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           Search results for: ACTIVE SITE LABELING REAGENTS Biotin Labeled Phe-Pro-Arg-Chloromethylketone   

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#22418280   2012/04/06 Save this To Up

Development of clickable active site-directed photoaffinity probes for γ-secretase.

We have developed clickable active site-directed photoaffinity probes for γ-secretase which incorporate a photoreactive benzophenone group and an alkyne handle for subsequent click chemistry mediated conjugation with azide-linked reporter tags for visualization (e.g., TAMRA-azide) or enrichment (e.g., biotin-azide) of labeled proteins. Specifically, we synthesized clickable analogs of L646 (2) and L505 (3) and validated specific labeling to presenilin-1N-terminal fragment (PS1-NTF), the active site aspartyl protease component within the γ-secretase complex. Additionally, we were able to identify signal peptide peptidase (SPP) by Western blot analysis. Furthermore, we analyzed the photo-labeled proteins in an unbiased fashion by click chemistry with TAMRA-azide followed by in-gel fluorescence detection. This approach expands the utility of γ-secretase inhibitor (GSI) photoaffinity probes in that labeled proteins can be tagged with any number of azide-linked reporters groups using a single clickable photoaffinity probe for target pull down and/or fluorescent imaging applications.

2948 related Products with: Development of clickable active site-directed photoaffinity probes for γ-secretase.

DNA (cytosine 5) methyltr Glycosylated Human PAI-1 Contact Factors: Human Fa Mouse PAI-1 (wild type ac Mouse PAI-1 (wild type ac Porcine PAI-1 (wild type Porcine PAI-1 (wild type Rabbit PAI-1 (wild type a Rabbit PAI-1 (wild type a Rat PAI-1 (wild type acti Rat PAI-1 (wild type acti Anti-BACE-1 (Memapsin-2,

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#22187146   2012/07/16 Save this To Up

Site-specific DOTA/europium-labeling of recombinant human relaxin-3 for receptor-ligand interaction studies.

Relaxin-3 (also known as INSL7) is a recently identified neuropeptide belonging to the insulin/relaxin superfamily. It has putative roles in the regulation of stress responses, food intake, and reproduction by activation of its cognate G-protein-coupled receptor RXFP3. It also binds and activates the relaxin family peptide receptors RXFP1 and RXFP4 in vitro. To obtain a europium-labeled relaxin-3 as tracer for studying the interaction of these receptors with various ligands, in the present work we propose a novel site-specific labeling strategy for the recombinant human relaxin-3 that has been previously prepared in our laboratory. First, the N-terminal 6 × His-tag of the single-chain relaxin-3 precursor was removed by Aeromonas aminopeptidase and all of the primary amines of the resultant peptide were reversibly blocked by citroconic anhydride. Second, the A-chain N-terminus of the blocked peptide was released by endoproteinase Asp-N cleavage that removed the linker peptide between the B- and A-chains. Third, an alkyne moiety was introduced to the newly released A-chain N-terminus by reaction with the highly active primary amine-specific N-hydroxysuccinimide ester. Fourth, after removal of the reversible blockage under mild acidic condition, europium-loaded DOTA with an azide moiety was introduced to the two-chain relaxin-3 carrying the alkyne moiety through click chemistry. Using this site-specific labeling strategy, homogeneous monoeuropium-labeled human relaxin-3 could be obtained with good overall yield. In contrast, conventional random labeling resulted in a complex mixture that was poorly resolved because human relaxin-3 has four primary amine moieties that all react with the modification reagent. Both saturation and competition binding assays demonstrated that the DOTA/Eu(3+)-labeled relaxin-3 retained high binding affinity for human RXFP3, RXFP4, and RXFP1 and was therefore a suitable non-radioactive and stable tracer to study the interaction of various natural or designed ligands with these receptors. Using this site-specific labeling strategy, other functional probes, such as fluorescent dyes, biotin, or nanoparticles could also be introduced to the A-chain N-terminal of the recombinant human relaxin-3. Additionally, we improved the time-resolved fluorescence assay for the DOTA-bound europium ion which paves the way for the use of DOTA as a lanthanide chelator for protein and peptide labeling in future studies.

1081 related Products with: Site-specific DOTA/europium-labeling of recombinant human relaxin-3 for receptor-ligand interaction studies.

