Development and precise characterization of phospho-site-specific antibody of Ser(357) of IRS-1: elimination of cross reactivity with adjacent Ser(358)

Biochemical and Biophysical Research Communications
Rizwana Sanaullah WaraichRainer Lehmann

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). Th...Continue Reading

References

Aug 10, 2002·American Journal of Physiology. Endocrinology and Metabolism·Morris F White
Jun 8, 2004·Archives of Pharmacal Research·Yong Hee Lee, Morris F White
Mar 1, 2005·Biochimie·Philippe GualJean-François Tanti
May 25, 2007·The FEBS Journal·Nousheen ZaidiHubert Kalbacher
Jun 20, 2007·Biochemical and Biophysical Research Communications·Nousheen ZaidiHubert Kalbacher

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