Antibody data
- Antibody Data
- Antigen structure
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- Comments [0]
- Validations
- Western blot [1]
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Validation data
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- Product number
- GTX25796 - Provider product page
- Provider
- GeneTex
- Proper citation
- GeneTex Cat#GTX25796, RRID:AB_380691
- Product name
- PKC gamma (phospho Thr655) antibody
- Antibody type
- Polyclonal
- Reactivity
- Human, Mouse
- Host
- Rabbit
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Supportive validation
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- GeneTex (provider)
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- Experimental details
- Lysates prepared from HeLa cells stimulated with PMA were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-11) or treated with lambda phosphatase (12), blocked with a 3% low-fat milk-TBST buffer overnight at 4¢XC, and incubated with 0.50 ?g/ml PKCgamma [pT655] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 11, 12), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphothreonine containing peptide (3), the phosphopeptide immunogen (4), or, the phosphopeptide corresponding to the immunogen from other PKC isoforms (5-10). After washing, membranes were incubated with goat F(ab)2 anti-rabbit IgG alkaline phosphatase and signals were detected. The data show that the peptide corresponding to PKCgamma [pT655] blocks the antibody signal while the peptides corresponding to PKC isoforms betaI [pT642], delta [pS645], epsilon [pT710] and eta [pT655] do not completely block the antibody signal, thereby demonstrating the specificity of the antibody. However, peptides corresponding to PKC isoforms alpha [pT638] and betaII [pT641] do block the antibody signal, indicating that the PKCgamma [pT655] antibody may cross-react with their respective sites. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.