title: Development and precise characterization of phospho-site-specific antibody of Ser(357) of IRS-1: elimination of cross reactivity with adjacent Ser(358).
abstract: 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-Ser(357) IRS-1 antibody. While determining the specificity of p-Ser(357) antiserum we came across the cross reactivity of the antiserum with adjacent Ser(358) 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 Ser(357)/Ser(358)/Ser(357/358). Immuno-purified-p-Ser(357) did not react with IRS-1 Ala(357) and IRS-1 Ala(357/358). In conclusion, the present study describes generation and characterization of p-Ser(357) IRS-1 antibody, which reacts with IRS-1 in site specific and phosphorylation state-dependent manner without showing cross reactivity to adjacent Ser(358). This antibody can be effectively used to further clarify the inhibitory role of Ser(357) in insulin signal transduction.
authors: Department of Internal Medicine IV, Endocrinology, Metabolism, Pathobiochemistry and Clinical Chemistry, University Hospital Tuebingen, Germany.
authors: Rizwana Sanaullah
authors: RS
authors: Waraich
authors: Nousheen
authors: N
authors: Zaidi
authors: Klaus
authors: K
authors: Moeschel
authors: Alexander
authors: A
authors: Beck
authors: Cora
authors: C
authors: Weigert
authors: Wolfgang
authors: W
authors: Voelter
authors: Hubert
authors: H
authors: Kalbacher
authors: Rainer
authors: R
authors: Lehmann
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D045163:Antibodies, Phospho-Specific / Q000276:immunology*; D000918:Antibody Specificity; D002460:Cell Line; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D051381:Rats; D012694:Serine / Q000235:genetics / Q000276:immunology*; D044967:Serum / Q000276:immunology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D045163:Antibodies, Phospho-Specific; C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; D012694:Serine