Data System (ADS)

The paper proposes a new algorithm for supervised dimensionality reduction, called Laplacian Discriminant Projection based on Affinity Propagation (APLDP). APLDP defines three scatter matrices using similarities based on representative exemplars which are found by Affinity Propagation Clustering. After linear transformation, the considered pairwise samples within the same exemplar subset and the same class are as close as possible, while those exemplars between classes are as far as possible. The experiments on several data sets demonstrate the competence of APLDP.

Chang, Xueping; Zheng, Zhonglong

403

Superior triacylglycerol (TAG) accumulation in starchless mutants of Scenedesmus obliquus: (II) evaluation of TAG yield and productivity in controlled photobioreactors  

PubMed Central

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.

2014-01-01

404

Molecular cloning, purification and functional implications of recombinant GST tagged hGMCSF cytokine.  

PubMed

We report the cloning, purification and cell proliferative activity of a novel recombinant GST tagged human granulocyte macrophage colony stimulating factor (GST-hGMCSF). The hGMCSF gene was PCR amplified from the cDNA of ACHN renal carcinoma cells and was cloned into the bacterial expression vector. The GST-hGMCSF was purified to homogeneity using glutathione agarose affinity chromatography and subsequently characterized by Western blot, circular dichroism (CD) and MALDI TOF-TOF analysis. Homology modelling studies revealed the possible binding domains of the recombinant cytokine with cognate receptor. The proliferation of THP-1, Raw 264.7, MCF-7 and U87MG cells upon GST-hGMCSF addition was found to be dose dependent. Hence, this functionally active recombinant cytokine has potential application in cancer therapy for stimulating facile growth recovery of normal cell population. PMID:23334834

Chaubey, Nidhi; Ghosh, Siddhartha Sankar

2013-03-01

405

Chemistry meets proteomics: the use of chemical tagging reactions for MS-based proteomics.  

PubMed

As proteomics matures from a purely descriptive to a function-oriented discipline of the life sciences, there is strong demand for novel methodologies that increase the depth of information that can be obtained from proteomic studies. MS has long played a central role for protein identification and characterization, often in combination with dedicated chemical modification reactions. Today, chemistry is helping to advance the field of proteomics in numerous ways. In this review, we focus on those methodologies that have a significant impact for the large-scale study of proteins and peptides. This includes approaches that allow the introduction of affinity tags for the enrichment of subclasses of peptides or proteins and strategies for in vitro stable isotope labeling for quantification purposes, among others. Particular attention is given to the study of PTMs where recent advancements have been promising, but many interesting targets are not yet being addressed. PMID:16972287

Leitner, Alexander; Lindner, Wolfgang

2006-10-01

406

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

SciTech Connect

We present a rare example of a biologically active G protein coupled receptor (GPCR) whose purity and identity were verified by mass spectrometry after being purified to near homogeneity from its native system. An overexpression vector was constructed to encode the Saccharomyces cerevisiae GPCR -factor receptor (Ste2p, the STE2 gene product) containing a 9-amino acid sequence of rhodopsin that served as an epitope/affinity tag. In the construct, two glycosylation sites and two cysteine residues were removed to aid future structural and functional studies. The receptor was expressed in yeast cells and was detected as a single band in a western blot indicating the absence of glycosylation. Tests of the epitope-tagged, mutated receptor showed it maintained its full biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5 % n-dodecyl maltoside (DM). Approximately 120 g of purified -factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (Kd) of the purified -factor receptor in DM micelles was 28 nM as compared to Kd = 12.7 nM for Ste2p in cell membranes, and approximately 40 % of the purified receptor was correctly folded as judged by ligand saturation binding. About 50 % of the receptor sequence was retrieved from MALDITOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the -factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.

