ROCK2, GST-tag Recombinant

Catalog #

40086

$410 *

Size: 10 µg

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Description

Recombinant human ROCK2 (Rho-associated protein kinase 2), encompassing amino acids 5-554. The constructs contain an N-terminal GST-tag. The protein was affinity purified and is active.

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Synonyms

KIAA0619, ROCK-II, ROKalpha

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Product Data Gallery

4-20% SDS-PAGE Coomassie Staining

Specific Activity

Product Info

Storage and Usage

Citations

Species

Human

Construct

ROCK2 (GST-5-554)

Host Species/Expression System

Sf9

Purity

≥90%

Format

Aqueous buffer solution

Formulation

50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM Glutathione, 0.1 mM EDTA, 0.25 mM DTT, 0.1 mM PMSF, and 25% glycerol

88 kDa

Amino Acids

5-554

Specific Activity

370 pmol/min/µg

Genbank #

NM_004850

UniProt #

O75116

Tag(s)

N-terminal GST-tag

Background

ROCK2 is a ubiquitously expressed serine/threonine kinase localized in the nucleus that regulates cytokinesis, smooth muscle contraction, the formation of actin stress fibers and focal adhesions, and the activation of the c-fos serum response element (1). ROCK2 is an immediate downstream target of the small GTPase RhoA. ROCK2 may play a pivotal role in cardiovascular diseases such as vasospastic angina, ischemic stroke, and heart failure. Inhibition of ROCKs by statins or other selective inhibitors leads to the upregulation and activation of endothelial nitric oxide synthase (eNOS) and reduction of vascular inflammation and atherosclerosis (2).

References

1. Zhao, Y M. et al. Dev Biol. 2004 Nov 1;275(1):183-91.
2. Noma, K. et al. Am J Physiol Cell Physiol. 2006 Mar;290(3):C661-8.

Storage/Stability

At least 6 months at -80°C

Assay Conditions

ROCK2 activity was measured by using S6K Substrate (KRRRLASLR), diluted in distilled water to a working concentration of 1 mg/ml, in a [33P]-ATP based assay. Reaction was initiated by mixing increasing amounts of ROCK2 with 1250 pmoles of [33P]-ATP in 5 mM MOPS, pH 7.2, 2.5 mM β-glycerol-phosphate, 5 mM MgCl₂, 1 mM EGTA, 0.4 mM EDTA, 50 ng/µl BSA, 50 µM DTT and substrate at a final concentration of 200 µg/ml.

The reaction was initiated by addition of 5 µl of [33P]-ATP Assay Cocktail (50 µM of [33P]-ATP with 50 µM ATP) to 20 µl of Reaction Mix (10 µl of diluted ROCK2 + 5 µl of Substrate Solution + 5 µl of distilled water), followed by a 15-minute incubation at 30°C. The reaction was terminated by spotting the reaction mixture on phosphocellulose P81 paper, air-dry and three 10-minute washes with 1% phosphoric acid solution. Radioactivity was measured in a scintillation counter. The corrected activity (RLU) was calculated by removing the blank value for each sample. The Kinase Specific Activity was calculated as follows: RLU / [(specific activity of [33P]-ATP in cpm/pmol)*(Reaction time in min)*(Enzyme amount in µg or mg)] * [(Reaction Volume) / (Spot Volume)]. The blank was determined from a “no substrate” sample by replacing the substrate solution with an equal volume of distilled water.

Instructions for Use

Thaw on ice and gently mix prior to use. DO NOT VORTEX. Perform a quick spin before opening. Aliquot into small volumes and flash freeze for long term storage. Avoid multiple freeze/thaw cycles.

Applications

Useful for the study of enzyme kinetics, screening inhibitors, and selectivity profiling.

Shipping Temperature

-80°C

Warnings

Avoid freeze/thaw cycles

Scientific Category

Kinase/Serine-Threonine

Regulatory Status

Research Use Only

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Datasheets/Protocols

40086 Lot 240315

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