Peptide Competition and Phosphatase Treatment. Lysates prepared from Jurkat cells left unstimulated (1) or stimulated with H2O2 (2-6) and mouse OKT-5 lymphocytes left unstimulated (7) or stimulated with H2O2 (8) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (Lanes 1-5, 7 and 8) or treated with L-phosphatase (6), blocked with a 3% Milk-TBST buffer for one hour at room temperature, and incubated with Syk [pY317] antibody for two hours at room temperature in a 3% Milk-TBST buffer, following prior incubation with: no peptide (1, 2, 6), the non-phosphopeptide corresponding to the immunogen (3), a generic phosphotyrosine-containing peptide (4), or, the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F(ab and quote;)2 anti-rabbit IgG HRP conjugate (Cat. no. ALI4404) and bands were detected using the Pierce SuperSignal™ method. The data show that the signal was induced upon H2O2 treatment. The data also show peptide corresponding to Syk [pY317] blocks the antibody signal, and that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.
|Tested species reactivity||Human, Mouse|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antiserum was produced against a chemically synthesized phosphopeptide derived from a region of mouse Syk that contains tyrosine 317 (tyrosine 323 in the human sequence).|
|Purification||Antigen affinity chromatography|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 1mg/ml BSA, 50% glycerol|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Western Blot (WB)||Assay Dependent|
* 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.
SYK is a positive effector of BCR stimulated responses. It couples the B cell antigen receptor (BCR) to the mobilization of calcium ions either through a phosphoinositide 3 kinase dependent pathway, when not phosphorylated on tyrosines of the linker region, or through a phospholipase C gamma dependent pathway, when phosphorylated on Tyr 342 and Tyr 346. Thus the differential phosphorylation of SYK can determine the pathway by which BCR is coupled to the regulation of intracellular calcium ions.
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