Phosphorylation of specific tyrosine residues is an important post-translational modification for regulating the activity of most proteins. Stimulation of a variety of cell signaling pathways activates the receptor and non-receptor tyrosine kinases that mediate these protein modifications. Antibodies that can detect phosphotyrosine residues are excellent tools for characterizing changes in the post-translational state of a broad range of phosphotyrosine-containing proteins. Immunoprecipitation of proteins of interest, followed by detection of phosphotyrosine using anti-phosphotyrosine antibody is commonly used to correlate changes in tyrosine phosphorylation state with alterations in protein activity.
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Anti-Phosphotyrosine was generated from a panel of phosphotyrosine-containing peptide immunogens designed from human protein sequences. All peptide sequences used are highly conserved phosphotyrosine motifs.
Western blot of HeLa cells treated with pervanadate (1 mM) for 30 min. Phosphotyrosine containing proteins were immunoprecipitated with rabbit polyclonal anti-Phosphotyrosine:Agarose (Lane 1) or with no antibody agarose beads (Lane 2), and blots were made that included the whole lysate (Lane 3). The blots were probed with mouse monoclonal anti-Phosphotyrosine (PM3751) to detect phosphotyrosine containing proteins.
Bar graph showing rabbit polyclonal anti-Phosphotyrosine (PP2221) binding to a variety of phosphotyrosine containing peptides, but no binding to unphosphorylated peptide (beta-Catenin (a.a. 29-45).
Agarose-conjugated rabbit polyclonal, affinity-purified antibody is supplied as 50% slurry of antibody:agarose and phosphate-buffered saline with 0.05% sodium azide. Store at 4°C. Stable for 1 year.
The products are are safely shipped at ambient temperature for both domestic and international shipments. Each product is guaranteed to match the specifications as indicated on the corresponding technical data sheet. Please store at -20C upon arrival for long term storage.
This agarose-conjugated antibody can be used to immunoprecipitate or affinity purify tyrosine phosphorylated proteins from cell and tissue lysates. The conjugate has been shown to immunoprecipitate tyrosine phosphorylated proteins from EGF treated A431 cells, as well as pervanadate treated HeLa, Jurkat, and endothelial cells. For initial immunoprecipitation tests, 25-50 μl of the conjugate is recommended per 500 of lysate. Each user should determine the optimal volume for their experimental model.
*All molecular weights (MW) are confirmed by comparison to Bio-Rad Rainbow Markers and to western blot
mobilities of known proteins with similar MW.