RANK Ligand Soluble, Huma Bone Morphogenetic Protei Growth Differentiation Fa Macrophage Colony Stimula RANK Ligand Soluble, Huma RANK Ligand Soluble, Huma RANK Ligand Soluble, Huma RANK Ligand Soluble, Huma RANK Ligand Soluble, Huma RANK Ligand Soluble, Huma Recombinant Human 4-1BB R Recombinant Human ALT1 GP

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#21044582   2010/11/03 Save this To Up

Movements of individual BKCa channels in live cell membrane monitored by site-specific labeling using quantum dots.

The movements of BK(Ca) channels were investigated in live cells using quantum dots (QDs). The extracellular N-terminus was metabolically tagged with biotin, labeled with streptavidin-conjugated QDs and then monitored using real-time time-lapse imaging in COS-7 cells and cultured neurons. By tracking hundreds of channels, we were able to determine the characteristics of channel movements quantitatively. Channels in COS-7 cells exhibited a confined diffusion in an area of 1.915 μm(2), with an initial diffusion coefficient of 0.033 μm(2)/s. In neurons, the channel movements were more heterogeneous and highly dependent on subcellular location. While the channels in soma diffused slowly without clear confinement, axodendritic channels showed more rapid and pseudo-one-dimensional movements. Intriguingly, the channel movement in somata was drastically increased by the neuronal β4 subunit, in contrast to the channels in the axodendritic area where the mobility were significantly decreased. Thus, our results demonstrate that the membrane mobility of BK(Ca) channels can be greatly influenced by the expression system used, subunit composition, and subcellular location. This QD-based, single-molecule tracking technique can be utilized to investigate the cellular mechanisms that determine the mobility as well as the localization of various membrane proteins in live cells.

2007 related Products with: Movements of individual BKCa channels in live cell membrane monitored by site-specific labeling using quantum dots.

3,3 dioctadecyloxacarbocy Cell Explorer™ Live Cel Cell Explorer™ Live Cel Cell Explorer™ Live Cel Cell Explorer™ Live Cel Cell Explorer™ Live Cel Cell Explorer™ Live Cel BYL-719 Mechanisms: PI3K- Cell cycle antibody array Cell Cycle Control Phosph Cell Cycle Phospho-Specif Nuclear Membrane Receptor

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#19810697   2009/11/20 Save this To Up

Expedited solid-phase synthesis of fluorescently labeled and biotinylated aminoalkane diphenyl phosphonate affinity probes for chymotrypsin- and elastase-like serine proteases.

In this study, we report on a novel, expedited solid-phase approach for the synthesis of biotinylated and fluorescently tagged irreversible affinity based probes for the chymotrypsin and elastase-like serine proteases. The novel solid-phase biotinylation or fluorescent labeling of the aminoalkane diphenyl phosphonate warhead using commercially available Biotin-PEG-NovaTag or EDANS NovaTag resin permits rapid, facile synthesis of these reagents. We demonstrate the kinetic evaluation and utilization of a number of these irreversible inactivators for chymotrypsin-like (chymotrypsin/human cathepsin G) and elastase-like serine proteases. Encouragingly, these compounds display comparable potency against their target proteases as their N-benzyloxycarbonyl (Cbz)-protected parent compounds, from which they were derived, and function as efficient active site-directed inactivators of their target proteases. We subsequently applied the biotinylated reagents for the sensitive detection of protease species via Western blot, showing that the inactivation of the protease was specifically mediated through the active site serine. Furthermore, we also demonstrate the successful detection of serine protease species with the fluorescently labeled derivatives "in-gel", thus avoiding the need for downstream Western blotting. Finally, we also show the utility of biotinylated and pegylated affinity probes for the isolation/enrichment of serine protease species, via capture with immobilized streptavidin, and their subsequent identification via de novo sequencing. Given their selectivity of action against the serine proteases, we believe that these reagents can be exploited for the direct, rapid, and selective identification of these enzymes from biological milieu containing multiple protease subclasses.

2207 related Products with: Expedited solid-phase synthesis of fluorescently labeled and biotinylated aminoalkane diphenyl phosphonate affinity probes for chymotrypsin- and elastase-like serine proteases.

