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7508
HTScan® ROCK2 Kinase Assay Kit
Cellular Assay Kits

HTScan® ROCK2 Kinase Assay Kit #7508

This product is discontinued

Kinase Assay - Radiometric Image 1

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.

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Kinase Assay - DELFIA Image 2

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.)

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Kinase Assay - DELFIA Image 3

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.)

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Kinase Assay - DELFIA Image 4

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.)

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Kinase Assay - DELFIA Image 5

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.)

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Product Includes Quantity (with Count) Solution Color
ATP (10 mM) 9804 1 x 1 ml
Phospho-PLK (Ser137) Antibody 1 x 30 µl
Kinase Buffer (10X) 9802 1 x 15 ml

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 Formula:

Peptide substrate, Biotin-PLK (Ser137): 1,945 Daltons. GST-ROCK2 Kinase: 88 kDa.

Peptide Core Sequence:

RRPS*YRK

ROCK (Rho-associated kinase), a family of serine/threonine kinases, is an important downstream target of Rho-GTPase 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).

  1. Nakagawa, O. et al. (1996) FEBS Lett. 392, 189-193.
  2. Lee, J.H. et al. (2004) J. Cell. Biol. 167, 327-337.
  3. Sebbagh, M. et al. (2005) J. Exp. Med. 201, 465-471.
  4. Amano, M. et al. (1996) J. Biol. Chem. 271, 20246-20249.
  5. Kureishi, Y. et al. (1997) J. Biol. Chem. 272, 12257-12260.
  6. Totsukawa, G. et al. (2000) J. Cell Biol. 150, 797-806.
Entrez-Gene Id
9475
Swiss-Prot Acc.
O75116
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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