Peptide biotinylation with amine-reactive esters: differential side chain reactivity.
AuthorsMiller BT, Collins TJ, Rogers ME, Kurosky A
JournalPeptides
PubMed ID9437720
N-hydroxysuccinimide (NHS) esters of biotin are reported to react specifically with amino groups of peptides and proteins. However, we have found that these reagents can readily acylate other functional groups in specific peptide sequences under relatively mild conditions. We have extended our inquiry of sequence-dependent acylation by evaluating the reactivity ... More
Enumeration of antigen-specific IgE responses at the single-cell level by an ELISA plaque assay.
AuthorsChen SS
JournalJ Immunol Methods
PubMed ID1703186
In vitro elicitation and enumeration of antigen-specific IgE-secreting cells in antigen-primed murine lymphocyte cultures can be reproducibly and accurately quantitated. Rat anti-mouse IgE monoclonal antibodies (Mab anti-epsilon) were developed. Antigen-specific IgE-secreting cells can be detected on antigen or MAb anti-epsilon coated nitrocellulose discs pasted to 24-well culture plates. This IgE ... More
A novel fluorescent toxin to detect and investigate Kv1.3 channel up-regulation in chronically activated T lymphocytes.
AuthorsBeeton C, Wulff H, Singh S, Botsko S, Crossley G, Gutman GA, Cahalan MD, Pennington M, Chandy KG
JournalJ Biol Chem
PubMed ID12511563
T lymphocytes with unusually high expression of the voltage-gated Kv1.3 channel (Kv1.3(high) cells) have been implicated in the pathogenesis of experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. We have developed a fluoresceinated analog of ShK (ShK-F6CA), the most potent known inhibitor of Kv1.3, for detection of Kv1.3(high) cells ... More
Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques.
AuthorsMusil LS, Goodenough DA
JournalJ Cell Biol
PubMed ID1659577
'We previously demonstrated that the gap junction protein connexin43 is translated as a 42-kD protein (connexin43-NP) that is efficiently phosphorylated to a 46,000-Mr species (connexin43-P2) in gap junctional communication-competent, but not in communication-deficient, cells. In this study, we used a combination of metabolic radiolabeling and immunoprecipitation to investigate the assembly ... More
Localized suppression of RhoA activity by Tyr31/118-phosphorylated paxillin in cell adhesion and migration.
AuthorsTsubouchi A, Sakakura J, Yagi R, Mazaki Y, Schaefer E, Yano H, Sabe H
JournalJ Cell Biol
PubMed ID12446743
'RhoA activity is transiently inhibited at the initial phase of integrin engagement, when Cdc42- and/or Rac1-mediated membrane spreading and ruffling predominantly occur. Paxillin, an integrin-assembly protein, has four major tyrosine phosphorylation sites, and the phosphorylation of Tyr31 and Tyr118 correlates with cell adhesion and migration. We found that mutation of ... More
Fast solid support detection of PCR amplified viral DNA sequences using radioiodinated or hapten labelled primers.
AuthorsSauvaigo S, Fouqué B, Roget A, Livache T, Bazin H, Chypre C, Téoule R
JournalNucleic Acids Res
PubMed ID2162518
'Oligonucleotides with novel modifications have been synthesized and incorporated into enzymatically amplified DNA sequences. They allow the fast detection of viral DNA sequences after two rounds of amplification. The hybrids formed are immobilized by affinity on coated tubes and detected by direct beta (32P) or gamma (125I) counting or by ... More
Aggretin, a heterodimeric C-type lectin from Calloselasma rhodostoma (malayan pit viper), stimulates platelets by binding to alpha 2beta 1 integrin and glycoprotein Ib, activating Syk and phospholipase Cgamma 2, but does not involve the glycoprotein VI/Fc receptor gamma chain collagen receptor.
AuthorsNavdaev A, Clemetson JM, Polgar J, Kehrel BE, Glauner M, Magnenat E, Wells TN, Clemetson KJ
JournalJ Biol Chem
PubMed ID11287424
'Aggretin, a potent platelet activator, was isolated from Calloselasma rhodostoma venom, and 30-amino acid N-terminal sequences of both subunits were determined. Aggretin belongs to the heterodimeric snake C-type lectin family and is thought to activate platelets by binding to platelet glycoprotein alpha(2)beta(1). We now show that binding to glycoprotein (GP) ... More
Topologically restricted appearance in the developing chick retinotectal system of Bravo, a neural surface protein: experimental modulation by environmental cues.
Authorsde la Rosa EJ, Kayyem JF, Roman JM, Stierhof YD, Dreyer WJ, Schwarz U
JournalJ Cell Biol
PubMed ID2269667
'A novel neural surface protein, Bravo, shows a pattern of topological restriction in the embryonic chick retinotectal system. Bravo is present on the developing optic fibers in the retina; however, retinal axons in the tectum do not display Bravo. The appearance of Bravo in vitro is modulated by environmental cues. ... More
Trafficking of matrix collagens through bone-resorbing osteoclasts.
AuthorsNesbitt SA, Horton MA
JournalScience
PubMed ID9092478
'An intracellular pathway for proteins liberated from mineralized matrix during resorption was identified in osteoclasts. Analysis by confocal microscopy of sites of active bone resorption showed that released matrix proteins, including degraded type I collagen, were endocytosed along the ruffled border within the resorption compartment and transcytosed through the osteoclast ... More
BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip.
AuthorsNieba L, Nieba-Axmann SE, Persson A, Hämäläinen M, Edebratt F, Hansson A, Lidholm J, Magnusson K, Karlsson AF, Plückthun A
JournalAnal Biochem
PubMed ID9344407
'While BIACORE instruments are routinely used for kinetic measurements and for the determination of binding constants, the immobilization of a ligand onto the sensor chip surface has to be individually optimized for every system. We show here that the histidine (His) tag, routinely used in protein purification and in detection ... More
Nonisotopic detection of RNA in an enzyme immunoassay using a monoclonal antibody against DNA-RNA hybrids.