Lee, Byung-Kwon [University of Tennessee, Knoxville (UTK); Jung, Kyung-Sik [University of Tennessee, Knoxville (UTK); Son, Cagdas D [ORNL; Kim, Heejung [University of Tennessee, Knoxville (UTK); Verberkmoes, Nathan C [ORNL; Arshava, Boris [College of Staten Island; Naider, Fred [College of Staten Island; Becker, Jeffrey Marvin [ORNL

2007-01-01

407

Discovery of Dermorphin-Based Affinity Labels with Subnanomolar Affinity for Mu Opioid Receptors+  

PubMed Central

A series of potent electrophilic affinity labels (IC50 = 0.1-5 nM) containing either a bromoacetamide or isothiocyanate based on the mu opioid receptor (MOR) selective peptide dermorphin were prepared. All four analogs exhibited wash resistant inhibition of [3H]DAMGO binding at subnanomolar to nanomolar concentrations, suggesting that these analogs bind covalently to MOR. To our knowledge these peptides are the highest affinity peptide-based affinity labels for MOR reported to date.

Sinha, Bhaswati; Cao, Zhengyu; Murray, Thomas F.; Aldrich, Jane V.

2009-01-01

408

A low-power CMOS integrated circuit for field-powered radio frequency identification tags  

Microsoft Academic Search

Cheap, compact radio frequency identification (RFID) tags will make a wide range of new applications cost-effective. Minimum cost can be achieved only in a passive tag (that acquires operating power from the interrogating RF field). A compact tag form factor demands a small tag antenna, that in turn demands either external components or a high-frequency RF carrier for effective tag

D. Friedman; H. Heinreich; D.-W. Duan

1997-01-01

409

Self-cleavable stimulus responsive tags for protein purification without chromatography.  

PubMed

A simple method to purify recombinant proteins is described by fusing a target protein with an intein and an elastin-like polypeptide that only requires NaCl, dithiothreitol, and a syringe filter to isolate the target protein from Escherichia coli lysate. This tripartite fusion system enables rapid isolation of the target protein without the need for affinity chromatography for purification or proteases for cleavage of the target protein from the fusion. The elastin-like polypeptide tag imparts reversible phase transition behavior to the tripartite fusion so that the fusion protein can be selectively aggregated in cell lysate by the addition of NaCl. The aggregates are isolated by microfiltration and resolubilized by reversal of the phase transition in low ionic strength buffer. After resolubilizing the fusion protein, the intein is activated to cleave the target protein from the elastin-intein tag, and the target protein is then isolated from the elastin-intein fusion by an additional phase transition cycle. PMID:16089436

Ge, Xin; Yang, Daniel S C; Trabbic-Carlson, Kimberly; Kim, Bumjoon; Chilkoti, Ashutosh; Filipe, Carlos D M

2005-08-17

410

Optimized immobilization of lectins using self-assembled monolayers on polysilicon encoded materials for cell tagging.  

PubMed

Self-assembled monolayers (SAMs) have been used for the preparation of functional microtools consisting of encoded polysilicon barcodes biofunctionalized with proteins of the lectin family. These hybrid microtools exploit the lectins ability for recognizing specific carbohydrates of the cell membrane to give an efficient system for cell tagging. This work describes how the control of the methodology for SAM formation on polysilicon surfaces followed by lectin immobilization has a crucial influence on the microtool biofunction. Several parameters (silanization time, silane molar concentration, type of solvent or deposition methodology) have been studied to establish optimal function. Furthermore, silanes incorporating different terminal groups, such as aldehyde, activated ester or epoxide groups were tested in order to analyze their chemical coupling with the biomolecules, as well as their influence on the biofunctionality of the immobilized protein. Two different lectins - wheat germ agglutinin (WGA) and phytohemagglutinin (PHA-L) - were immobilized, because they have different and specific cell recognition behaviour and exhibit different cell toxicity. In this way we can assess the effect of intrinsic bulk toxicity with that of the cell compatibility once immobilized as well as the importance of cell affinity. A variety of nanometrical techniques were used to characterize the active surfaces, and lectin immobilization was quantified using ultraviolet-visible absorption spectroscopy (UV-vis) and optical waveguide light mode spectroscopy (OWLS). Once the best protocol was found, WGA and PHA were immobilized on polysilicon coded barcodes, and these microtools showed excellent cell tagging on living mouse embryos when WGA was used. PMID:24462779

Penon, Oriol; Siapkas, Dimitrios; Novo, Sergi; Durán, Sara; Oncins, Gerard; Errachid, Abdelhamid; Barrios, Lleonard; Nogués, Carme; Duch, Marta; Plaza, José Antonio; Pérez-García, Lluïsa

2014-04-01

411

Biotinylated diaminopyridine: an approach to tagging oligosaccharides and exploring their biology.  

