Purified from rabbit serum by sequential epitope-specific chromatography. The antibody has been negatively preadsorbed using i) a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated paxillin, and ii) a generic tyrosine phosphorylated peptide to remove antibody that is reactive with phosphotyrosine, irrespective of the sequence. The final product is generated by affinity chromatography using a paxillin-derived peptide that is phosphorylated at tyrosine 31.
Western blot - Anti-Paxillin (phospho Y31) antibody (ab4832)
Extracts prepared from NMµMG cells transfected with either the wild-type EGFP-tagged paxillin or a site directed mutant, in which tyrosine 31 was changed to phenylalanine, were resolved on a 10% Tris-glycine gel and transferred to nitrocellulose. Membranes were blocked with 1% BSA, followed by incubation with 0.5 µg/mL anti-paxillin [pY31] antibody. After washing, membranes were incubated with goat F(ab')2 anti-rabbit IgG alkaline phosphatase and the signal was detected by chemiluminescence using the Tropix WesternStar detection method and Kodak BioMax ultraclear film. The data show detection of paxillin phosphorylation with the wild-type but not the Y31F mutant recombinant proteins. Note that the recombinant EGFP-tagged paxillin proteins run at a higher Mr as opposed to the endogenous protein and that in both cell types that the antibody recognizes the endogenous wild-type paxillin protein, which migrates at the expected Mr of 68 kDa. Ext