Lck [pS158] Antibody

Lck [pS158] Antibody | catalog# XBP-4159

Background

Lck (p56lck), a member of the Src family of non-receptor tyrosine protein kinases, is expressed predominantly in T cells. Lck function is critical both for T cell development in the thymus and activation of mature T cells in the periphery by antigen. The activity of Lck is known to be regulated by phosphorylation of two conserved tyrosine residues, tyrosine 505 (equivalent to tyrosine 529 in c-Src) and tyrosine 394 (equivalent to tyrosine 418 in c-Src). The effects of phosphorylation at serine 158 are still under investigation.

Description

Full length untagged recombinant human Lck protein was added to control cell extract which does not contain Lck and then resolved by SDS-PAGE on a 10% Tris-glycine gel. The proteins then were transferred to PVDF and incubated with 0.50 μg/mL Lck [pS158] antibody, following prior incubation with: (1) no peptide, (2) the non-phosphorylated peptide corresponding to the phosphopeptide, (3) a generic phosphoserine-containing peptide, and (4) the phosphopeptide immunogen. After washing, membranes were incubated with goat F(ab')2 anti-rabbit IgG alkaline phosphatase and bands were detected using the Tropix WesternStar™ detection method.

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Specifications

Formulation

Rabbit polyclonal immunoglobulin in phosphate buffer, pH 7.4, with 1.0 mg/mL BSA (IgG, protease-free) as a carrier.

Clonality/Clone

This is a polyclonal antibody.

Specificity

Human Lck. Mouse Lck [pS158] has not been tested.

Source

Lck antibody was produced against a chemically synthesized phosphopeptide derived from the region of Lck that contains serine 158 (based on Swiss Protein database, accession number P06239). The sequence is conserved in human and mouse.

Purification

Lck antibody was purified from rabbit serum by sequential epitope-specific chromatography. The antibody has been negatively preadsorbed using (i) a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated Lck, and (ii) a generic serine phosphorylated peptide to remove antibody that is reactive with phosphoserine (irrespective of the sequence). The final product is generated by affinity chromatography using a Lck-derived peptide that is phosphorylated at serine 158.

Host

Lck [pS158] antibody was raised in rabbit.

Please use anti-rabbit secondary antibodies.

Application

For Western blot applications, we recommend using the antibody at 0.1-1.0 μg/mL. At 0.50 μg/mL, the dilution provides 100 mL working solution, which at 10 mL/blot allows 10 blots to be performed.

Tested Application

Buffer

Rabbit polyclonal immunoglobulin in phosphate buffer, pH 7.4, with 1.0 mg/mL BSA (IgG, protease-free) as a carrier.

Storage

Store at -80˚C. Upon initial thawing, apportion into working aliquots and Store at -80˚C. Avoid repeated freeze-thaw cycles to prevent denaturing the antibody.

Positive Control

Full length untagged recombinant human Lck protein.]

Species Reactivity

Reference

Fujimaki, W., et al. (2001) Functional uncoupling of TCR engagement and Lck activation in anergic human thymic CD4+ T cells. J. Biol. Chem. 276(20):17455-17460.
Ellery, J.M., et al. (2000) Activation of the interleukin 2 receptor: a possible role for tyrosine phosphatases. Cell Signal 12(6):367-373.
Gervais, F.G. and A. Veillette (1997) Reconstitution of interactions between protein-tyrosine phosphatase CD45 and tyrosine-protein kinase p56(lck) in nonlymphoid cells. J. Biol. Chem. 272(19):12754-12761.
Soula, M., et al. (1993) Anti-CD3 and phorbol ester induce distinct phosphorylated sites in the SH2 domain of p56lck. J. Biol. Chem. 268(36):27420-27427.]