PubMed

Fluorescent tagging of free oligosaccharides by reductive amination permits sensitive detection and fractionation of these molecules. To expand the scope of this approach, we have synthesized a fluorescent reagent, 2-amino-(6-amidobiotinyl)pyridine. This reagent can tag oligosaccharides under nondegradative conditions with high efficiency. The resulting adducts show excellent fractionation by reverse-phase HPLC with sensitive detection in the low picomole range. When combined with sequential exoglycosidase digestion, stepwise sequencing of the sugar chains is possible. The biotinyl group can also be used to recover the sugar chain from reaction mixtures. The high-affinity interaction of the biotinyl group with multivalent avidin or streptavidin can be used to create the functional equivalent of neoglycoproteins carrying multiple copies of oligosaccharides of defined structure. These complexes allow the production of IgG antibodies directed against the oligosaccharide chain. They can also harness the power of (strept)avidin-biotin technology for the detection and isolation of oligosaccharide-specific receptors from native sources of recombinant libraries. PMID:8265652

Rothenberg, B E; Hayes, B K; Toomre, D; Manzi, A E; Varki, A

1993-12-15

412

A versatile drug delivery system using streptavidin-tagged pegylated liposomes and biotinylated biomaterials.  

PubMed

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

Chen, Ming-Han; Soda, Yasushi; Izawa, Kiyoko; Kobayashi, Seiichiro; Tani, Kenzaburo; Maruyama, Kazuo; Tojo, Arinobu; Asano, Shigetaka

2013-09-15

413

Generation and analysis of recombinant Bunyamwera orthobunyaviruses expressing V5 epitope-tagged L proteins  

PubMed Central

The L protein of Bunyamwera virus (BUNV; family Bunyaviridae) is an RNA-dependent RNA polymerase, 2238?aa in length, that catalyses transcription and replication of the negative-sense, tripartite RNA genome. To learn more about the molecular interactions of the L protein and to monitor its intracellular distribution we inserted a 14?aa V5 epitope derived from parainfluenza virus type 5, against which high-affinity antibodies are available, into different regions of the protein. Insertion of the epitope at positions 1935 or 2046 resulted in recombinant L proteins that retained functionality in a minireplicon assay. Two viable recombinant viruses, rBUNL4V5 and rBUNL5V5, expressing the tagged L protein were rescued by reverse genetics, and characterized with respect to their plaque size, growth kinetics and protein synthesis profile. The recombinant viruses behaved similarly to wild-type (wt) BUNV in BHK-21 cells, but formed smaller plaques and grew to lower titres in Vero E6 cells compared with wt BUNV. Immunofluorescent staining of infected cells showed the L protein to have a punctate to reticular distribution in the cytoplasm, and cell fractionation studies indicated that the L protein was present in both soluble and microsomal fractions. Co-immunoprecipitation and confocal microscopic assays confirmed an interaction between BUNV L and N proteins. The recombinant viruses expressing tagged L protein will be highly valuable reagents for the detailed dissection of the role of the BUNV L protein in virus replication.

Shi, Xiaohong; Elliott, Richard M.

2009-01-01

414

Reconciling Knowledge in Social Tagging Web Services  

NASA Astrophysics Data System (ADS)

Sometimes we want to search for new information about topics but we can not find relevant results using our own knowledge (for example, our personal bookmarks). A potential solution could be the use of knowledge from other users to find what we are searching for. This solution implies that we can achieve some agreement on implicit semantics used by the other users. We call it Reconciliation of Knowledge. The aim of this paper is to show an agent-based method which lets us reconcile two different knowledge basis (associated with tagging systems) into a common language, obtaining a new one that allows the reconcilitiation of (part of) this knowledge. The agents use Formal Concept Analysis concepts and tools and it has been implemented on the JADE multiagent platform.

