Anti-phosphotyrosine-HRP antibody is an HRP labeled tyrosine phosphorylation specific antibody that is part of the Signal-Seeker™ product line. APY03-HRP phosphotyrosine antibody recognizes a wide range of tyrosine phosphorylated proteins in cells treated with kinase inhibitors, hydrogen peroxide (H2O2) and sodium vanadate (Figure 1) and has been validated for western blot applications. APY03 anti-phosphotyrosine antibody 27B10 has been tested in western blot, immunofluorescence, immunoprecipitation, and ELISA with excellent specificity and low background with strong potent signals.
APY03 is a mouse monoclonal antibody 27B10 that recognizes phosphorylated tyrosine residues. APY03 was raised against a proprietary mixture of phosphotyrosine peptides conjugated to KLH. APY03 can detect less than 10 ng of phosphotyrosine-labeled bovine serum albumin (see Certificate of Analysis [COA]). APY03 is purified by protein G affinity chromatography and is supplied as a lyophilized white powder. Each Lot of antibody is quality controlled to provide a high batch to batch consistency. The Lot specific µg per tube can be found in the Lot specific COA documents. APY03-HRP shows high specificity to phosphotyrosine proteins and does not cross-react with phosphoserine/ threonine proteins a western blot assay (Figures 1).
Figure 1: Western Blot demonstration of APY03-HRP phosphotyrosine specificity
A431 cells were either treated (+) or untreated (-) with Calyculin A (CalyA: a serine/threonine phosphatase inhibitor, 50nM for 1 hour). NIH3T3 cells were either treated or untreated with H2O2-activated sodium orthovanadate (Vanadate: a specific tyrosine phosphatase inhibitor, 100 µM for 10 minutes). 10 µg of each lysate was resolved in SDS-PAGE and proteins were transferred to PVDF membrane. APY03-HRP (1:6000) was used to detect tyrosine phosphorylated proteins. Western blot was developed with SuperSignal West Dura chemiluminescent reagent (Thermo Scientific) and exposure time was 10 seconds. As shown in Fig.1, a wide range of tyrosine phosphorylated proteins were detected in NIH3T3 cells treated with orthovanadate but not in Calyculin A treated A431 cells.
To see the full Western blot protocol, see the product datasheet.
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Signal-Seeker™ Phosphotyrosine Detection Kit (Cat. # BK160)
Signal-Seeker™ Phosphotyrosine Affinity Beads (Cat.# APY03-beads)
Signal-Seeker™: BlastR™ Rapid Lysate Prep Kit (Cat. # BLR01)
For product Datasheets and MSDSs please click on the PDF links below.
Sample Size Datasheet (Cat. APY03-HRP-S):
Certificate of Analysis: Lot 011
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