Revision 1

#7758Store at -80C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
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UniProt ID:

#P06239

Entrez-Gene Id:

3932

Product Information

Storage

Antibodies are supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Do not aliquot the antibodies. Peptides are supplied at 6 µM in 0.001% DMSO.
Enzyme is supplied in 50 mM Tris-HCl, pH7.5; 150 mM NaCl, 0.25 mM DTT, 0.1mM EGTA, 0.1 mM EDTA, 0.1 mM PMSF, 25% glycerol, 7 mM glutathione.
Store at –80°C.
Keep enzymes on ice during use.
Avoid repeated freeze-thaw cycles.

Source / Purification

The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing a fragment of human Lck (Met1-Pro509) (GenBank accession No. NM_005356) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.

Product Description

The kit provides a means of performing enzymatic assays with active human Lck kinase. It includes active Lck kinase (supplied as a GST fusion protein), a biotinylated substrate peptide and a phospho-tyrosine monoclonal antibody for detection of the phosphorylated form of the substrate peptide.
Molecular Formula Peptide substrate, Biotin-FHIT (Tyr114): 2,150 Daltons, GST-Lck Kinase: 84 kDa.

Background

The Src family of protein tyrosine kinases, which includes Src, Lyn, Fyn, Yes, Lck, Blk, and Hck, are important in the regulation of growth and differentiation of eukaryotic cells (1). Src activity is regulated by tyrosine phosphorylation at two sites, but with opposing effects. While phosphorylation at Tyr416 in the activation loop of the kinase domain upregulates enzyme activity, phosphorylation at Tyr527 in the carboxy-terminal tail by Csk renders the enzyme less active (2).
Lck is essential for T-lymphocyte activation and differentiation (3,4). Phosphorylation of Tyr505 in the carboxy-terminal tail of Lck downregulates its catalytic activity, while phosphorylation of Tyr394 leads to an increase in Lck activity (5).

  1. Thomas, S.M. and Brugge, J.S. (1997) Annu Rev Cell Dev Biol 13, 513-609.
  2. Hunter, T. (1987) Cell 49, 1-4.
  3. Molina, T.J. et al. (1992) Nature 357, 161-164.
  4. Straus, D.B. and Weiss, A. (1992) Cell 70, 585-593.
  5. Chow, L.M. et al. (1993) Nature 365, 156-160.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
仅供研究使用。不得用于诊断流程。
Revision 1
#7758

HTScan® Lck Kinase Assay Kit

HTScan® Lck Kinase Assay Kit: Image 1 Expand Image
图 3. Dose dependence curve of Lck kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide #1365 by Lck kinase. 在 50 µl 反应体积中，每个反应孔在 25ºC 下用数量递增的 Lck 和 1.5 µM 底物肽反应 30 分钟。（DELFIA® 是 PerkinElmer, Inc. 的注册商标）
HTScan® Lck Kinase Assay Kit: Image 2 Expand Image
图 5. Staurosporine inhibition of Lck kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of Lck substrate peptide (#1365) by Lck kinase. 在 50 µl 反应体积中，每个反应孔在室温下用 50 ng Lck、1.5 µM 底物肽、50 µM ATP 和数量递增的 staurosporine 反应 30 分钟。（DELFIA® 是 PerkinElmer, Inc. 的注册商标）
HTScan® Lck Kinase Assay Kit: Image 3 Expand Image
图 4. Peptide concentration dependence of Lck kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1365) by Lck kinase. 在 50 µl 反应体积中，每个反应孔在室温下用 50 ng Lck 和浓度递增的底物肽反应 30 分钟。（DELFIA® 是 PerkinElmer, Inc. 的注册商标）
HTScan® Lck Kinase Assay Kit: Image 4 Expand Image
图 2. Time course of Lck kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of Lck substrate peptide (#1365) by Lck kinase. 在 50 µl 反应体积中，每个反应孔用 50 ng 和 1.5 µM 底物肽。（DELFIA® 是 PerkinElmer, Inc. 的注册商标）
HTScan® Lck Kinase Assay Kit: Image 5 Expand Image
图 1. 使用以下反应条件在放射分析中测量 Lck 激酶活性：5 mM MOPS、pH 7.2、2.5 mM β-甘油磷酸酯、1 mM EGTA、0.4 mM EDTA、5 mM MgCl2、0.05 mM DTT、50 μM ATP，底物：Poly (EY 4:1) 400 ng/μL，和变量的 Lck 激酶。
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Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
仅供研究使用。不得用于诊断流程。