Aranda-Corral, Gonzalo A.; Borrego-Díaz, Joaquín

415

Development of a flexible tag microlab  

NASA Astrophysics Data System (ADS)

The objective of this ongoing work is the development of a microlab on flexible tag, capable to monitor the quality of the food, during transport, storage and vending. The idea is to bring together different sensor technologies that will be integrated into a data communication environment for online food monitoring during the logistics chain. The proposed solution is the concept of silicon chips and microcomponents assembled and integrated on top of a flexible substrate acting mainly as a passive interconnect structure. Three technologies have been identified as necessary to get the final integration: a) Substrate technology. This technology refers to the realisation of the flexible substrate with the metallic interconnections. b) Assembly technology to integrate the discrete components on the flexible substrate. The conventional processes are wire bonding, flip chip, and adhesive bonding. c) Encapsulation technology and windows opening over the gas sensitive areas. The first flexible tag prototype integrates two different metal oxide sensor arrays with a commercial microprocessor. The dimensions are 43 mm long, 22 mm wide and about 2 mm thick and two metal levels are necessary for the interconnect. The strategy undertaken by the groups involved in this work, consists in the evaluation of different approaches, that combine diverse process sequences and materials, with the final aim of identifying the best solution. Regarding the substrate technology, the approach realized using Pyralux copper-clad laminated composites, constructed of DuPont Kapton polyimide film with copper foil on both sides, as flexible substrate will be described in this paper. The cupper interconnections are generated by standard photolithography and wet etching and the vias definition in Kapton is performed by femtosecond laser ablation. On the other hand, the assembly technology based on the use of anisotropically conductive adhesives will be also illustrated.

Abad, Estefania; Raffa, Vittoria Simona; Mazzolai, Barbara; Marco, Santiago; Krenkow, Angelika; Becker, Thomas

2005-07-01

416

Genomic Signature Tags (GSTs): A System for Profiling Genomic DNA  

Microsoft Academic Search

Genomic signature tags (GSTs) are the products of a method we have developed for identifying and quantitatively analyzing genomic DNAs. The DNA is initially fragmented with a type II restriction enzyme. An oligonucleotide adaptor containing a recognition site for MmeI, a type IIS restriction enzyme, is then used to release 21-bp tags from fixed positions in the DNA relative to

John J. Dunn; Sean R. McCorkle; Laura A. Praissman; Geoffrey Hind; Daniel van der Lelie; Wadie F. Bahou; Dmitri V. Gnatenko; Maureen K. Krause

2008-01-01

417

Collaborative tagging as a knowledge organisation and resource discovery tool  

Microsoft Academic Search

Purpose – The purpose of the paper is to provide an overview of the collaborative tagging phenomenon and explore some of the reasons for its emergence. Design\\/methodology\\/approach – The paper reviews the related literature and discusses some of the problems associated with, and the potential of, collaborative tagging approaches for knowledge organisation and general resource discovery. A definition of controlled

George Macgregor; Emma McCulloch

2006-01-01

418

Material tagging for improved inspection and process control  

Microsoft Academic Search

Researchers have developed a unique nondestructive inspection concept for the assessment of advanced composites and other hard-to-test materials. The technique known as 'Tagging' involves adding a small amount of tiny ferromagnetic particles to a material during manufacture such that the resulting electromagnetic signature can be related to material condition. In the most simple applications, the tagging signal can reveal important

W. G. Clark Jr.

1991-01-01

419

Harmonic radar transceiver design: miniature tags for insect tracking  

Microsoft Academic Search

The design and operation along with verifying measurements of a harmonic radar transceiver, or tag, developed for insect tracking are presented. A short length of wire formed the antenna while a beam lead Schottky diode across a resonant loop formed the frequency doubler circuit yielding a total tag mass of less than 3 mg. Simulators using the method-of-moments for the

Bruce G. Colpitts; Gilles Boiteau

2004-01-01

420

HIP-tags architecture implementation for the Internet of things  

Microsoft Academic Search

This paper describes a possible implementation for the innovative and highly secure networking architecture dedicated to the Internet of Things (IoT). We propose an infrastructure that works with a new type of tags, supporting the upcoming standard Host Identity Protocol (HIP). Our main concern is to ensure RFID tags privacy, while enabling things to things communications.

