Phospho-Lck (Tyr505) Antibody [P3K14]

Application: Reactivity: Human, Mouse, Rat

Lane 1: Jurkat, Lane 2: Jurkat (phosphatase treated)

Usage Information

Dilution
WB1:1000

Application
WB

Reactivity
Human, Mouse, Rat

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW
56 kDa

Datasheet & SDS

Biological Description

Specificity
Phospho-Lck (Tyr505) Antibody [P3K14] detects endogenous levels of total Lck protein only when it is phosphorylated at Tyr505.

Clone
P3K14

Synonym(s)
Tyrosine-protein kinase Lck; Leukocyte C-terminal Src kinase; LSK; Lymphocyte-specific protein tyrosine kinase; Proto-oncogene Lck; T cell-specific protein-tyrosine kinase; p56-LCK; LCK

Background
Phospho-Lck at Tyr505 represents the inactive form of Lck, a Src family tyrosine kinase essential for initiating T cell receptor signaling in T lymphocytes. Lck possesses N-terminal myristoylation and palmitoylation sites that anchor it to the plasma membrane and Golgi apparatus, SH3 and SH2 domains for protein interactions, and a kinase domain flanked by regulatory tyrosines Tyr394 and Tyr505 in the activation loop and C-terminal tail, respectively. Phosphorylation at Tyr505 by C-terminal Src kinase (Csk) induces intramolecular binding of the phosphotyrosine tail to the SH2 domain, clamping the kinase into a closed conformation that inhibits catalytic activity and substrate access, maintaining Lck in a primed but inactive state in resting T cells. Dephosphorylation of Tyr505 by CD45 phosphatase exposes the activation loop, permitting trans-autophosphorylation at Tyr394 that stabilizes an open conformation and enhances kinase activity toward immunoreceptor tyrosine-based activation motifs (ITAMs) on CD3ζ chains upon TCR engagement. Activated Lck then recruits and phosphorylates Zap-70 via its tandem SH2 domains on phospho-ITAMs, propagating signals through LAT adaptor phosphorylation to assemble multimolecular complexes that activate phospholipase Cγ1, calcium fluxes, ERK, NF-κB, and NFAT pathways, driving cytokine production, proliferation, and differentiation. Tyr505 phosphorylation predominates in unstimulated states, with TCR stimulation triggering localized dephosphorylation and Tyr394 phosphorylation primarily at the plasma membrane near engaged TCR clusters, where about 20% of Lck molecules undergo conformational opening.

Background

Phospho-Lck at Tyr505 represents the inactive form of Lck, a Src family tyrosine kinase essential for initiating T cell receptor signaling in T lymphocytes. Lck possesses N-terminal myristoylation and palmitoylation sites that anchor it to the plasma membrane and Golgi apparatus, SH3 and SH2 domains for protein interactions, and a kinase domain flanked by regulatory tyrosines Tyr394 and Tyr505 in the activation loop and C-terminal tail, respectively. Phosphorylation at Tyr505 by C-terminal Src kinase (Csk) induces intramolecular binding of the phosphotyrosine tail to the SH2 domain, clamping the kinase into a closed conformation that inhibits catalytic activity and substrate access, maintaining Lck in a primed but inactive state in resting T cells. Dephosphorylation of Tyr505 by CD45 phosphatase exposes the activation loop, permitting trans-autophosphorylation at Tyr394 that stabilizes an open conformation and enhances kinase activity toward immunoreceptor tyrosine-based activation motifs (ITAMs) on CD3ζ chains upon TCR engagement. Activated Lck then recruits and phosphorylates Zap-70 via its tandem SH2 domains on phospho-ITAMs, propagating signals through LAT adaptor phosphorylation to assemble multimolecular complexes that activate phospholipase Cγ1, calcium fluxes, ERK, NF-κB, and NFAT pathways, driving cytokine production, proliferation, and differentiation. Tyr505 phosphorylation predominates in unstimulated states, with TCR stimulation triggering localized dephosphorylation and Tyr394 phosphorylation primarily at the plasma membrane near engaged TCR clusters, where about 20% of Lck molecules undergo conformational opening.

References
https://pubmed.ncbi.nlm.nih.gov/29262519/ https://pubmed.ncbi.nlm.nih.gov/28096507/

Reference Table for Selecting PVDF Membrane Pore Size Specifications

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%