AuthorsCoutlee F, Yolken RH, Viscidi RP
JournalAnal Biochem
PubMed ID2479292
'A sensitive nonisotopic solution hybridization assay for detection of RNA is described and characterized using a pSP65 plasmid model system. The assay procedure is based on a hybridization reaction in solution between a biotinylated DNA probe and a target RNA. The biotin-labeled hybrids are captured on a microtiter plate coated ... More
Optimizing antibody immobilization strategies for the construction of protein microarrays.
AuthorsPeluso P, Wilson DS, Do D, Tran H, Venkatasubbaiah M, Quincy D, Heidecker B, Poindexter K, Tolani N, Phelan M, Witte K, Jung LS, Wagner P, Nock S
JournalAnal Biochem
PubMed ID12531195
'Antibody microarrays have the potential to revolutionize protein expression profiling. The intensity of specific signal produced on a feature of such an array is related to the amount of analyte that is captured from the biological mixture by the immobilized antibody (the "capture agent"). This in turn is a function ... More
Modulation of TGF-beta type 1 receptor: flow cytometric detection with biotinylated TGF-beta.
AuthorsNewman W, Beall LD, Bertolini DR, Cone JL
JournalJ Cell Physiol
PubMed ID2550480
'Transforming growth factor beta type 1 (TGF-beta 1) was reacted with NHS-biotin to yield a derivative of TGF-beta 1 which was biotinylated on lysine residues. The biotinylated form of TGF-beta 1 was separated from the unreacted material by reverse phase chromatography. In three separate bioassays, the derivatized peptide was as ... More
Sphingolipids and glycoproteins are differentially trafficked to the Chlamydia trachomatis inclusion.
AuthorsScidmore MA, Fischer ER, Hackstadt T
JournalJ Cell Biol
PubMed ID8707822
'Chlamydia trachomatis is an obligate intracellular pathogen that multiples within the confines of a membrane-bound vacuole called an inclusion. Approximately 40-50% of the sphingomyelin synthesized from exogenously added NBD-ceramide is specifically transported from the Golgi apparatus to the chlamydial inclusion (Hackstadt, T., M.A. Scidmore, and D.D. Rockey. 1995. Proc. Natl. ... More
Homogeneous functional insulin receptor from 3T3-L1 adipocytes. Purification using N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin and avidin-sepharose.
AuthorsKohanski RA, Lane MD
JournalJ Biol Chem
PubMed ID3886647
'Insulin receptor was purified 10,000-fold from cultured mouse 3T3-L1 adipocytes in 35% overall yield. The specific activities of 125I-insulin binding and autophosphorylation increased in parallel, following the initial Triton X-100 extraction of membranes. The isolation protocol, performed entirely at pH 8.45, entailed adsorption by avidin-Sepharose CL-4B of a complex formed ... More
Membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers.
AuthorsDanieli T, Pelletier SL, Henis YI, White JM
JournalJ Cell Biol
PubMed ID8636231
'In this study we tested the hypothesis that fusion mediated by the influenza virus hemagglutinin (HA) is a cooperative event. To so this we characterized 3T3 cell lines that express HA at nine different defined surface densities. HA densities ranged from 1.0 to 12.6 x 10(3) HA trimers/microns2 as determined ... More
Anterograde tracing of retinal axons in the avian embryo with low molecular weight derivatives of biotin.
AuthorsHalfter W
JournalDev Biol
PubMed ID2433173
'Several reactive biotin esters were injected into the eyes of chick and quail embryos at various stages of development. Four of the biotin esters reacted with molecules of the eye tissue and were detected with light and electron microscopy in fluorescein isothiocyanate and peroxidase-avidin incubated sections and whole mounts. Intra ... More
Identification and characterization of O-biotinylated hydroxy amino acid residues in peptides.
AuthorsMiller BT, Rogers ME, Smith JS, Kurosky A
JournalAnal Biochem
PubMed ID8080081
'Recent findings have indicated that certain hydroxyl groups of peptides have enhanced intrinsic chemical reactivity and can be O-acylated by N-hydroxysuccinimide esters of biotin. Analytical procedures are described for the identification of O-acylated derivatives of seryl, threonyl, and tyrosyl residues after reaction with the N-hydroxysuccinimide ester of biotin containing the ... More
Synthesis of a highly fluorescent beta-diketone-europium chelate and its utility in time-resolved fluoroimmunoassay of serum total thyroxine.
AuthorsWu FB, Han SQ, Zhang C, He YF
JournalAnal Chem
PubMed ID12463376
'A new highly fluorescent beta-diketone-europium chelate was synthesized and employed as a tracer to develop a time-resolved fluoroimmunoassay (TRFIA) for detection of serum total thyroxine (T4). The tetradentate beta-diketone chelator, 1,10-bis(thiophene-2''-yl)-4,4,5,5,6,6,7,7-octafluorodecane-1,3,8,10-tetraone (BTOT), was structurally composed of two units of thenoyltrifluoroacetone (TTA) derivatives but expressed fluorescence that was greatly enhanced, as ... More
The role of alpha and beta chains in ligand recognition by beta 7 integrins.
AuthorsHiggins JM, Cernadas M, Tan K, Irie A, Wang J, Takada Y, Brenner MB
JournalJ Biol Chem
PubMed ID10837471
'Integrins alpha(E)beta(7) and alpha(4)beta(7) are involved in localization of leukocytes at mucosal sites. Although both alpha(E)beta(7) and alpha(4)beta(7) utilize the beta(7) chain, they have distinct binding specificities for E-cadherin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1), respectively. We found that mutation of the metal ion-dependent adhesion site (MIDAS) in the ... More
Interaction of dihydrofolate reductase with methotrexate: ensemble and single-molecule kinetics.
