ROCK1, GST-tag Recombinant

Catalog #

40085

$410 *

Size: 10 µg

Qty

Bulk/Custom

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Description

Recombinant human ROCK1 (Rho associated coiled-coil containing protein kinase 1), encompassing amino acids 17-535. This construct contains an N-terminal GST tag. The recombinant protein was affinity purified and kinase active.

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Synonyms

P160ROCK, ROCK-I, ROKbeta

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Product Data Gallery

4-20% SDS-PAGE Coomassie Staining

Specific Activity

Product Info

Storage and Usage

Citations

Species

Human

Host Species/Expression System

Sf9 insect cells

Purity

≥80%

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, 25% glycerol

85 kDa

Amino Acids

17–535

Specific Activity

65 pmol/min/µg

Genbank #

NM_005406

UniProt #

Q13464

Tag(s)

N-terminal GST-tag

Background

ROCK1 is a ubiquitously expressed serine/threonine kinase that is a downstream target of the small GTPase RhoA. ROCK1 is involved in diverse cellular functions, including smooth muscle contraction, actin cytoskeleton organization, cell adhesion and motility, and gene expression (1). ROCK1 contributes to the development of cardiac fibrosis and induction of fibrogenic cytokines in cardiomyocytes in response to pathological stimuli. ROCK1 knockout mice exhibit reduced perivascular and interstitial fibrosis which is associated with reduced expression of a variety of extracellular matrix (ECM) proteins and fibrogenic cytokines (2).

References

1. Zhao, Y M. et al. Dev Biol. 2004 Nov 1,275(1):183-91.
2. Zhang, C. et al. FASEB J. 2006 May,20(7):916-25.

Storage/Stability

At least 6 months at -80°C

Assay Conditions

Kinase activity was measured using the ADP-Glo™ Kinase Assay kit (Promega; Cat# V9101) which quantifies the amount of ADP produced. The ADP-Glo™ Reagent is added to terminate the reaction and deplete the remaining ATP. The Kinase Detection Reagent is then added to convert ADP to ATP and to measure the newly synthesized ATP using a luciferase reaction.

ROCK1 activity was measured by using S6K substrate (KRRRLASLR) diluted in water to a final concentration of 1 mg/ml. Reaction was initiated by mixing increasing amounts of the ROCK1 with 25 µM ATP in 40 mM Tris-HCl, pH 7.4, 20 mM MgCl₂, 0.1 mg/ml BSA prepared with 250 µM fresh DTT and the 20 µg/mL substrate.

After a 40-minute incubation at 37°C, the reaction was terminated by addition of the AMP-Glo™ Reagent followed by a subsequent 40 minute incubation at room temperature. Kinase Detection Reagent was then added and incubated for another 30 minutes. Detection of luminescence was measured using the Luminescence Module Protocol on GloMax®-Multi Microplate reader. The corrected activity (RLU) was calculated by removing the blank value for each sample divided by the (specific activity of ADP in RLU/pmol)*(Reaction time in min)*(Enzyme amount in µg or mg). The blank was determined from a “no kinase” sample by replacing the enzyme working solution with an equal volume of Kinase Dilution Buffer IX (1X).

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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40085 Lot 230112

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ROCK1, GST-tag Recombinant

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