Revision 1

#7508Store 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:

#O75116

Entrez-Gene Id:

9475

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. Enzymes are supplied in 50 mM Tris-HCL (pH 8.0), 100 mM NaCl, 5 mM DTT, 15 mM reduced glutathione and 20% glycerol.
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 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.

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

Background

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). ROCK1 is cleaved by caspase-3 at a conserved DETD1113/G sequence resulting in loss of its C-terminal inhibitory domain (3). ROCK2 is directly cleaved by granzyme B (grB). Cleavage activates ROCK and leads to phosphorylation of myosin light chain (MLC) and inhibition of myosin phosphatase (4). This phosphorylation may account for the mechanism by which Rho regulates cytokinesis, cell motility, cell membrane blebbing during apoptosis, and smooth muscle contraction (5-7).

  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. Sebbagh, M. et al. (2001) Nat Cell Biol 3, 346-52.
  5. Amano, M. et al. (1996) J. Biol. Chem. 271, 20246-20249.
  6. Kureishi, Y. et al. (1997) J. Biol. Chem. 272, 12257-12260.
  7. Totsukawa, G. et al. (2000) J. Cell Biol. 150, 797-806.

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.

限制使用

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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
#7508

HTScan® ROCK2 Kinase Assay Kit

HTScan® ROCK2 Kinase Assay Kit: Image 1 Expand Image
图 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. 在 50 µl 反应体积中，每个反应孔在室温下用数量递增的 ROCK2 和 1.5 µM 底物肽反应 30 分钟。（DELFIA® 是 PerkinElmer, Inc. 的注册商标）
HTScan® ROCK2 Kinase Assay Kit: Image 2 Expand Image
图 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. 在 50 µl 反应体积中，每个反应孔在室温下用 50 ng ROCK2、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® ROCK2 Kinase Assay Kit: Image 3 Expand Image
图 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. 在 50 µl 反应体积中，每个反应孔在室温下用 50 ng ROCK2 和浓度递增的底物肽反应 30 分钟。（DELFIA® 是 PerkinElmer, Inc. 的注册商标）
HTScan® ROCK2 Kinase Assay Kit: Image 4 Expand Image
图 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. 在 50 µl 反应体积中，每个反应孔用 50 ng ROCK2 和 1.5 µM 底物肽。（DELFIA® 是 PerkinElmer, Inc. 的注册商标）
HTScan® ROCK2 Kinase Assay Kit: Image 5 Expand Image
图 1. 使用以下反应条件在放射测定法中测量 ROCK2 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：S6K 底物肽（KRRRLASLR）400 ng/μL，以及指标数量的重组 ROCK2。
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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。