Development and precise characterization of phospho-site-specific antibody of Ser(357) of IRS-1: elimination of cross reactivity with adjacent Ser(358) |
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Authors: | Waraich Rizwana Sanaullah Zaidi Nousheen Moeschel Klaus Beck Alexander Weigert Cora Voelter Wolfgang Kalbacher Hubert Lehmann Rainer |
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Affiliation: | a Department of Internal Medicine IV, Endocrinology, Metabolism, Pathobiochemistry and Clinical Chemistry, University Hospital Tuebingen, Germany b Medical and Natural Sciences, Research Centre, University of Tubingen, Ob dem Himmelreich 7, 72074 Tuebingen, Germany c Interfacultary Institute of Biochemistry, University of Tuebingen, Germany |
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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-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. |
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Keywords: | IRS-1, insulin receptor substrate-1 PTB, phosphotyrosine binding PKC, protein kinase C HPLC, high-performance liquid chromatography BHKIR, baby hamster kidney cells stably expressing the human insulin receptor GST, glutathione S-transferase p-Ser, phospho-serine |
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