CD3 zeta antibody

• Catalog Number: orb179875
• Category: Antibodies
• Description: Mouse monoclonal antibody to CD3 zeta
• Clonality: Monoclonal
• Clone Number: EM-17
• Tested applications: FC, WB
• Reactivity: Human, Mouse
• Isotype: Mouse IgG1
• Immunogen: A phospho specific peptide corresponding to the amino acids surrounding tyrosine 153 of mouse CD3 zeta linked to KLH
• Concentration: 1 mg/ml
• Dilution range: Western blotting: Recommended dilution: 2 - 5 μg/ml; positive control: Jurkat cells lysate treated with pervanadate, splenocyte lysate of Balb/c or F1 mouse treated with pervanadate, non-reducing conditions recommended. Flow cytometry: Intracellular staining; recommended dilution: 1-4 μg/ml; positive control: Jurkat cells treated with pervanadate.
• Purity: Purified by protein-A affinity chromatography.
• Conjugation: Unconjugated
• Target: CD3 zeta (pY153)
• Entrez: 919
• UniProt ID: P20963
• Storage: Store at 2-8°C. Do not freeze.
• 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: Western blotting: Recommended dilution: 2 - 5 μg/ml; positive control: Jurkat cells lysate treated with pervanadate, splenocyte lysate of Balb/c or F1 mouse treated with pervanadate, non-reducing conditions recommended. Flow cytometry: Intracellular staining; recommended dilution: 1-4 μg/ml; positive control: Jurkat cells treated with pervanadate.
• Expiration Date: 12 months from date of receipt.

Western blotting analysis using monoclonal antibodies EM-26 (anti-CD3 zeta phospho-Tyr72) and EM-17 (anti-CD3 zeta phospho-Tyr153) reacting with particular phosphorylated human CD3 zeta mutants. The Y1F and Y6F mutatants lack phosphotyrosine 72 and 153, respectively.

Anti-Hu CD3 zeta (pY153) purified antibody (clone EM-17) 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 (pY153) purified antibody (concentration in sample 1 µg/ml, red-filled histogram) binds specifically to phosphorylated tyrosine 153 (pY153) 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 (pY153) Purified (clone EM-17) specificity verification by WB. The specificity of EM-17 antibody to phosphorylated Tyr 153 (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-17 (1 µg/ml). Subclass-specific secondary antibody IRDye 680LT Goat-anti-Mouse IgG (red) was used for fluorescent Western blot detection.