Western blot analysis of extracts from serum-starved Jurkat cells, untreated (-) or treated with either H2O2 (11 mM, 1 min; +) or with cross-linked anti-CD3 plus anti-CD28 (10 μg/mL each, 5 min; +), using Phospho-CD3ζ (Tyr142) Antibody (upper) and CD3ζ Antibody (lower).
Western blot analysis of extracts from serum-starved Jurkat cells, treated with either isotype control (-) or with cross-linked anti-CD3 plus anti-CD28 (10 ug/mL each, 5 min; +), using Phospho-CD3ζ (Tyr142) Antibody. The phospho-specificity of the antibody was verified by pre-incubating the antibody with no peptide (left), CD3ζ (Tyr142) non-phosphopeptide (middle), and CD3ζ (Tyr142) phosphopeptide (right).
Western blot analysis of extracts from serum-starved Jurkat cells, untreated (-) or treated with H2O2 (11 mM, 1 min; +), using Phospho-CD3ζ (Tyr142) Antibody (upper) and CD3ζ Antibody (lower). H2O2-treated lysates were treated with calf intestinal phosphatase and Lambda phosphatase (CIP/λ phosphatase; +) as indicated.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
Phospho-CD3ζ (Tyr142) Antibody recognizes endogenous levels of CD3ζ protein only when phosphorylated at Tyr142. This antibody cross-reacts with a 65 kDa phosphoprotein of unknown identity.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr142 of human CD3ζ protein. Antibodies are purified by peptide affinity chromatography.
When T cells encounter antigens via the T cell receptor (TCR), information about the quantity and quality of antigens is relayed to the intracellular signal transduction machinery (1). This activation process depends mainly on CD3 (Cluster of Differentiation 3), a multiunit protein complex that directly associates with the TCR. CD3 is composed of four polypeptides: ζ, γ, ε and δ. Each of these polypeptides contains at least one immunoreceptor tyrosine-based activation motif (ITAM) (2). Engagement of TCR complex with foreign antigens induces tyrosine phosphorylation in the ITAM motifs and phosphorylated ITAMs function as docking sites for signaling molecules such as ZAP-70 and p85 subunit of PI-3 kinase (3,4). TCR ligation also induces a conformational change in CD3ε, such that a proline region is exposed and then associates with the adaptor protein Nck (5).
The CD3ζ invariant chain is a type-I transmembrane protein that exists in the TCR signaling complex as a disulfide-linked homodimer (6). The cytoplasmic tail of each CD3ζ monomer contains three distinct ITAM motifs, each containing two tyrosine residues. Phosphorylation of CD3ζ ITAM tyrosine residues, including Y142, is driven by recruitment of the Lck and Fyn tyrosine kinases to the TCR (7). Lck/Fyn-mediated ITAM phosphorylation creates docking sites that promote the SH2 domain-dependent recruitment and activation of ZAP-70 (8-10), which drives amplification of signaling events downstream of the TCR that facilitate T cell activation (10). Phosphorylation of a pool of p16 CD3ζ leads to the generation of p21 and p23 species, which differ in the degree of ITAM phosphorylation. It has been proposed that the ratio of p21/p23 contributes to regulating the amplitude of T cell activation (11). CD3ζ plays an important role in the assembly and surface expression of the TCR complex. Indeed, research studies have demonstrated that CD3ζ is degraded in response to Ag-dependent TCR stimulation as a mechanism to tightly control T cell activation (12).
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