Product Pathways - HTScan Kinase Assay Kits
HTScan® ROCK2 Kinase Assay Kit #7508
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 |
|---|---|
| ATP (10 mM) # 9804 | 1 milliliters |
| ROCK2 Kinase | 5 micrograms |
| CREB (Ser133) Biotinylated Peptide # 1331 | 1.25 milliliters |
| Phospho-PKA Substrate (RRXS/T) (100G7E) Rabbit mAb # 9624 | 30 microliters |
| Kinase Buffer (10X) # 9802 | 15 milliliters |
Description
The kit provides a means of performing kinase activity assays with recombinant human ROCK2 kinase. It includes active ROCK2 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-PLK (Ser137): 1,945 Daltons. GST-ROCK2 Kinase: 88 kDa.
Peptide Core Sequence
RRPS*YRK
Kinase Assay - Radiometric
Figure 1. ROCK2 kinase activity was measured in a radiometric assay using the following reaction conditions: 5 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, 0.05 mM DTT, 50 μM ATP, Substrate: S6K substrate peptide (KRRRLASLR) 400 ng/μL, and variable amounts of recombinant ROCK2.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of ROCK2 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of substrate peptide (#1300) by ROCK2 kinase. In a 50 µl reaction, increasing amounts of ROCK2 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 ROCK2 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of ROCK2 substrate peptide (#1300) by ROCK2 kinase. In a 50 µl reaction, 50 ng ROCK2, 1.5 µM substrate peptide, 50 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 ROCK2 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of substrate peptide (#1300) by ROCK2 kinase. In a 50 µl reaction, 50 ng of ROCK2 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 ROCK2 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibodty #5070 to detect phosphorylation of ROCK2 substrate peptide (#1300) by ROCK2 kinase. In a 50 µl reaction, 50 ng ROCK2 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 human ROCK2 (Pro6-Ser553) (GenBank Accession No. NM_004850) 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-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 was used for detection. The quality of the biotinylated peptide was evaluated by reverse-phase HPLC and by mass spectrometry.Purified ROCK2 kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain. ROCK2 kinase activity was determined using a radiometric assay [Fig.1]. Time course [Fig.2], kinase dose-dependency [Fig.3] and substrate dose-dependency [Fig.4] assays were performed to verify ROCK2 activity using the ROCK2 substrate peptide provided in this kit. ROCK2 sensitivity to the inhibitor staurosporine was measured using the ROCK2 substrate peptide provided in this kit [Fig.5].
Background
ROCK (Rho-associated kinase), a family of serine/threonine kinases, is an important downstream target of GTPase Rho and plays an important role in Rho-mediated signaling. Two isoforms of ROCK have been identified (ROCK1 and ROCK2). ROCK is composed of N-terminal catalytic, coiled-coil and C-terminal PH (pleckstrin homology) domains. The C-terminus of ROCK negatively regulates its kinase activity (1,2). Caspase-3-induced cleavage of ROCK1 and direct cleavage of ROCK2 by granzyme B (grB) activates ROCK and leads to phosphorylation of myosin light chain and inhibition of myosin phosphatase (3). This phosphorylation may account for the mechanism by which Rho regulates cytokinesis, cell motility, cell membrane blebbing during apoptosis and smooth muscle contraction (4-6).
- Nakagawa, O. et al. (1996) FEBS Lett. 392, 189-193.
- Lee, J.H. et al. (2004) J. Cell. Biol. 167, 327-337.
- Sebbagh, M. et al. (2005) J. Exp. Med. 201, 465-471.
- Amano, M. et al. (1996) J. Biol. Chem. 271, 20246-20249.
- Kureishi, Y. et al. (1997) J. Biol. Chem. 272, 12257-12260.
- Totsukawa, G. et al. (2000) J. Cell Biol. 150, 797-806.
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
- 9624 Phospho-PKA Substrate (RRXS/T) (100G7E) Rabbit mAb
- 1331 CREB (Ser133) 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.