'The thermodynamics and kinetics of the interaction of dihydrofolate reductase (DHFR) with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods. DHFR was modified to permit the covalent addition of a fluorescent molecule, Alexa 488, and a biotin at the N terminus of the molecule. The fluorescent molecule ... More
Endocytosis in filter-grown Madin-Darby canine kidney cells.
AuthorsBomsel M, Prydz K, Parton RG, Gruenberg J, Simons K
JournalJ Cell Biol
PubMed ID2689455
'In this paper, we have characterized the apical and basolateral endocytic pathways of epithelial MDCK cells grown on filters. The three-dimensional organization of the endocytic compartments was analyzed by confocal microscopy after internalization of a fluorescent fluid-phase marker from either side of the cell layer. After 5 min of internalization, ... More
The biotin/avidin-mediated microtiter plate lectin assay with the use of chemically modified glycoprotein ligand.
AuthorsDuk M, Lisowska E, Wu JH, Wu AM
JournalAnal Biochem
PubMed ID7810865
'The conditions of a simple and sensitive microtiter plate lectin assay with biotinylated TF- and/or Tn-reactive lectins and ExtrAvidin/alkaline phosphatase conjugate are described. As ligand for lectin binding, chemically modified glycophorin A from human erythrocytes was used. The TF and Tn receptors present in glycophorin A in cryptic form were ... More
Conformation coupled enzyme catalysis: single-molecule and transient kinetics investigation of dihydrofolate reductase.
'Ensemble kinetics and single-molecule fluorescence microscopy were used to study conformational transitions associated with enzyme catalysis by dihydrofolate reductase (DHFR). The active site loop of DHFR was labeled with a fluorescence quencher, QSY35, at amino acid position 17, and the fluorescent probe, Alexa555, at amino acid 37, by introducing cysteines ... More
Purification and characterization of membrane proteins.
AuthorsKraehenbuhl JP, Bonnard C
JournalMethods Enzymol
PubMed ID2167430
One- and two-step non-competitive avidin-biotin immunoassays for monomeric and heterodimeric antigen.
AuthorsVilja P
JournalJ Immunol Methods
PubMed ID1995714
'In this study of one-step and two different two-step non-competitive avidin-biotin assays (NABAs) were developed for the measurement of a monomeric antigen (lactoferrin, LF) using polyclonal antibodies and the detection of a heterodimeric antigen (lutropin. LH) using monoclonal antibodies. The assays were based on the use of performed complexes of ... More
Ligand binding regulates the directed movement of beta1 integrins on fibroblasts.
AuthorsFelsenfeld DP, Choquet D, Sheetz MP
JournalNature
PubMed ID8837776
'To enable cells to crawl, adhesion receptors such as integrins must bind to extracellular molecules and simultaneously interact with force-generating components of the cytoskeleton. We show here that the binding of extracellular ligand in living cells induces the attachment of beta1 integrins to the retrograde-moving cytoskeleton. Unliganded integrins are not ... More
Novel biotinylated nucleotide--analogs for labeling and colorimetric detection of DNA.
AuthorsGebeyehu G, Rao PY, SooChan P, Simms DA, Klevan L
JournalNucleic Acids Res
PubMed ID3588302
'A series of dATP and dCTP nucleotide analogs have been synthesized which are modified by attachment of aliphatic linkers containing a functional group to the amino-nitrogen at the hydrogen bonding positions of the bases, that is, at the 6-position of adenine and the 4-position of cytosine. These nucleotides are incorporated ... More
The biotin-(strept)avidin system: principles and applications in biotechnology.
AuthorsDiamandis EP, Christopoulos TK
JournalClin Chem
PubMed ID2032315
'The biotin-(strept)avidin system has been used for many years in a variety of different applications. Here we present a general overview of the system, describe its components and advantages, and show how the system is used in various applications, with emphasis on immunological and nucleic acid hybridization assays. This system ... More
Transferrin-binding protein complex is the receptor for transferrin uptake in Trypanosoma brucei.
AuthorsSteverding D, Stierhof YD, Fuchs H, Tauber R, Overath P
JournalJ Cell Biol
PubMed ID8522581
'In Trypanosoma brucei, the products of two genes, ESAG 6 and ESAG 7, located upstream of the variant surface glycoprotein gene in a polycistronic expression site form a glycosylphosphatidylinositol-anchored transferrin-binding protein (TFBP) complex. It is shown by gel filtration and membrane-binding experiments that the TFBP complex is heterodimeric and binds ... More
Avidin binding of carboxyl-substituted biotin and analogues.
AuthorsHofmann K, Titus G, Montibeller JA, Finn FM
JournalBiochemistry
PubMed ID7041971
Coupling of monoclonal antibodies with biotin.
AuthorsHaugland RP, You WW
JournalMethods Mol Biol
PubMed ID7550685
Coupling of antibodies with biotin.
AuthorsHaugland RP, You WW
JournalMethods Mol Biol
PubMed ID9664374
Preparation of fluorescent, cross-linking, and biotinylated calmodulin derivatives and their use in studies of calmodulin-activated phosphodiesterase and protein phosphatase.
AuthorsKincaid RL, Billingsley ML, Vaughan M
JournalMethods Enzymol
PubMed ID2842624
Preparation of modified tubulins.
AuthorsHyman A, Drechsel D, Kellogg D, Salser S, Sawin K, Steffen P, Wordeman L, Mitchison T
JournalMethods Enzymol
PubMed ID2034137
Quantitative analysis of polymerase chain reaction (PCR) products using primers labeled with biotin and a fluorescent dye.
