Anti-Hu CD3 zeta (pY111) Purified

• Catalog Number: orb222767
• Category: Antibodies
• Description: Mouse monoclonal antibody to CD3 zeta (Phospho-Tyr111)
• Clonality: Monoclonal
• Clone Number: EM-55
• Tested applications: FC, WB
• Reactivity: Human, Mouse
• Isotype: Mouse IgG1
• Immunogen: BP1 (pTyr111/123) - KLH
• Antibody Type: Primary Antibody
• Concentration: 1 mg/ml
• Dilution range: Flow cytometry: Intracellular staining; recommended dilution: 2-6 μg/ml. Western blotting: Recommended dilution: 1 µg/ml; non-reducing conditions.
• Purity: Purified by protein-A affinity chromatography.
• Conjugation: Unconjugated
• Target: CD3 zeta (pY111)
• Entrez: 919
• UniProt ID: P20963
• Storage: Maintain refrigerated at 2-8°C for up to 2 weeks. For long term storage store at -20°C in small aliquots to prevent freeze-thaw cycles.
• Buffer/Preservatives: Phosphate buffered saline (PBS), pH 7.4, 15 mM sodium azide
• Alternative names: anti CD 3 antibody, anti Cd247 antibody, anti CD24
• Note: For research use only
• Application notes: Flow cytometry: Intracellular staining; recommended dilution: 2-6 μg/ml.Western blotting: Recommended dilution: 1 µg/ml; non-reducing conditions.
• Expiration Date: 12 months from date of receipt.

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 with mouse mAb anti-pY111 mCD3zeta (clone EM-55). 1: Wt mCD3zeta pervanadate non-stimulated; 2: Wt mCD3zeta pervanadate stimulated; 3: mut. mCD3zeta Y/F2-6; 4: mut. mCD3zeta Y/F1 and 3-6; 5: mut. mCD3zeta Y/F1-2 and 4-6 (thus only pY111 remains native); 6: mut. mCD3zeta Y/F1-3 and 5-6; 7: mut. mCD3zeta Y/F1-4 and 6; 8: mut. mCD3zeta Y/F1-5.

Anti-Hu CD3 zeta (pY111) purified antibody (clone EM-55) 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-Hu CD3 zeta (pY111) purified antibody (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 isotype control PE (MOPC-21) under same conditions (concentration in sample 1 µg/ml, black-dashed histogram).

Anti-Hu CD3 zeta (pY111) Purified (clone EM-55) 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.