Anti-phosphotyrosine antibody is a tyrosine phosphorylation specific antibody that is part of the Signal-Seeker™ product line. APY03 anti-phosphotyrosine mouse monoclonal antibody clone 27B10 has been tested in western blot, immunofluorescence, immunoprecipitation and ELISA with excellent specificity and low background with strong potent signals. APY03 phosphotyrosine antibody recognizes a wide range of tyrosine phosphorylated proteins in cells treated with kinase inhibitors, hydrogen peroxide (H2O2) and sodium vanadate (Figure 1).
APY03 is a mouse monoclonal antibody 27B10 that recognizes phosphorytated tyrosine residues. APY03 was raised against a proprietary mixture of phosphotyrosine peptides conjugated to KLH. APY03 can detect 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 at www.cytoskeleton.com. APY03 shows high specificity to phosphotyrosine peptides and does not cross-react with phosphoserine/threonine peptides in a western blot or an ELISA assay (Figures 1 & 4).
Figure 1: Western Blot using Phosphotyrosine Antibody
APY03 is highly specific for p-Tyr and does not recognize p-Ser or p-Thr tagged proteins. To see the full Western blot protocol, see the product datasheet.
Figure 2: Immunoprecipitation using Phosphotyrosine Antibody
APY03 was able to enrich a wide range of tyrosine-phosphorylated proteins from NIH3T3 cells treated with H2O2-activated orthovanadate. No signal was detected with Protein G bead control without APY03. To see the full Immunoprecipitation protocol, see the product datasheet.
Human epidermoid carcinoma A431 cells, untreated (3A) or treated (3B) with EGF (100 ng/ml for 3 minutes), and NIH3T3, untreated (3C) or treated (3D) with H2O2-activated sodium orthovanadate (100 µM for 10 minutes), were stained as described in the method. Phosphotyrosine and nuclei were visualized in green fluorescence and blue DAPI staining, respectively. To see the full Immunofluorescence protocol, see the product datasheet.
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