Production and Characterization of Polyclonal Generic Phosphotyrosine‐specific Antibodies

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Reversible protein‐tyrosine phosphorylation plays critical roles in cell regulation under normal and pathological conditions, which has made generic phosphotyrosine (pY) antibodies invaluable tools for biomedical research. A recent study that compared the specificities of three widely‐used monoclonal pY antibodies has raised concerns about strong sequence motif selectivity and low overall coverage rates. These issues can potentially introduce significant bias in pY site identification and quantification. In this study we describe a novel strategy of generating a pool of polyclonal antibodies from a large set of physiological pY‐site sequences. Over 400 peptides spanning 7 to 20 amino acids in length, each featuring 1 to 7 phosphorylation sites and representing over 1000 phosphosites, were used to immunize over 100 rabbits. The sera from these rabbits were subjected to ammonium sulphate fractionation, pooled, and then affinity‐purified on pY‐agarose columns to produce the PYK antibody preparations. The specificity of the purified PYK antibody was assessed with phosphopeptide microarrays, including the Jerini Peptide Technologies Phosphatase Peptide Microarray, which features 6,099 peptides representing diverse human pY‐sites. The rabbit polyclonal PYK phosphotyrosine antibody proved to perform better than the well known 4G10, PY20 and PY100 mouse monoclonal antibodies. PYK was extremely stable to repeated freeze thaw, and found to be 4‐fold or more sensitive for detection of phosphopeptides on arrays and for proteins following Western blotting of EGF‐treated A431 cell lysates than the monoclonal antibodies. Like the other monoclonal pY antibodies, PYK reactivities with phosphothreonine and phosphoserine were over 200‐ and 300‐fold less, respectively, when compared to phosphotyrosine on phosphopeptide microarrays. PYK also detected a larger number of diverse pY‐containing peptides than 4G10, PY20 and PY100 on phosphopeptide microarrays, and unlike these other antibodies, acidic amino acid residues surrounding the pY‐sites were not negative determinants for antibody recognition. Since acidic amino acids are important positive determinants for substrate recognition for most protein‐tyrosine kinases and flank most protein‐tyrosine phosphosites, our findings indicate that the PYK antibody may be more useful for enrichment and analysis of physiological pY‐sites than the commonly used mouse monoclonal antibodies for such purposes. Further purification of the PYK antibody over an IgG‐agarose column also allowed development of preparation of a reporter antibody that was suitable for use in sandwich antibody microarrays to assess changes in the tyrosine phosphorylation status of over 500 signaling proteins simultaneously with as little as 50 μg of crude lysate protein from cells and tissues.Support or Funding InformationSupported by Kinexus Bioinformatics Corporation.