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Validation of antibody specificity using RNA Interference
Author(s) -
Sullivan Yvonne B.,
Narahari Janaki,
Hughes Douglas E.,
Webb Brian L.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1070.25
Subject(s) - antibody , western blot , gene knockdown , blot , microbiology and biotechnology , rna interference , small interfering rna , transfection , immunoprecipitation , chemistry , biology , rna , immunology , biochemistry , gene
The utility of an antibody as a research tool is dependant on the antibody recognizing the proper protein target. Most often antibody function is determined by Western blot analysis, with recognition of a band of the appropriate molecular weight denoting proper function. In this study we used siRNA‐mediated protein knockdown to evaluate antibody specificity. Lysates from siRNA‐transfected cells were probed using antibodies from Thermo Scientific SuperSignal® siRNA/Antibody kits as well as other commercially available antibodies. Interestingly, we observed for several targets that some commercially available antibodies recognized a band of the appropriate molecular weight that was not the correct protein, as judged by lack of siRNA‐mediated protein knockdown. Other commercially available antibodies required considerable Western blot optimization. The antibodies in the Thermo Scientific SuperSignal® siRNA/Antibody Modules all detected a protein band that was specifically reduced with corresponding siRNA treatment. These studies highlight the value of siRNA‐validation of antibodies for Western blotting and other protein detection applications.