Pascal Urien; Simon Elrharbi; Dorice Nyamy; Hervé Chabanne; Thomas Icart; François Lecocq; Cyrille Pépin; Khalifa Toumi; Mathieu Bouet; Guy Pujolle; Patrice Krzanik; Jean-Ferdinand Susini

2009-01-01

421

SAGE2Splice: Unmapped SAGE Tags Reveal Novel Splice Junctions  

Microsoft Academic Search

Serial analysis of gene expression (SAGE) not only is a method for profiling the global expression of genes, but also offers the opportunity for the discovery of novel transcripts. SAGE tags are mapped to known transcripts to determine the gene of origin. Tags that map neither to a known transcript nor to the genome were hypothesized to span a splice

Byron Yu-Lin Kuo; Ying Chen; Slavita Bohacec; Öjvind Johansson; Wyeth W. Wasserman; Elizabeth M. Simpson

2006-01-01

422

Relative Position of Coded Wire Tags in Paddlefish Rostrums  

Microsoft Academic Search

Paddlefish Polyodon spathula were tagged with 2-mm-long coded wire tags (CWTs) inserted 2 mm into the distal end of the rostrum to determine if the relative position of CWTs changed as the rostrum grew. Thirty fish were randomly selected weekly for measurements of body length (anterior edge of eye to tail fork), rostrum length (anterior eye to distal end of

D. Scott Waters; Christopher S. Guy; Christopher P. Clouse

1997-01-01

423

Inventory Management using Passive RFID Tags: A Survey  

Microsoft Academic Search

Radio Frequency Identification (RFID) systems have emerged as an affordable solution for object identification. They are a cheap and error proof alternative to traditional object identification techniques such as bar codes and visual recognition. The problem is to identify objects attached with passive tags. If there are multiple objects within the range of the tag reader, then all objects send

Cherian Abraham; Vinay Ahuja; Arnab Kumar Ghosh; Praveen Pakanati

424

QUANTITATION OF MAST CELLS AND COLLAGEN FIBERS IN SKIN TAGS  

PubMed Central

Background: Skin tags are common benign skin tumors usually occurring on the neck and major flexors of elder people. Aims: The aim of this study is to perform quantitation of mast cells and collagen fibers in skin tags and normal skin in diabetics and nondiabetics, to find a possible correlation between mast cells and collagen fibers in the pathogenesis of skin tags. Methods: Thirty participants with skin tags were divided into two groups (15 diabetic and 15 nondiabetic). Three biopsies were obtained from one anatomical site: A large skin tag, a small skin tag, and adjacent normal skin. Mast cells stained with Bismarck brown were counted manually in ten different fields of each section with magnification ×1000 and the average count was correlated with the percentage of mean collagen area in five fields done by the image analyzer. Results: A statistically significant correlation between mast cell count and percentage of collagen mean area was detected in both studied groups (except in large skin tags of the nondiabetic group). Conclusion: The positive correlation between mast cell count and percentage of collagen mean area suggests the critical role of mast cells in the etiogenesis of skin tags through its interaction with fibroblasts.

El Safoury, Omar Soliman; Fawzy, Marwa M; El Maadawa, Zeinab M; Mohamed, Dalia H

2009-01-01

425

Amplified energy transfer in conjugated polymer nanoparticle tags and sensors  

Microsoft Academic Search

Nanoparticles primarily consisting of pi-conjugated polymers have emerged as extraordinarily bright fluorescent tags with potential applications in biological imaging and sensing. As fluorescent tags, conjugated polymer nanoparticles possess a number of advantageous properties, such as small particle size, extraordinary fluorescence brightness, excellent photostability, and high emission rate. Exciton diffusion occurring in the nanoparticles results in amplified energy transfer, doubling the

Zhiyuan Tian; Jiangbo Yu; Changfeng Wu; Craig Szymanski; Jason McNeill

2010-01-01

426

Gas tagging and cover gas combination for nuclear reactor  

DOEpatents

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.

