Product Pathways - HTScan Kinase Assay Kits
HTScan® TSK2 Kinase Assay Kit #7630
Cell Signaling Technology offers a full line of protein kinases, substrates, antibody detection reagents and HTScan® kits. Browse our "Reagents for High-Throughput Screening" product listing or contact us at drugdiscovery@cellsignal.com.
| Kit Includes | Quantity |
|---|---|
| Phospho-PLK (Ser137) Antibody # 5070 | 30 microliters |
| Kinase Buffer (10X) # 9802 | 15 milliliters |
| ATP (10 mM) # 9804 | 1 milliliters |
| PLK (Ser137) Biotinylated Peptide # 1300 | 1.25 milliliters |
| TSK2 Kinase # 7629 | 5 micrograms |
Description
The kit provides a means of performing kinase activity assays with recombinant human TSK2 kinase. It includes active TSK2 kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a phospho-serine/threonine antibody for detection of the phosphorylated form of the substrate peptide.
Molecular Weights
Peptide substrate, Biotin-peptide: 1,646 Daltons. GST-TSK2 Kinase domain: 74 kDa.
Peptide Core Sequence
RRS*LL
Kinase Assay - Radiometric
Figure 1. TSK2 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 µM Na-orthovanadate, 1.2 mM DTT, 1 µM ATP, 2.5 µg/50 µl PEG20,000, Substrate: Histone H2A, variable, and recombinant TSK2: variable.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of TSK2 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of substrate peptide (#1300) by TSK2 kinase. In a 50 µl reaction, increasing amounts of TSK2 and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
Figure 5. Staurosporine inhibition of TSK2 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of TSK2 substrate peptide (#1300) by TSK2 kinase. In a 50 µl reaction, 50 ng TSK2, 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.)
Kinase Assay - DELFIA
Figure 4. Peptide concentration dependence of TSK2 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of substrate peptide (#1300) by TSK2 kinase. In a 50 µl reaction, 50 ng of TSK2 and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
Figure 2. Time course of TSK2 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of TSK2 substrate peptide (#1300) by TSK2 kinase. In a 50 µl reaction, 50 ng TSK2 and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Source / Purification
The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing full-length human TSK2 (Met1-Thr358) (GenBank Accession No. NM_053006) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.
Quality Control
The substrate peptide was selected using our Serine/Threonine Kinase Substrate Screening Kit #7400. Phospho-PLK (Ser137) antibody #5070 was used for detection. The quality of the biotinylated peptide was evaluated by reverse-phase HPLC and by mass spectrometry.Purified TSK2 kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain and Western blot. The specific activity of the TSK2 kinase was determined using a radiometric filter binding assay [Fig.1]. Time course [Fig.2], kinase dose dependency [Fig.3] and substrate dose dependency [Fig.4] assays were performed to verify TSK2 activity using the TSK2 substrate peptide provided in this kit. TSK2 sensitivity to the inhibitor staurosporine was measured using the TSK2 substrate peptide provided in this kit [Fig.5].
Background
Testis-specific kinases (TSKs) are serine/threonine kinases that may play a role in sperm function. Three TSKs, TSK1, TSK2 and TSK3 have been identified. Open reading frames (ORF) within the TSK gene code for the 3 isoforms: 367 amino acids for TSK1, 358 amino acids for TSK2 and 275 amino acids for TSK3. The kinase domains are located near the NH2-terminal regions of the TSK proteins and contain conserved amino acid residues characteristic of a serine/threonine kinase. While TSK1 and TSK2 both have a 100 amino acid domain located C-terminally to the kinase domain, TSK3 is composed essentially of a serine/threonine kinase domain. TSK1 and TSK2 messenger RNA is expressed exclusively in the testis of mature males. An in vitro kinase assay of immunoprecipitates containing either TSK1 or TSK2 revealed serine phosphorylation of a coprecipitating protein of approximately 65 kDa. This substrate protein is also expressed exclusively in the testis as demonstrated by northern and dot blot analyses (1,2). While TSK1 and TSK2 form detergent resistant complexes during immunoprecipitation, TSK3 does not associate with either protein. Expression of TSK3 is induced at puberty, is restricted to the interstitial Leydig cells of postpubertal males, persists throughout adulthood and is implicated in regulating androgenesis and/or Leydig cell proliferation (4).
- Bielke, W. et al. (1994) Gene 139, 235-239.
- Hao, Z. et al. (2004) Mol Hum Reprod 10, 433-444.
- Kueng, P. et al. (1997) J Cell Biol 139, 1851-1859.
- Zuercher, G. et al. (2000) Mech Dev 93, 175-177.
Application References
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
Companion Products
- 7629 TSK2 Kinase
- 5070 Phospho-PLK (Ser137) Antibody
- 1300 PLK (Ser137) Biotinylated Peptide
- 9953 Staurosporine
This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