AuthorsLandgraf A, Reckmann B, Pingoud A
JournalAnal Biochem
PubMed ID1908194
PCR primers covalently labeled with biotin and a fluorescent dye allow immobilization and separation of the products which can be quantitatively analyzed subsequently. The procedure we have developed circumvents electrophoretic separation and radioactive labeling. Exact quantitative analysis of reaction products is feasible during the logarithmic phase of amplification when Taq ... More
Nonradioactive labeling of synthetic oligonucleotide probes with terminal deoxynucleotidyl transferase.
AuthorsKumar A, Tchen P, Roullet F, Cohen J
JournalAnal Biochem
PubMed ID2837921
Synthetic oligonucleotides were tailed at the 3' end using terminal deoxynucleotidyl transferase. Nucleotide triphosphates with free primary amines at the end of side chains were compared for their tailing efficiency and/or detection sensitivity, using biotin-11-dUTP as a reference. Free primary amines were tagged with activated biotin or fluorescein isothiocyanate. The ... More
Biotin-protein bond: instability and structural modification to provide stability for in vivo applications.
AuthorsMock DM, Bogusiewicz A,
JournalMethods Mol Biol
PubMed ID18287661
Biotinylation of proteins is a powerful tool for investigating biological phenomenon, both in vitro and in vivo. Biotinylating reagents that form covalent bonds with several types of amino acid residues are commercially available. However, most, if not all, of these commercially available biotinylating agents produce biotin-protein bonds that are susceptible ... More
Apyrase immobilized on paramagnetic beads used to improve detection limits in bioluminometric ATP monitoring.
AuthorsNyrén P
JournalJ Biolumin Chemilumin
PubMed ID8154301
A novel technique that is useful for the control of the luminescence output in a bioluminometric assay has been developed. The method relies on the immobilization on paramagnetic beads of an enzyme that is capable of catalysing the hydrolysis or oxidation of the substrate for the luminescence reaction. This technique ... More
Elevated intrinsic reactivity of seryl hydroxyl groups within the linear peptide triads His-Xaa-Ser or Ser-Xaa-His.
AuthorsMiller BT, Kurosky A
JournalBiochem Biophys Res Commun
PubMed ID8216328
Chemical modification studies of peptide hormones and random peptides have revealed that seryl hydroxyl groups had enhanced reactivity toward acylating reagents when they occurred in the linear triads His-Xaa-Ser or Ser-Xaa-His (Xaa = any amino acid). O-acylation of serine within these triads was achieved by reaction with N-hydroxysuccinimide esters of ... More
Nonradioactive quantification of glucocorticoid receptor expression during differentiation of human monocytic cells (U937).
AuthorsRoux S, Térouanne B, Defacque H, Vachier I, Loubatière J, Nicolas JC
JournalAnal Biochem
PubMed ID7668385
We describe a method for relative quantification of specific mRNA using a nonradioactive assay based on DNA strand competition between identical sequences of biotin- and fluorescein-labeled amplicon (probe) and unlabeled amplicon (target) during hybridization. As the target quantity increased, that of the double-labeled probe decreased in accordance with the mass ... More
Selective electrofusion of conjugated cells in flow.
AuthorsBakker Schut TC, Kraan YM, Barlag W, de Leij L, de Grooth BG, Greve J
JournalBiophys J
PubMed ID8218887
Using a modified flow cytometer we have induced electrofusion of K562 and L1210 cells in flow. The two cell types are stained with two different fluorescent membrane probes, DiO and DiI, to facilitate optical recognition, and then coupled through an avidin-biotin bridge. In the flow cytometer, the hydrodynamically focused cells ... More
Antibody microarray-based profiling of complex specimens: systematic evaluation of labeling strategies.
Antibody microarrays have often had limited success in detection of low abundant proteins in complex specimens. Signal amplification systems improve this situation, but still are quite laborious and expensive. However, the issue of sensitivity is more likely a matter of kinetically appropriate microarray design as demonstrated previously. Hence, we re-examined ... More
Specific biarsenical labeling of cell surface proteins allows fluorescent- and biotin-tagging of amyloid precursor protein and prion proteins.
AuthorsTaguchi Y, Shi ZD, Ruddy B, Dorward DW, Greene L, Baron GS,
JournalMol Biol Cell
PubMed ID18987338
Fluorescent tagging is a powerful tool for imaging proteins in living cells. However, the steric effects imposed by fluorescent tags impair the behavior of many proteins. Here, we report a novel technique, Instant with DTT, EDT, And Low temperature (IDEAL)-labeling, for rapid and specific FlAsH-labeling of tetracysteine-tagged cell surface proteins ... More
Biotinylation reagents for the study of cell surface proteins.
AuthorsElia G,
JournalProteomics
PubMed ID18763706
The extraordinarily stable, non-covalent interaction between avidin and biotin is one of the most commonly exploited tools in chemistry and biology. Methods for derivatization with biotin of a variety of molecules (in particular, proteins) have been introduced, in order to allow their efficient recovery, immobilization and detection with avidin-based reagents. ... More
A microplate assay for analysis of solution-phase glycosyltransferase reactions: determination of kinetic constants.
AuthorsMengeling BJ, Smith PL, Stults NL, Smith DF, Baenziger JU
JournalAnal Biochem
PubMed ID1812792
We have developed a sensitive and simple method for assaying glycosyltransferase activities. This method makes use of solution-phase transferase reactions followed by capture to a microplate well coated with a substrate-specific monoclonal antibody. Sugar incorporation is quantitated by binding a saccharide-specific lectin and using bioluminescent aequorin for a reporter molecule. ... More
Engineering novel cell surface receptors for virus-mediated gene transfer.
