CD3 zeta (pY153) Antibody

SKU: orb179875

Description

Mouse monoclonal antibody to CD3 zeta

Images & Validation

• Tested Applications: FC, WB
• Reactivity: Human, Mouse
• 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.

Key Properties

• Antibody Type: Primary Antibody
• Clonality: Monoclonal
• Isotype: Mouse IgG1
• Clone No.: EM-17
• Immunogen: A phospho specific peptide corresponding to the amino acids surrounding tyrosine 153 of mouse CD3 zeta linked to KLH
• Target: CD3 zeta (pY153)
• Purification: Purified by protein-A affinity chromatography.
• Conjugation: Unconjugated

Storage & Handling

• 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
• Concentration: 1 mg/ml
• Disclaimer: For research use only

Alternative Names

CD3 zeta chain, CD247, T3Z, TCRz, IMD25

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.

Quick Database Links

Gene Symbol

CD3 zeta (pY153)

UniProt

P20963

NCBI Gene ID

919