Purified anti-Lck Phospho (Tyr394) Antibody

Pricing & Availability
Clone
A18002D (See other available formats)
Regulatory Status
RUO
Other Names
Lsk, p56lck, LCK Proto-Oncogene, Src Family Tyrosine Kinase, Tyrosine-protein kinase LCK, Leukocyte C-terminal Src kinase, LSK, Lymphocyte cell-specific protein-tyrosine kinase, T cell-specific protein-tyrosine kinase
Isotype
Mouse IgG1, κ
A18002D_PURE_Lck-Phospho_Antibody_1_092619
Whole cell extracts (15 µg protein) from serum starved Jurkat cells untreated (-) or treated (+) with 5 mM H2O2 for 3 minutes were resolved on a 4-12% Bis-Tris gel, transferred to a PVDF membrane, and probed with 0.25 µg/mL (1:2500 dilution) of Purified anti-Lck Phospho (Tyr394) (clone A18002D) for 2 hours at room temperature. Proteins were visualized by chemiluminescence detection using HRP Goat anti-mouse IgG Antibody (Cat. No. 405306) at a 1:3000 dilution. Equal Lck loading was confirmed by probing membranes with Purified anti-Lck Antibody, clone LCK-01 (Cat. No. 628302) at a 1:1000 dilution. Lane M: Molecular Weight marker.
  • A18002D_PURE_Lck-Phospho_Antibody_1_092619
    Whole cell extracts (15 µg protein) from serum starved Jurkat cells untreated (-) or treated (+) with 5 mM H2O2 for 3 minutes were resolved on a 4-12% Bis-Tris gel, transferred to a PVDF membrane, and probed with 0.25 µg/mL (1:2500 dilution) of Purified anti-Lck Phospho (Tyr394) (clone A18002D) for 2 hours at room temperature. Proteins were visualized by chemiluminescence detection using HRP Goat anti-mouse IgG Antibody (Cat. No. 405306) at a 1:3000 dilution. Equal Lck loading was confirmed by probing membranes with Purified anti-Lck Antibody, clone LCK-01 (Cat. No. 628302) at a 1:1000 dilution. Lane M: Molecular Weight marker.
  • A18002D_PURE_Lck-Phospho_Antibody_2_092619
    Whole cell extracts (15 µg protein) from serum starved EL4 cells untreated (-) or treated (+) with 5 mM H2O2 for 3 minutes were resolved on a 4-12% Bis-Tris gel, transferred to a PVDF membrane, and probed with 0.25 µg/mL (1:2500 dilution) of Purified anti-Lck Phospho (Tyr394) (clone A18002D) for 2 hours at room temperature. Proteins were visualized by chemiluminescence detection using HRP Goat anti-mouse IgG Antibody (Cat. No. 405306) at a 1:3000 dilution. Equal Lck loading was confirmed by probing membranes with an anti-Lck Antibody. Lane M: Molecular Weight marker.
  • A18002D_PURE_Lck-Phospho_Antibody_3_092619
    Whole cell extracts (15 µg protein) from Jurkat and J.Cam1.6 cells untreated (-) or treated (+) with 1 µM staurosporine for 3 hours were resolved on a 4-12% Bis-Tris gel, transferred to a PVDF membrane, and probed with 0.25 µg/mL (1:2500 dilution) of Purified anti-Lck Phospho (Tyr394) (clone A18002D) for 2 hours at room temperature. Proteins were visualized by chemiluminescence detection using HRP Goat anti-mouse IgG Antibody (Cat. No. 405306) at a 1:3000 dilution. Equal Lck loading was confirmed by probing membranes with an anti-Lck Antibody. Lane M: Molecular Weight marker.
  • A18002D_PURE_Lck-Phospho_Antibody_4_092619
    Jurkat cells untreated (panel A) or treated (panel B) with 5 mM H2O2 for 3 minutes were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with Triton X-100 for 10 minutes, and blocked with 5% FBS for 60 minutes. Cells were then intracellularly stained with 1.0 µg/mL (1:500 dilution) dilution of Purified anti-Lck Phospho (Tyr394) (clone A18002D) for two hours at room temperature, followed by incubation with Alexa Fluor® 594 goat anti-mouse IgG (Cat. No. 405326) at 2.0 µg/mL. Nuclei were counterstained with DAPI, and the image was captured with a 60X objective.
  • A18002D_PURE_Lck-Phospho_Antibody_5_092619
    Human peripheral blood lymphocytes were treated with (left), or without (right) H2O2 for 5 minutes, fixed with Fixation Buffer (Cat. No. 420801), permeabilized with True-Phos™ Perm Buffer (Cat. No. 425401), then intracellularly stained with purified anti-LCK Phospho (Tyr394) (clone A18002D), followed by anti-mouse IgG, κ PE, and then surface stained with CD3 APC.
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933101 25 µg $112.00
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933102 100 µg $265.00
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Description

