Anti-CD3 zeta (phospho Y111) Antibody [EM-55] (A86526)

Western blotting analysis of cell lyzates of HEK293T/17 cells transiently transfected with expression vectors harboring genes for mCD3zeta wild type and six different mCD3zeta mutants, where particular ITAM tyrosines were substituted with phenylalanines. The lysates (non-reducing conditions) were immunoblotted using Anti-CD3 zeta (phospho Y111) Antibody [EM-55] (A86526). 1: Wt mCD3zeta pervanadate non-stimulated2: Wt mCD3zeta pervanadate stimulated3: mut. mCD3zeta Y/F2-64: mut. mCD3zeta Y/F1 and 3-65: mut. mCD3zeta Y/F1-2 and 4-6 (thus only pY111 remains native)6: mut. mCD3zeta Y/F1-3 and 5-67: mut. mCD3zeta Y/F1-4 and 6 8: mut. mCD3zeta Y/F1-5

Anti-CD3 zeta (phospho Y111) Antibody [EM-55] (A86526) works in Flow Cytometry application. Analysis of the antibody staining was performed on Jurkat cells treated or untreated with pervanadate (PV) prior to the fixation and permeabilization of cell suspension with cold methanol. Anti-CD3 zeta (phospho Y111) Antibody [EM-55] (A86526), (concentration in sample 1 µg/ml, red-filled histogram) binds specifically to phosphorylated tyrosine 111 (pY111) of CD3 zeta chain in PV treated, methanol permeabilized Jurkat cells (upper panel), but not to untreated methanol permeabilized control cells (lower panel). Level of non-specific binding was assessed using Mouse IgG1 kappa Isotype Control [MOPC-21] (PE) (A86199) under same conditions (concentration in sample 1 µg/ml, black-dashed histogram).

Anti-CD3 zeta (phospho Y111) Antibody [EM-55] (A86526) specificity verification by WB. The specificity of EM-55 antibody to phosphorylated Tyr 111 (CD3 zeta chain) was assessed by analysis of binding signals in HEK293T transfected with CD3 zeta/ZAP-70 construct followed by pervanadate (PV) treatment in comparison to the series of control cells - PV untreated transfectants, and both PV treated and untreated mock HEK293T cells. Western blotting analysis was performed on whole cell extracts (RIPA lysis buffer with PhosSTOP and pervanadate), mixed and heated (100°C, 5 min) with non-reducing SDS-loading buffer. Samples were resolved using 15% Tris-glycine SDS gel electrophoresis. Nitrocellulose membrane blot was probed with mouse IgG1 monoclonal antibody EM-55 (1 µg/ml). Subclass-specific secondary antibody IRDye 680LT Goat-anti-Mouse IgG (red) was used for fluorescent Western blot detection.