Development and precise characterization of phospho-site-specific antibody of Ser357 of IRS-1: Elimination of cross reactivity with adjacent Ser358

Antibodies that recognize specifically phosphorylated sites on proteins are widely utilized for studying the regulation and biological function of phosphoproteins. The proposed strategy is a powerful, analytical tool allowing the generation of phospho-site specific antibodies albeit adjacent phosphorylation sites are present. Here, we demonstrate the assessment and elimination of cross reactivity of phospho-site-specific-Ser357 IRS-1 antibody. While determining the specificity of p-Ser357 antiserum we came across the cross reactivity of the antiserum with adjacent Ser358 which was successfully abolished by an improved immuno-purification method. The specificity of the purified antiserum was then verified by indirect ELISA, results of ELISA were also mirrored in the experiments carried out in BHK-IR cells using different mutants of IRS-1 carrying mutations at either Ser357/Ser358/Ser357/358. Immuno-purified-p-Ser357 did not react with IRS-1 Ala357 and IRS-1 Ala357/358. In conclusion, the present study describes generation and characterization of p-Ser357 IRS-1 antibody, which reacts with IRS-1 in site specific and phosphorylation state-dependent manner without showing cross reactivity to adjacent Ser358. This antibody can be effectively used to further clarify the inhibitory role of Ser357 in insulin signal transduction.