The absence of viral receptors is a major barrier to efficient gene transfer in many cells. To overcome this barrier, we developed an artificial receptor based on expression of a novel sugar. We fed cells an unnatural monosaccharide, a modified mannosamine that replaced the acetyl group with a levulinate group ... More
In-situ monitoring of protein labeling reactions by matrix-assisted laser desorption/ionization mass spectrometry.
AuthorsLu J, Zenobi R
JournalFresenius J Anal Chem
PubMed ID11225811
Taking the labeling reaction of horse heart cytochrome c or ubiquitin with biotinamidocaproate N-hydroxysucchinimide ester (biotin-NHS) as test cases, this report demonstrates the usefulness of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for in-situ monitoring of the labeling process and for determining the composition of the labeled products without the need ... More
Electrochemiluminescence enzyme immunoassays for TNT and pentaerythritol tetranitrate.
AuthorsWilson R, Clavering C, Hutchinson A
JournalAnal Chem
PubMed ID14632142
Electrochemiluminescence enzyme immunoassays for 2,4,6-trinitrotoluene (TNT) and pentaerythritol tetranitrate (PETN) are described. The latter is, to the best of our knowledge, the first report of an immunoassay for PETN. Haptens corresponding to these explosives were covalently attached to high-affinity dextran-coated paramagnetic beads. The beads were mixed with the corresponding Fab ... More
Novel chemical method for the preparation of nucleic acids for nonisotopic hybridization.
AuthorsViscidi RP, Connelly CJ, Yolken RH
JournalJ Clin Microbiol
PubMed ID2939103
A novel chemical method was used to prepare biotin-labeled nucleic acids for nonisotopic hybridization. The method involves the transamination of unpaired cytosine residues in polynucleotides with sodium bisulfite and ethylenediamine. Primary amino groups on the cytosine derivatives are then reacted with biotinyl-e-aminocaproic acid N-hydroxysuccinimide ester. Biotinylated probes hybridized with 1 ... More
Simultaneous cytometric analysis for the expression of cytoplasmic and surface antigens in activated T cells.
AuthorsHayden GE, Walker KZ, Miller JF, Wotherspoon JS, Raison RL
JournalCytometry
PubMed ID2842118
A method of two-colour immunofluorescence staining has been developed to allow the simultaneous analysis of both surface and cytoplasmic antigens. This involves the use of direct fluorochrome antibody conjugates for cell-surface antigen staining, followed by cell permeabilization and the staining of cytoplasmic antigens with biotinylated antibodies and streptavidin-fluorochrome conjugates. Fluorochrome-antibody ... More
Preclinical evaluation of biotin labeling for red cell survival testing.
AuthorsHoffmann-Fezer G, Trastl C, Beisker W, Berg D, Obermaier J, Kessler W, Mysliwietz J, Schumm M, Filser J, Thierfelder S
JournalAnn Hematol
PubMed ID9200996
Biotin labeling of red cells was tested in dogs as a preclinical study for cell survival. Red cells were labeled with either spacered Biotin-X-NHS (BxNHS) or water-soluble biotin compounds. After reinfusion, biotinylated red cells were detected in small blood samples (5 microliters) with flow cytometry. Improved BxNHS labeling allows an ... More
Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots.
AuthorsLeary JJ, Brigati DJ, Ward DC
JournalProc Natl Acad Sci U S A
PubMed ID6191328
Biotin-labelled DNA probes, prepared by nick-translation in the presence of biotinylated analogs of TTP, are hybridized to DNA or RNA immobilized on nitrocellulose filters. After removal of residual probe, the filters are incubated for 2--5 min with a preformed complex made with avidin-DH (or streptavidin) and biotinylated polymers of intestinal ... More
Synthesis and hybridization of a series of biotinylated oligonucleotides.
AuthorsCook AF, Vuocolo E, Brakel CL
JournalNucleic Acids Res
PubMed ID3375076
A series of oligonucleotides containing biotin-11-dUMP at various positions were synthesized and compared in quantitative, colorimetric hybridization-detection studies. A deoxyuridine phosphoramidite containing a protected allylamino sidearm was synthesized and used in standard, automated synthesis cycles to prepare oligonucleotides with allylamino residues at various positions within a standard 17-base sequence. Biotin ... More
Oxazolidinones mechanism of action: inhibition of the first peptide bond formation.
AuthorsPatel U, Yan YP, Hobbs FW, Kaczmarczyk J, Slee AM, Pompliano DL, Kurilla MG, Bobkova EV
JournalJ Biol Chem
PubMed ID11483595
Oxazolidinones are potent inhibitors of bacterial protein biosynthesis. Previous studies have demonstrated that this new class of antimicrobial agent blocks translation by inhibiting initiation complex formation, while post-initiation translation by polysomes and poly(U)-dependent translation is not a target for these compounds. We found that oxazolidinones inhibit translation of natural mRNA ... More
The tumor-sensitive calmodulin-like protein is a specific light chain of human unconventional myosin X.
AuthorsRogers MS, Strehler EE
JournalJ Biol Chem
PubMed ID11278607
Human calmodulin-like protein (CLP) is an epithelial-specific Ca(2+)-binding protein whose expression is strongly down-regulated in cancers. Like calmodulin, CLP is thought to regulate cellular processes via Ca(2+)-dependent interactions with specific target proteins. Using gel overlays, we identified a approximately 210-kDa protein binding specifically and in a Ca(2+)-dependent manner to CLP, ... More
Topology of the integral membrane form of Escherichia coli SecA protein reveals multiple periplasmically exposed regions and modulation by ATP binding.
AuthorsRamamurthy V, Oliver D
JournalJ Biol Chem
PubMed ID9287332
SecA insertion and integration into the Escherichia coli inner membrane is a critical step for the catalysis of protein translocation across this layer. To understand this step further, SecA topology was investigated. To determine which regions of SecA are periplasmically exposed, right-side out membrane vesicles were prepared from strains synthesizing ... More
tRNA-mediated labelling of proteins with biotin. A nonradioactive method for the detection of cell-free translation products.