Androgen Receptor (Phosph Androgen Receptor (Phosph Rabbit Anti-Human Androge Rabbit Anti-Human Androge Elastase Inhibitor, SPCK; Androgen Receptor (Ab 650 AZD-3514 Mechanisms: Andr 17β-Acetoxy-2α-bromo-5 (5α,16β)-N-Acetyl-16-[2 (5α,16β)-N-Acetyl-16-ac 5α-N-Acetyl-2'H-androst- 5α-N-Acetyl-2'H-androst-

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#17545978   2007/06/04 Save this To Up

Tandem orthogonal proteolysis-activity-based protein profiling (TOP-ABPP)--a general method for mapping sites of probe modification in proteomes.

Activity-based protein profiling (ABPP) utilizes active site-directed chemical probes to monitor the functional state of enzymes directly in native biological systems. Identification of the specific sites of probe labeling on enzymes remains a major challenge in ABPP experiments. In this protocol, we describe an advanced ABPP platform that utilizes a tandem orthogonal proteolysis (TOP) strategy coupled with mass spectrometric analysis to simultaneously identify probe-labeled proteins together with their exact sites of probe modification. Elucidation of probe modification sites reveals fundamental insights into the molecular basis of specific probe-protein interactions. The TOP-ABPP method can be applied to any type of proteomic sample, including those derived from in vitro or in vivo labeling experiments, and is compatible with a variety of chemical probe structures. Completion of the entire protocol, including chemical synthesis of key reagents, requires approximately 8-10 days.

2806 related Products with: Tandem orthogonal proteolysis-activity-based protein profiling (TOP-ABPP)--a general method for mapping sites of probe modification in proteomes.

EnzyChrom™ Kinase Assay pCAMBIA0105.1R Vector, (G HIV 1 intergase antigen. Bone Morphogenetic Protei Human Macrophage Inflamma Human Macrophage Inflamma Mouse Macrophage Inflamma Mouse Macrophage Inflamma Recombinant Human Inhibin Recombinant Human Inhibin Cell Meter™ Fluorimetri Cell Meter™ Fluorimetri

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#11554721   2001/09/13 Save this To Up

Biotin derivatives of D-Phe-Pro-Arg-CH2Cl for active-site-specific labeling of thrombin and other serine proteinases.

Biotin derivatives of peptide chloromethyl ketones have ideal properties for specific labeling of the catalytic sites of serine proteinases but have not been widely used as probes because of the difficulty of synthesis and their instability. To make the reagents more accessible, a simple, economical method was developed for preparation of three biotin derivatives of the thrombin-specific inhibitor D-Phe-Pro-Arg-CH2Cl containing increasing lengths of the spacer connecting biotin. Reaction of the peptide with biotin-succinimidyl esters and purification by conventional chromatography yielded the compounds in 91-96% purity. The biotin-labeled inhibitors bound avidin with stoichiometries of 0.88-1.02 mol biotin compound/mol avidin subunits and irreversibly inactivated human thrombin with stoichiometries of 0.89-1.10 mol inhibitor/mol thrombin. Comparison of the three inhibitors by Western blotting indicated that a > or = 7- to 14-atom spacer was needed for sensitive (approximately 10 ng) detection of thrombin, with the derivative lacking a spacer only weakly detected because of its greatly reduced affinity for avidin. Application of the compounds to identify catalytically active products of factor Xa-catalyzed human prethrombin 1 activation in the absence of the protein cofactor, factor Va, allowed the direct observation of transient, low levels of the active intermediate, meizothrombin des-fragment 1, in addition to thrombin. Formation of this intermediate is concluded to reflect an intrinsic property of factor Xa activation of prethrombin 1 that is modulated by factor Va. The methods developed for preparation and characterization of the biotin-labeled inhibitors may be applicable to other tripeptide chloromethyl ketones, and the reagents can be employed for labeling of serine proteinases of diverse substrate specificity.

2710 related Products with: Biotin derivatives of D-Phe-Pro-Arg-CH2Cl for active-site-specific labeling of thrombin and other serine proteinases.

MOUSE ANTI BOVINE ROTAVIR MOUSE ANTI BORRELIA BURGD NATIVE HUMAN PROLACTIN, P RABBIT ANTI GSK3 BETA (pS ChromaLink™ Biotin Labe Rabbit Anti-PAR-1 Thrombi Rabbit Anti-Nkx2.5 Cardia 10x ELISA WASH BUFFER, Pr 10X PHOSPHATE BUFFERED SA PERMANENT AQUEOUS MOUNTIN Rat Visceral adipose spec CAR,CAR,Constitutive acti

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#9575231   1998/06/12 Save this To Up

Determination of external loop topology in the serotonin transporter by site-directed chemical labeling.

