کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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1934287 | 1050637 | 2008 | 6 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Development and precise characterization of phospho-site-specific antibody of Ser357 of IRS-1: Elimination of cross reactivity with adjacent Ser358 Development and precise characterization of phospho-site-specific antibody of Ser357 of IRS-1: Elimination of cross reactivity with adjacent Ser358](/preview/png/1934287.png)
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.
Journal: Biochemical and Biophysical Research Communications - Volume 376, Issue 1, 7 November 2008, Pages 26–31