AuthorsKurzchalia TV, Wiedmann M, Breter H, Zimmermann W, Bauschke E, Rapoport TA
JournalEur J Biochem
PubMed ID3350017
We have developed a new method for the rapid and sensitive detection of cell-free translation products. Biotinylated lysine is incorporated into newly synthesized proteins by means of lysyl-tRNA that is modified in the epsilon-position. After electrophoresis in a dodecyl sulfate gel and blotting onto nitrocellulose, the translation products can be ... More
Identification and isolation of a 45-kDa calcium-dependent lactoferrin receptor from rat hepatocytes.
AuthorsBennatt DJ, McAbee DD
JournalBiochemistry
PubMed ID9204883
Isolated rat hepatocytes bind, internalize, and degrade bovine lactoferrin (Lf) via high-affinity Ca2+-dependent sites [<10(6) sites/cell; McAbee et al., (1993) Biochemistry 32, 13749-13760]. In this study, we identified a 45-kDa Ca2+-dependent Lf binding protein on rat hepatocytes by three independent approaches. First, dithiobis(sulfosuccimidylproprionate) (DTSSP) cross-linked 125I-Lf to a 45-kDa adduct ... More
Calcium-dependent conformation of a mouse macrophage calcium-type lectin. Carbohydrate binding activity is stabilized by an antibody specific for a calcium-dependent epitope.
AuthorsKimura T, Imai Y, Irimura T
JournalJ Biol Chem
PubMed ID7541793
We established monoclonal antibodies (mAbs) against the mouse macrophage galactose/N-acetylgalactosamine-specific lectin (MMGL) that is a 42-kDa calcium-dependent lectin, using a solid phase carbohydrate binding assay as a novel strategy for screening mAbs. The specificity of six mAbs were investigated by antibody binding to native or recombinant forms (rML) of MMGL, ... More
Single Molecule Detection Technologies in Miniaturized High Throughput Screening: Fluorescence Correlation Spectroscopy.
AuthorsMoore KJ, Turconi S, Ashman S, Ruediger M, Haupts U, Emerick V, Pope AJ
JournalJ Biomol Screen
PubMed ID10838431
Fluorescence assay technologies used for miniaturized high throughput screening are broadly divided into two classes. Macroscopic fluorescence techniques (encompassing conventional fluorescence intensity, anisotropy [also often referred to as fluorescence polarization] and energy transfer) monitor the assay volume- and time-averaged fluorescence output from the ensemble of emitting fluorophores. In contrast, single-molecule ... More
In vitro reconstituted Dictyostelium discoideum early endosome fusion is regulated by Rab7 but proceeds in the absence of ATP-Mg2+ from the bulk solution.
AuthorsLaurent O, Bruckert F, Adessi C, Satre M
JournalJ Biol Chem
PubMed ID9422733
We characterized the in vitro fusion of endosomal compartments from Dictyostelium discoideum. Fusion activity was restricted to early compartments, was dependent on cytosolic proteins, and was activated by GTP and guanosine 5'-O(3-thio)triphosphate (GTPgammaS). This stimulation suggests the involvement of a small G protein, which we propose to be Rab7 on ... More
The animal research kit (ARK) can be used in a multistep double staining method for human tissue specimens.
Authorsvan der Loos CM, Göbel H
JournalJ Histochem Cytochem
PubMed ID10990496
The newly developed Animal Research Kit (ARK) offers a simple and economic way of biotinylating mouse primary antibodies for background-free immunostaining of mouse and rat tissue specimens. Biotinylation involves the use of a biotinylated goat anti-mouse immunoglobulin Fab fragment mixed with a mouse primary antibody and subsequent blocking with normal ... More
Phorbol 12-myristate 13-acetate down-regulates Na,K-ATPase independent of its protein kinase C site: decrease in basolateral cell surface area.
AuthorsBeron J, Forster I, Beguin P, Geering K, Verrey F
JournalMol Biol Cell
PubMed ID9188092
The effect of protein kinase C (PKC) stimulation on the pump current (Ip) generated by the Na,K-ATPase was measured in A6 epithelia apically permeabilized with amphotericin B. Phorbol 12-myristate 13-acetate (PMA) produced a decrease in Ip carried by sodium pumps containing the endogenous Xenopus laevis or transfected Bufo marinus alpha ... More
Haptenylation of antibodies during affinity purification: a novel and convenient procedure to obtain labeled antibodies for quantification and double labeling.
AuthorsPitt JC, Lindemeier J, Habbes HW, Veh RW
JournalHistochem Cell Biol
PubMed ID9749965
Haptenylation of primary antibodies is a useful technique for multiple purposes. It is a technically straightforward procedure, as many haptens are available as N-hydroxysuccinimide esters or isothiocyanates. Unfortunately, the hapten group may become covalently attached to or close to the combining site of antibodies, lectins, or other ligand-binding proteins during ... More
Clathrin-mediated endocytosis and recycling of autocrine motility factor receptor to fibronectin fibrils is a limiting factor for NIH-3T3 cell motility.
AuthorsLe PU, Benlimame N, Lagana A, Raz A, Nabi IR
JournalJ Cell Sci
PubMed ID10954421
Autocrine motility factor receptor (AMF-R) is internalized via a clathrin-independent pathway to smooth endoplasmic reticulum tubules. This endocytic pathway is shown here to be inhibited by methyl-(beta)-cyclodextrin (m(beta)CD) implicating caveolae or caveolae-like structures in AMF internalization to smooth ER. AMF-R is also internalized via a clathrin-dependent pathway to a transferrin ... More
Comparison of biotinylated DNA and RNA probes for rapid detection of varicella-zoster virus genome by in situ hybridization.
