Peptide Competition and Phosphatase Treatment. Lysates prepared from Jurkat cells left untreated (1) or treated with H2O2 (2-6) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-6) or treated with YOP phosphatase (7), blocked with a 5% BSA-TBST buffer for one hour at room temperature, and incubated with ZAP-70 [pYpY315/319] antibody for two hours at room temperature in a 3% BSA-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) the phosphopeptide immunogen (5) or, the phosphopeptide corresponding to ZAP-70 [pY315] (6). 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 only the peptide corresponding to ZAP-70 [pYpY315/319] completely blocks the antibody signal. 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 the region of human ZAP-70 that contains tyrosines 315 and 319. The sequence is conserved in mouse.|
|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.
This gene encodes an enzyme belonging to the protein tyrosine kinase family, and it plays a role in T-cell development and lymphocyte activation. This enzyme, which is phosphorylated on tyrosine residues upon T-cell antigen receptor (TCR) stimulation, functions in the initial step of TCR-mediated signal transduction in combination with the Src family kinases, Lck and Fyn. This enzyme is also essential for thymocyte development. Mutations in this gene cause selective T-cell defect, a severe combined immunodeficiency disease characterized by a selective absence of CD8-positive T-cells. Two transcript variants that encode different isoforms have been found for this gene.
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