Exbio — Research products — Antibodies — CD and related antigens — Anti-Hu CD3 zeta (pY111) Purified

Anti-Hu CD3 zeta (pY111) Purified

Regulatory status

RUO

Antigen

CD3 zeta (pY111)

Clone

EM-55

Format

Purified

Reactivity

Mouse, Human

Application

WB, FC (QC tested)

Variant

0.1 mg

11-737-C100

In stock

147.40 USD

0.025 mg

11-737-C025

Delivery 1 week

73.70 USD

Variant

0.1 mg

11-737-C100

In stock

147.40 USD

0.025 mg

11-737-C025

Delivery 1 week

73.70 USD

Contact distributor

Product details

Description

Images

SDS download

Isotype

Mouse IgG1

Specificity

The mouse monoclonal antibody EM-55 recognizes phosphorylated intracellular tyrosine 111 of CD3 zeta chain (CD247), which is a component of TCR/CD3 complex expressed on T cells.

Application

WB, FC (QC tested)

Application details

Flow cytometry: Intracellular staining; recommended dilution: 2-6 μg/ml.
Western blotting: Recommended dilution: 1 µg/ml; non-reducing conditions.

Reactivity

Mouse, Human

Immunogen

BP1 (pTyr111/123) - KLH

Concentration

1 mg/ml

Preparation

Purified by protein-A affinity chromatography.

Formulation

Phosphate buffered saline (PBS), pH 7.4, 15 mM sodium azide

Storage and handling

Store at 2-8°C. Do not freeze.

The product is intended For Research Use Only. Diagnostic or therapeutic applications are strictly forbidden. Products shall not be used for resale or transfer to third parties either as a stand-alone product or as a manufacture component of another product without written consent of EXBIO Praha, a.s. EXBIO Praha, a.s. will not be held responsible for patent infringement or any other violations of intellectual property rights that may occur with the use of the products. Orders for all products are accepted subject to the Term and Conditions available at www.exbio.cz. EXBIO, EXBIO Logo, and all other trademarks are property of EXBIO Praha, a.s.

Other names

CD3 zeta chain, CD247, T3Z, TCRz, IMD25

Antigen description

CD3 complex is crucial in transducing antigen-recognition signals into the cytoplasm of T cells and in regulating the cell surface expression of the TCR complex. T cell activation through the antigen receptor (TCR) involves the cytoplasmic tails of the CD3 subunits CD3 gamma, CD3 delta, CD3 epsilon and CD3 zeta (CD247). These CD3 subunits are structurally related members of the immunoglobulins super family encoded by closely linked genes on human chromosome 11. The CD3 components have long cytoplasmic tails that associate with cytoplasmic signal transduction molecules. This association is mediated at least in part by a double tyrosine-based motif present in a single copy in the CD3 subunits. CD3 may play a role in TCR-induced growth arrest, cell survival and proliferation.

Entrez Gene ID 919

UniProt ID P20963

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, GAM APC, 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 purified (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% SDS-PAGE gel.

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.