AuthorsForghani B, Yu GJ, Hurst JW
JournalJ Clin Microbiol
PubMed ID1645371
We describe a general method for the production of nonisotopic DNA and RNA probes for the detection of the varicella-zoster virus (VZV) genome by in situ hybridization. VZV DNA was extracted from purified viral nucleocapsids, cleaved with restriction enzyme (RE) BamHI, and cloned into plasmid pBR322 by the standard vector ... More
Radiolabeled biotinyl peptides as useful reagents for the study of proteolytic enzymes.
AuthorsBasak A, Boudreault A, Jean F, Chrétien M, Lazure C
JournalAnal Biochem
PubMed ID8470802
A radiometric assay for studying the proteolytic activity of endopeptidases using a radiolabeled biotinyl peptide substrate is described. The method relies on the use of a peptidyl substrate incorporating susceptible bonds located between a biotinyl group at one end and a radioiodinated group at the other end. Two tyrosine-containing peptidyl ... More
N-Methyl-D-aspartate receptors are clustered and immobilized on dendrites of living cortical neurons.
AuthorsBenke TA, Jones OT, Collingridge GL, Angelides KJ
JournalProc Natl Acad Sci U S A
PubMed ID7689230
The response of nerve cells to synaptic inputs and the propagation of this activation is critically dependent on the cell-surface distribution of ion channels. In the hippocampus, Ca2+ influx through N-methyl-D-aspartate receptors (NMDAR) and/or voltage-dependent calcium channels on dendrites is thought to be critically involved in long-term potentiation, neurite outgrowth, ... More
The Ca2+ dependence of human Fc gamma receptor-initiated phagocytosis.
AuthorsEdberg JC, Lin CT, Lau D, Unkeless JC, Kimberly RP
JournalJ Biol Chem
PubMed ID7673212
Differing roles for [Ca2+]i transients in Fc gamma R-mediated phagocytosis have been suggested based on the observations that antibody-opsonized erythrocyte phagocytosis by human neutrophils shows a [Ca2+]i dependence, while that by murine macrophages appears [Ca2+]i-independent. To explore whether this difference might reflect different receptor isoforms or different cell types, we ... More
Avidin-biotin affinity chromatography: application to the isolation of human placental insulin receptor.
AuthorsFinn FM, Titus G, Horstman D, Hofmann K
JournalProc Natl Acad Sci U S A
PubMed ID6390435
The ligand N alpha, B1-(6-biotinylamido)hexanoyl-insulin was attached noncovalently to Sepharose 4B immobilized succinoylavidin to form an insulin-affinity resin. This resin was used to isolate highly purified insulin receptor from human placental tissue by a four step process involving (i) preparation of a crude membrane fraction, (ii) solubilization with Triton X-100, ... More
Imaging of endosome fusion in BHK fibroblasts based on a novel fluorimetric avidin-biotin binding assay.
AuthorsEmans N, Biwersi J, Verkman AS
JournalBiophys J
PubMed ID8527685
A fluorescence assay of in vivo endosome fusion was developed and applied to define the kinetics of endosome fusion in baby hamster kidney (BHK) fibroblasts. The assay is based on an approximately 10-fold enhancement of the green fluorescence of BODIPY-avidin upon biotin binding. The BODIPY-avidin fluorescence enhancement occurred in < ... More
Biotinylated human beta-endorphins as probes for the opioid receptor.
AuthorsHochhaus G, Gibson BW, Sadée W
JournalJ Biol Chem
PubMed ID2826453
The reaction of human beta-endorphin and biotinyl N-hydroxysuccinimide with or without spacer arm, afforded a series of products that were separated by high performance liquid chromatography (HPLC). Liquid secondary ion mass spectrometry of the biotinylated products and their tryptic digests produced abundant protonated molecular ions (MH+), which specified the number ... More
Outer membrane monolayer domains from two-dimensional surface scanning resistance measurements.
AuthorsSuzuki K, Sterba RE, Sheetz MP
JournalBiophys J
PubMed ID10866970
Cellular plasma membranes have domains that are defined, in most cases, by cytoskeletal elements. The outer half of the bilayer may also contain domains that organize glycosylphosphatidylinositol (GPI)-linked proteins. To define outer membrane barriers, we measured the resistive force on membrane bound beads as they were scanned across the plasma ... More
Fiber-optic evanescent wave biosensor for the detection of oligonucleotides.
AuthorsAbel AP, Weller MG, Duveneck GL, Ehrat M, Widmer HM
JournalAnal Chem
PubMed ID8794925
An automated optical biosensor system based on fluorescence excitation and detection in the evanescent field of a quartz fiber was used to detect 16-mer oligonucleotides in DNA hybridization assays. A biotinylated capture probe was immobilized on the fiber surface via avidin or streptavidin. The hybridization with fluorescein-labeled complementary strands was ... More
Diverging pathways for lipopolysaccharide and CD14 in human monocytes.
AuthorsAntal-Szalmás P, Poppelier MJ, Broekhuizen R, Verhoef J, van Strijp JA, van Kessel KP
JournalCytometry
PubMed ID11084613
BACKGROUND: CD14 is considered to be the major endotoxin (lipopolysaccharide [LPS]) binding molecule on human monocytes. It initiates cellular response, but its role in the clearance of LPS is not well understood. Under conditions that ensure totally CD14-dependent LPS binding on human monocytes, the internalization mechanisms of LPS and CD14 ... More
Immunogenicity of biotinylated hapten-avidin complexes.
AuthorsScott D, Nitecki DE, Kindler H, Goodman JW
JournalMol Immunol
PubMed ID6440008
The efficacy of avidin as a carrier for the generation of anti-hapten antibodies was assessed in mice by immunization with complexes of avidin and synthetic peptides containing biotin and an epsilon-dinitrophenyl (DNP) lysine residue. The synthetic haptens were constructed with 0, 1 or 2 6-aminocaproyl groups as spacers between the ... More
Real-time fluorescence measurement of cell-free endosome fusion: regulation by second messengers.