The transmembrane topology of the serotonin transporter (SERT) has been examined by measuring the reactivity of selected lysine and cysteine residues with extracellular reagents. An impermeant biotinylating reagent, sulfosuccinimidyl 2-(biotinamido)ethyl-1, 3-dithiopropionate (NHS-SS-biotin), was shown to label SERT transiently expressed in cultured cells. Replacement of four lysine residues that were predicted to lie in external hydrophilic loops (eK-less) largely prevented the biotinylation reaction. Likewise, the cysteine-specific biotinylation reagent N-biotinylaminoethylmethanethiosulfonate (MTSEA-biotin) labeled wild type SERT but not a mutant in which Cys-109, predicted to lie in the first external loop, was replaced with alanine. These two mutant transporters reacted with the biotinylating reagents in digitonin-permeabilized cells, demonstrating that the abundant lysine and cysteine residues predicted to lie in intracellular hydrophilic domains were reactive but not accessible in intact cells. Mutants containing a single external lysine at positions 111, 194, 243, 319, 399, 490, and 571 reacted more readily with NHS-SS-biotin than did the eK-less mutant. Similarly, mutants with a single cysteine at positions 109, 310, 406, 489, and 564 reacted more readily with MTSEA-biotin than did the C109A mutant. All of these mutants were active and therefore likely to be folded correctly. These results support the original transmembrane topology and argue against an alternative topology proposed recently for the related glycine and gamma-aminobutyric acid transporters.

1924 related Products with: Determination of external loop topology in the serotonin transporter by site-directed chemical labeling.

Directed In Vivo Angiogen BYL-719 Mechanisms: PI3K- Horizontal Laminar Flow Horizontal Laminar Flow Horizontal Laminar Flow Vertical Laminar Flow Cle Vertical Laminar Flow Cle Horizontal Laminar Flow Vertical Laminar Flow Cle Vertical Laminar Flow Cle Thermal Shaker with cooli Vertical Laminar Flow Cle

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#7773729   1995/07/10 Save this To Up

Evidence that meizothrombin is an intermediate product in the clotting of whole blood.

Meizothrombin is an intermediate that is produced during the conversion of prothrombin to thrombin in systems composed of purified factor Xa and factor Va that are quantitatively assembled on an anionic phospholipid surface. The biological significance of this intermediate has recently been challenged by the apparent absence of meizothrombin during clotting of sodium citrate-anticoagulated plasma. We analyzed the formation of thrombin during coagulation of nonanticoagulated, unchilled, minimally manipulated whole blood in glass tubes. The process was stopped at 0, 3, 5, and 7 minutes by the addition of biotinylated peptidyl chloromethyl-ketone active-site labeling reagents. Plasma/serum was separated by centrifugation, and labeled species were extracted by immunoadsorption with a polyclonal anti-prothrombin antibody. The purified prothrombin-derived species were separated by SDS-polyacrylamide gradient gel electrophoresis and visualized on a chemiluminescent avidin blot. Meizothrombin appeared as an intermediate product of this reaction and persisted with some increase through the 7-minute time point. We also observed incorporation of the active-site label into a species of lower molecular weight consistent with the B1 chain of beta- and/or gamma-thrombin. These degraded forms of thrombin have not been previously demonstrated in a biologically relevant preparation. Our data clearly establish the generation of meizothrombin as an intermediate product of thrombin generation during whole-blood clotting. The data also represent the first experimental evidence for the generation of beta- and gamma-thrombin in a biologically relevant environment and time scale.

2907 related Products with: Evidence that meizothrombin is an intermediate product in the clotting of whole blood.

Interleukin-34 IL34 (N-t Interleukin-34 IL34 anti MOUSE ANTI BOVINE ROTAVIR Anti AGO2 Human, Monoclon Anti AGO2 Mouse, Monoclon Anti AGO2 Human, Monoclon Anti AGO2 Mouse, Monoclon HIV1 integrase antibody, Goat Anti-Human Laforin ( anti H inh human blood an MOUSE ANTI BORRELIA BURGD interleukin 17 receptor C

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