The Src family tyrosine kinase p56Lck (Lck) is a non-receptor tyrosine kinase that plays a critical role in T cell selection and maturation within the thymus, and also in the function of mature T cells. Lck, which is constitutively bound to cytosolic domains of CD4 and CD8 surface receptors, plays an essential role in T cell receptor (TCR) signaling. Engagement of the TCR with peptide antigen-loaded MHC complexes results in the recruitment of CD4- and CD8-bound Lck to the TCR/CD3 signaling complex. Lck then transphosphorylates TCR-gamma chains and CD3 subunits, thereby activating the TCR/CD3 signaling pathway and leading to the recruitment and subsequent phosphorylation of Zap70 by Lck. Lck also plays an important role in interleukin-2 signaling that regulates the T cell proliferative response. Phosphorylation of Lck by CSK at tyrosine 505 negatively regulates the kinase, and is proposed to generate a closed, inactive conformation of the protein. Conversely, phosphorylation at tyrosine 394 in the activation loop of Lck greatly stimulates enzymatic activity. In many types of cancer, Lck is a proliferative and anti-apoptotic driver, and has been proposed as a target for therapeutic intervention.

Technical data sheet

Product Details

Verified Reactivity
Human, Mouse
Antibody Type
Monoclonal
Host Species
Mouse
Immunogen
Synthetic peptide corresponding to human Src phosphorylated at tyrosine 419
Formulation
Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide.
Preparation
The antibody was purified by affinity chromatography.
Concentration
0.5 mg/mL
Storage & Handling
The antibody solution should be stored undiluted between 2°C and 8°C.
Application

WB - Quality tested
ICC, ICFC - Verified
Recommended Usage

Each lot of this antibody is quality control tested by Western blotting. For Western blotting, the suggested use of this reagent is 0.1 - 1.0 µg/mL. For immunocytochemistry, a concentration range of 1.0 - 5.0 μg/mL is recommended. For intracellular flow cytometry using our True-Phos™ Perm Buffer in Cell Suspensions Protocol, the suggested use of this reagent is ≤ 0.5 µg per million cells in 100 µL volume. It is recommended that the reagent be titrated for optimal performance for each application.

Application Notes

This clone has not been tested for IP.

This clone may recognize other Src family members.

This clone was ICC tested on PFA-fixed cells using both methanol and Triton X-100 permeabilization methods. Both permeabilazation methods were compatible with staining. Triton X-100 permeabilization produces stronger staining.

During ICC product development testing, this clone produced strong nuclear staining in a small subpopulation (<1%) of both unstimulated and stimulated cells. The source of this staining is not clear but we do not believe it is Lck-dependent.

This clone was developed using a synthetic peptide corresponding to human Src phosphorylated at tyrosine 419, which displays high sequence homology with the region surrounding Lck tyrosine 394. To confirm specificity to Lck Phospho (Tyr394), the cell line J.Cam1.6 was used. This cell line is a clonal derivative of Jurkat cells that harbors a truncated LCK allele with defective protein expression (Straus, et al. 1992. Cell. 70:585).

Product Citations
  1. Lam W, et al. 2023. Front Pharmacol. 13:1095186. PubMed
  2. Sasai M, et al. 2021. J Exp Med. 218:. PubMed
RRID
AB_2814639 (BioLegend Cat. No. 933101)
AB_2814639 (BioLegend Cat. No. 933102)

Antigen Details

Structure
Lck is a 509 amino acid protein with a predicted molecular weight of 58 kD
Distribution

Plasma-membrane associated/T cells

Function
Protein Tyrosine Kinase
Cell Type
T cells
Biology Area
Cell Biology, Signal Transduction
Molecular Family
Protein Kinases/Phosphatase
Antigen References
  1. Phillipsen L, et al. 2017. Sci Signal. 10:eaaf4736.
  2. Moogk D, et al. 2016. J Immunol. 197:644.
  3. Klammt C, et al. 2015. Nat Immunol. 16:961.
  4. Cancer Genome Atlas Network. 2015. Cell. 161:1681.
  5. Casas J, et al. 2014. Nat Commun. 5:5624
  6. McNeill L, et al. 2007. Immunnity. 27:425.
  7. Lefebvre DC, et al. 2003. Biochim Biophys Acta. 1650:40.
  8. Kabouridis PS. et al. 2003. Biochem J. 371:907.
  9. Abraham N, et al. 1990. Mol. Cell. Biol. 10:5197.
Gene ID
3932 View all products for this Gene ID
UniProt
View information about Lck Phospho Tyr394 on UniProt.org
Go To Top Version: 1    Revision Date: 09/25/2019

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