AuthorsEmans N, Verkman AS
JournalBiophys J
PubMed ID8804631
A quantitative real-time assay of cell-free endosomal vesicle fusion was developed and applied to study fusion mechanisms in endosomes from baby hamster kidney (BHK-21) cells. The assay is based on an irreversible approximately 10-fold increase in BODIPY-avidin fluorescence on binding of biotinylated conjugates. BODIPY-avidin and biotin-dextran were internalized for 10 ... More
Metabolic behavior of cell surface biotinylated proteins.
AuthorsHare JF, Lee E
JournalBiochemistry
PubMed ID2713332
The turnover of proteins on the surface of cultured mammalian cells was measured by a new approach. Reactive free amino or sulfhydryl groups on surface-accessible proteins were derivatized with biotinyl reagents and the proteins solubilized from culture dishes with detergent. Solubilized, biotinylated proteins were then adsorbed onto streptavidin-agarose, released with ... More
Time-resolved delayed luminescence image microscopy using an europium ion chelate complex.
AuthorsMarriott G, Heidecker M, Diamandis EP, Yan-Marriott Y
JournalBiophys J
PubMed ID7811952
Improvements and extended applications of time-resolved delayed luminescence imaging microscopy (TR-DLIM) in cell biology are described. The emission properties of europium ion complexed to a fluorescent chelating group capable of labeling proteins are exploited to provide high contrast images of biotin labeled ligands through detection of the delayed emission. The ... More
Zymogen/enzyme discrimination using peptide chloromethyl ketones.
AuthorsWilliams EB, Krishnaswamy S, Mann KG
JournalJ Biol Chem
PubMed ID2708377
Glutamylglycinylarginyl chloromethyl ketone, tyrosylglycinylarginyl chloromethyl ketone, and phenylalanylprolylarginyl chloromethyl ketone have been labeled at their amino termini using fluorescein, rhodamine-X, lissamine-rhodamine, pyrene, and the 1,5-, 2,5-, and 2,6-dimethylaminonaphthalene-1-sulfonyl moieties. These peptidyl chloromethyl ketones have also been modified by incorporation of biotin and epsilon-amino caproyl biotin. The ability of these various ... More
Syntheses of biotinylated and dethiobiotinylated insulins.
The 600-MHz proton spectrum of dethiobiotin (prepared from d-biotin with Raney nickel) was measured in order to gain information pertaining to its stereochemical homogeneity. The spectrum demonstrated clearly that the material is a 6:1 mixture of two stereoisomers. The cis compound, corresponding to the stereochemistry of d-biotin, is the major ... More
Sequence preferences in cleavage of dsDNA and ssDNA by the extracellular Serratia marcescens endonuclease.
AuthorsMeiss G, Friedhoff P, Hahn M, Gimadutdinow O, Pingoud A
JournalBiochemistry
PubMed ID7547935
The preferred cleavage sites in dsDNA and ssDNA for the extracellular Serratia marcescens endonuclease (commercially available as BENZONASE) were identified by limited digestion of PCR-generated substrates. Two different dsDNA substrates were synthesized by using either radioactively or fluorescent dye labeled primers. ssDNA of identical sequence to one of the fluorescent ... More
A synapse-specific carbohydrate at the neuromuscular junction: association with both acetylcholinesterase and a glycolipid.
AuthorsScott LJ, Bacou F, Sanes JR
JournalJ Neurosci
PubMed ID3346730
With the aim of investigating the roles of carbohydrates in synapse formation, we have characterized a synapse-specific saccharide at the vertebrate neuromuscular junction. Two lectins of similar specificity (Dolichos biflorus agglutinin, DBA, and Vicia villosa-B4 agglutinin, VVA-B4) stain synaptic but not extrasynaptic regions of the rat muscle fiber surface and ... More
Trafficking and proteolytic release of epidermal growth factor receptor ligands are modulated by their membrane-anchoring domains.
AuthorsDong J, Wiley HS
JournalJ Biol Chem
PubMed ID10617651
Ligands that bind to the epidermal growth factor (EGF) receptor are initially synthesized as integral membrane proteins that are released from the cell surface by regulated proteolysis. To study the role of the membrane-anchoring domain in ligand release, we made two artificial ligands. The first possessed the membrane-anchoring domain from ... More
ATP dependence of NHE-1, the ubiquitous isoform of the Na+/H+ antiporter. Analysis of phosphorylation and subcellular localization.
AuthorsGoss GG, Woodside M, Wakabayashi S, Pouyssegur J, Waddell T, Downey GP, Grinstein S
JournalJ Biol Chem
PubMed ID8132605
ATP is not hydrolyzed during the transport cycle of the Na+/H+ exchanger (NHE), yet depletion of the nucleotide drastically reduces the rate of cation exchange. The mechanism underlying this inhibition was investigated in fibroblasts transfected with NHE-1, the growth factor-sensitive isoform of the antiport. NHE-1 was found to be phosphorylated ... More
A high-throughput nonisotopic protein truncation test.
AuthorsGite S, Lim M, Carlson R, Olejnik J, Zehnbauer B, Rothschild K
JournalNat Biotechnol
PubMed ID12524552
Nonsense or frameshift mutations, which result in a truncated gene product, are prevalent in a variety of disease-related genes, including APC (implicated in colorectal cancer), BRCA1 and BRCA2 (breast and ovarian cancer), PKD1 (polycystic kidney disease), NF1 and NF2 (neurofibromatosis), and DMD (Duchenne muscular dystrophy). Such chain-truncating mutations can be ... More