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

1985-01-01

427

Improved gas tagging and cover gas combination for nuclear reactor  

DOEpatents

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.

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

1983-09-26

428

Investigating language independence in HMM PoS\\/MSD-tagging  

Microsoft Academic Search

The paper presents an investigation of functional dependencies in morphosyntactic tagging using hidden Markov models. Starting from a well known fact that the HMM tagging paradigm relies on lexical knowledge acquired from training corpora and stored in form of transition and emission matrices, also called a language model, in the experiment, we apply the TnT trigram tagger on creating language

Z. Agic; M. Tadic; Z. Dovedan

2008-01-01

429

Apoferritin-Templated Synthesis of Encoded Metallic Phosphate Nanoparticle Tags  

SciTech Connect

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.

Liu, Guodong; Wu, Hong; Dohnalkova, Alice; Lin, Yuehe

2007-07-31

430

Distortion-invariant ID tags for object identification  

NASA Astrophysics Data System (ADS)

Active and passive optical identification (ID) tags and readers for remote identification and verification of objects are described. We focus our attention on the design of passive ID tags to achieve distortion-invariant authentication of the information included in the optical tag. A passive ID tag will consist of an optical phase code which can be placed in a visible part of an object for remote detection. We aim to authenticate the object even if the reader captures a distorted version of the code due to in-plane rotations. Distortion-invariance is achieved by both multiplexing the information included in the ID tag and the topology of the tag. For security purposes, double-phase encryption has already been shown as an appropriate technique to encode information. By using double-phase encryption, a signature is hidden in a phase-encoded ID tag not visible by visual inspection. Once the ID tag is captured by the reader and is decrypted, a correlation-based processor verifies the decoded information with a previously stored reference signal. The proposed system may have broad applications in transportation, homeland security, and inventory control.

Perez-Cabre, Elisabet; Javidi, Bahram

2004-11-01

431

The Benefit of Using Tag-Based Profiles  

Microsoft Academic Search

Collaborative tagging, i.e. the process of assigning metadata in the form of keywords to shared content by many users, has emerged as an important way to provide informa- tion about resources on the Web and elsewhere. Such key- words (tags) are used to enable the organization of infor- mation within personal information spaces, such as photo collections, but can also

Claudiu S. Firan; Wolfgang Nejdl; Raluca Paiu

2007-01-01

432

The Benefit of Using Tag-Based Profiles  

Microsoft Academic Search

Collaborative tagging, i.e. the process of assigning metadata in the form of keywords to shared content by many users, has emerged as an important way to provide information about resources on the Web and elsewhere. Such keywords (tags) are used to enable the organization of information within personal information spaces, such as photo collections, but can also be shared, allowing

Claudiu S. Firan; Wolfgang Nejdl; Raluca Paiu

2007-01-01

433

Gene-tagged chromosome translocations in eleven stocks of mice  

Microsoft Academic Search

Summary  The cytogenetic problems of correlating linkage groups in the house mouse with their chromosomes, and of establishing the\\u000a chromosomal independence of known linkage groups, call for the use of numerous genetically tagged translocations. Eleven new\\u000a translocations have been induced and tagged; they involve linkage groups I, II, III, V, VIII, IX and XI.

