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7802
HTScan® Csk Kinase Assay Kit

HTScan® Csk Kinase Assay Kit #7802

This product is discontinued

Kinase Assay - Radiometric Image 1

Figure 1. CSK kinase activity was measured in a radioisotopic filter binding assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 uM Na-orthovanadate, 1.2 mM DTT, ATP (variable), 200 ng/50 µl PEG20,000, Substrate: Poly(EY) 1 ug/50 µl, recombinant Csk: 4 Units/50 µl.

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Kinase Assay - Radiometric Image 2

Figure 3. Dose dependence curve of Csk kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1370) by Csk kinase. In a 50 µl reaction, increasing amounts of Csk and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

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Kinase Assay - Radiometric Image 3

Figure 5. Staurosporine inhibition of Csk kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of Csk substrate peptide (#1370) by Csk kinase. In a 50 µl reaction, 100 ng Csk kinase, 1.5 µM substrate peptide, 20 µM ATP and increasing amounts of staurosporine were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

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Kinase Assay - Radiometric Image 4

Figure 4. Peptide concentration dependence of Csk kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1370) by Csk kinase. In a 50 µl reaction, 100 ng of Csk and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

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Kinase Assay - Radiometric Image 5

Figure 2. Time course of Csk kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of Csk substrate peptide (#1370) by Csk kinase. In a 50 µl reaction, 100 ng Csk and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

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Product Includes Quantity (with Count) Solution Color
DTT (1000X) 1 x 80 µl
ATP (10 mM) 9804 1 x 1 ml

The kit provides a means of performing kinase activity assays with recombinant human Csk kinase. It includes active Csk kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a phospho-Tyrosine monoclonal antibody for detection of the phosphorylated form of the substrate peptide.

Molecular Formula:

Peptide substrate, Biotin-peptide: 2,290 Daltons, GST-Csk Kinase: 80 kDa.

Peptide Core Sequence:

LVY*LN

Carboxy-terminal Src kinase (Csk) is a ubiquitously expressed nonreceptor tyrosine kinase that negatively regulates the Src family kinases (SFKs) by phosphorylation of the SFK carboxy-terminal tyrosine (1,2). Phosphorylated carboxy-terminal tyrosine binds to the SH2 domain of SFK intramolecularly and leads to folding and inactivation of the SFK (3). This Csk-catalyzed SFK tyrosine phosphorylation is highly specific and exclusive. The SFK carboxy-terminal tyrosine is the only known physiological substrate of Csk (4).

Csk consists of an SH2, an SH3, and a kinase domain. There is evidence that the SH2 and SH3 domains are essential for the regulation of SFK, and Csk can be recruited to the membrane where SFKs are in an active state. This process is mediated by a Csk-binding protein (Cbp, also called PAG), which binds tightly to the SH2 domain of Csk (5). Activation of SFK by extracellular stimuli leads to the tyrosine phosphorylation of Cbp, generating docking sites for Csk. The recruitment of Csk forms a feedback mechanism for termination of SFK function (6).

  1. Nada, S. et al. (1991) Nature 351, 69-72.
  2. Nada, S. et al. (1993) Cell 73, 1125-1135.
  3. Lee, S. et al. (2003) Proc. Natl. Acad. Sci. USA 100, 14707-14712.
  4. Imamoto, A. and Soriano, P. (1993) Cell 73, 1117-1124.
  5. Kawabuchi, M. et al. (2000) Nature 404, 999-1003.
  6. Matsuoka, H. et al. (2004) J. Biol. Chem. 279, 5975-5983.
Entrez-Gene Id
1445
Swiss-Prot Acc.
P41240
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DELFIA is a registered trademark of PerkinElmer, Inc.

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