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Western blotting (WB) is used to analyze a target protein in a mixed sample with numerous proteins according to the specificity of antibody against its antigen. With SDS-PAGE’s high resolution and solid-phase immunoassay’s high sensitivity and specificity, western blotting has been one of the most common methods for protein analysis. SAB follows a stringent validation protocol and uses several validation approaches in western blotting to ensure antibody quality and phospho-specificity.
Validation Approaches Include:
Phosphatase treatment
Stimulus or inhibitor treatment
Blocking peptide control
Validation by various cell lines
Phosphatase treatment
Western blot analysis of extracts from Hela cells, treated with IFNa or calf intestinal phosphatase (CIP), using PTEN (Phospho-Ser380/Thr382/Thr383) Antibody #
11056.
Stimulus or inhibitor treatment
Western blot analysis of extracts from SK-BR-3 cells, untreated or insulin and EGF treated, and pretreated with U0126 and LY294002, using p44/42 MAP Kinase (Phospho-Tyr204) Antibody #
11246 (upper) or p44/42 MAP Kinase Antibody #
21238 (lower) .
Fluorescent Western Blotting
Western blot analysis of extracts from 293 cells, treated with Hydroxyurea or calf intestinal phosphatase
(CIP), using CDC2 (Phospho-Tyr15) Antibody . #
11244.
Blocking peptide control
Western blot analysis of extracts from Hela cells using p53 (Phospho-Ser315) Antibody #
11100 (Lane 2) and the same antibody preincubated with blocking peptide (Lane1).
Validation by various cell lines
Western blot analysis of extracts from various cells using PKM2 Antibody #
21578.
