Peptide Competition and Phosphatase Treatment. Lysates prepared from Jurkat cells stimulated with PMA were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-9) or treated with lambda phosphatase (10), blocked with a 5% BSA-TBST buffer overnight at 4°C, and incubated with 1:2000 dilution of Phospho-PKCeta (Thr655) antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 10), 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 (5-9). After washing, membranes were incubated with goat F(ab and quote;)2 anti-rabbit IgG HRP conjugate (Product # ALI4404) and bands were detected using the Pierce SuperSignal™ method. The data show that the peptide corresponding to PKCeta [pT655] blocks the antibody signal. The antibody signal was not blocked by the peptides corresponding to PKC isoforms phospho-alpha (Thr638), phospho-betaI (Thr642), phospho-betaI (ThrT641), phospho-gamma (Th655) and zeta (Thr560), thereby demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.
|Tested species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antiserum was produced against a chemically synthesized phosphopeptide derived from a region of human PKC-eta that contains threonine 655.|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:20|
|Western Blot (WB)||1:2000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Protein kinase C (PKC) is a family of serine- and threonine-specific protein kinases that can be activated by calcium and second messenger diacylglycerol. PKC family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. PKC family members also serve as major receptors for phorbol esters, a class of tumor promoters. Each member of the PKC family has a specific expression profile and is believed to play a distinct role in cells.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.