T. G. Cartes; Mary F. Lyon; Rita J. S. Phillips

1955-01-01

434

Balanced RFID Tag Antenna Mountable on Metallic Plates  

Microsoft Academic Search

A novel balanced tag antenna for a radio frequency identification (RFID) system is presented. The radiating elements of two planar inverted-F antennas (PIFAs) are inductively coupled by the feed loop with out of phase. The balanced structure provides smaller degradation of performances when an RFID tag is mounted on various sizes of metal plates. The HFSS simulator is employed to

Byunggil Yu; Sung-Joo Kim; Byungwoon Jung; Frances J. Harackiewicz; Myun-Joo Park; Byungje Lee

2006-01-01

435

Low-Cost, Ubiquitous RFID-Tag-Antenna-Based Sensing  

Microsoft Academic Search

Radio-frequency identification (RFID) has been well established as an effective technology for track and trace applications. In this paper, we go beyond the ID in RFID, and discuss the potential for RFID tags to be used as low-cost sensors by mapping a change in some physical parameter of interest to a controlled change in RFID tag antenna electrical properties. We

Rahul Bhattacharyya; Christian Floerkemeier; Sanjay Sarma

2010-01-01

436

Optimization of UHF RFID tag antennas using a genetic algorithm  

Microsoft Academic Search

UHF band (860~960 MHz) RFID tag strip-line antennas for non-metallic object and slotted patch RFID antenna for metallic objects have been optimized with a GA. The antennas are optimized for commercially available RFID tag IC chips. Different cell sizes of FDTD have been tried while the GA optimizes the symmetrical shape of RFID antennas

Goojo Kim; YouChung Chung

2006-01-01

437

A Slim RFID Tag Antenna Design for Metallic Object Applications  

Microsoft Academic Search

A slim radio frequency identification (RFID) tag antenna design for metallic objects application is proposed in this letter. It is designed based on a high-impedance surface (HIS) unit cell structure directly rather than adopting a large HIS ground plane. The antenna structure consists of metallic rectangular patches electrically connected through vias to the ground plane to form an RFID tag

Sung-Lin Chen; Ken-Huang Lin

2008-01-01

438

Distributed emulation for the design of active tag based systems  

Microsoft Academic Search

Several choices have to be made during the design process of active tag based systems. Since the number of properties that must be decided before production and wide-scale deployment is relatively high, the use of real experiments in the design phase may be prohibitive. We propose the use of emulation for performing large-scale experiments with active tag based systems easily

Razvan Beuran; Junya Nakata; Takashi Okada; Tetsuya Kawakami; Ken-ichi Chinen; Yasuo Tan; Yoichi Shinoda

2009-01-01

439

Distributed Emulator for a Pedestrian Tracking System Using Active Tags  

Microsoft Academic Search

In this paper we introduce a distributed emulator for a pedestrian tracking system using active tags that is currently being developed by the authors. The emulator works on StarBED which is a network testbed consisting of hundreds of PCs connected to each other by Ethernet. The three major components of the emulator (the processor emulator of the active tag micro-controller,

Junya NAKATA; Razvan Beuran; Tetsuya Kawakami; Ken-ichi Chinen; Yasuo Tan; Yoichi Shinoda

2008-01-01

440

ONE-STEP METAL-AFFINITY PURIFICATION OF HISTIDINE-TAGGED PROTEINS BY TEMPERATURE-TRIGGERED PRECIPITATION. (R829606)  

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

441

Expression of functional multidrug-resistance protein 1 in Saccharomyces cerevisiae: effects of N- and C-terminal affinity tags  

Microsoft Academic Search

Studies of the multidrug-resistance protein 1 (MRP1) have been hampered by the lack of a simple expression system allowing for rapid generation of mutants and yielding milligram amounts of protein. Here, we describe a Saccharomyces cerevisiae expression system that meets those conditions. MRP1 was expressed under the control of the constitutive PMA1 (yeast proton pump) promoter. The best conditions for

Sung Haeng Lee; Guillermo A Altenberg

2003-01-01

442

Mass spectrometry-based proteomic analysis of the epitope-tag affinity purified protein complexes in eukaryotes  

Microsoft Academic Search

In recent years, MS has been widely used to study protein complex in eukaryotes. The identifi- cation of interacting proteins of a particular target protein may help defining protein-protein interaction and proteins of unknown functions. To isolate protein complexes, high-speed ultra- centrifugation, sucrose density-gradient centrifugation, and coimmunoprecipitation have been widely used. However, the probability of getting nonspecific binding is comparatively

Ing-Feng Chang