Recombinant Human Active ROCK1 Protein, CF

The approximate molecular weight is 85 kDa and the average purity is 90%.

• Reactivity: Hu
• Applications: Bioactivity
• Format: Carrier-Free

Recombinant Human Active ROCK1 Protein, CF Summary

• Details of Functionality: The specific activity of ROCK1 is typically 49-67 nmol/min/mg using a synthetic peptide substrate.
• Source: Spodoptera frugiperda, Sf 9 (baculovirus)-derived human ROCK1 protein
  aa 17-535
• Accession #: NM_005406
• N-terminal Sequence: Using an N-terminal GST tag
• Protein/Peptide Type: Recombinant Proteins
• Gene: ROCK1
• Purity: >80%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Applications/Dilutions

• Dilutions:
  • Bioactivity
• SDS-PAGE: 85 kDa

Packaging, Storage & Formulations

• Storage: This product is stable at ≤ ‑70 °C for up to 1 year from the date of receipt. For optimal storage, aliquot into smaller quantities after centrifugation and store at recommended temperature. Avoid repeated freeze-thaw cycles.
• Buffer: Supplied in 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.
• Purity: >80%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
• Assay Procedure:
  • Active Kinase - Active ROCK1 (0.1 μg/μL) diluted with Kinase Dilution Buffer X (1X) and assayed as outlined in Figure 2. Note: These are suggested working dilutions and it is recommended that the researcher perform a serial dilution of active ROCK1 for optimal results.
  • Kinase Assay Buffer III (5X) - 200 mM Tris-HCl, pH 7.4, 100 mM MgCl₂, and 0.5 mg/mL BSA. Add fresh DTT prior to use to a final concentration of 250 μM.
  • Kinase Dilution Buffer IX (1X) - Kinase Assay Buffer III diluted at a 1:4 ratio (5X dilution) with cold water. Add fresh DTT to the aliquot prior to use to a final concentration of 50 μM.
  • ADP-Glo™ Kinase Assay Kit - 10 mM ATP Solution, 10 mM ADP Solution, ADP-Glo™ Reagent, and Kinase Detection Reagent.
  • Substrate - S6K synthetic peptide substrate (KRRRLASLR) diluted in distilled water to a final concentration of 1 mg/mL.
  1. Thaw the Active ROCK1, Kinase Assay Buffer III (5X), and Substrate on ice. Prepare a 15 μL enzyme dilution with Kinase Dilution Buffer IX (1X), in a pre-chilled 96-well plate.
  2. Prepare a Substrate/ATP mixture as follows (25 μM ATP example):
     a. 10 mM ATP Solution: 1 μL
     b. Kinase Assay Buffer III (5X): 79 μL
     c. Substrate at 1 mg/mL: 80 μL
  3. Transfer the following reaction components prepared in Step 2 to a 384-well opaque plate bringing the reaction volume up to 5 μL:
     a. 3 μL of diluted Active ROCK1
     b. 2 μL of Substrate/ATP mix as prepared in Step 2. This initiates the reaction.
  4. Set up the blank control as outlined in Step 2, excluding the addition of the kinase. Replace the kinase with an equal volume of Kinase Dilution Buffer IX (1X).
  5. Incubate at ambient temperature for 40 minutes.
  6. After the 40 minute incubation period, terminate the reaction and deplete the remaining ATP by adding 5 μL of ADP-Glo™ Reagent. Spin down and shake the 384-well plate. Then incubate the reaction mixture for another 40 minutes at ambient temperature.
  7. Add 10 μL of the Kinase Detection Reagent to the 384-well plate and incubate the reaction mixture for another 30 minutes at ambient temperature.
  8. Read the 384-well reaction plate using the Luminescence Module Protocol on a GloMax®-Multi Microplate Multimode Reader.
  9. Determine the corrected activity (RLU) by removing the blank control value (see Step 4) for each sample and calculate the kinase specific activity as outlined below.
     
     
     Calculation of Specific Activity of ADP (RLU/pmol)
     From ADP standard curve, determine RLU/pmol of ADP
     
     Kinase Specific Activity (SA) (pmol/min/μg or nmol/min/mg)
     Corrected RLU from reaction / [(SA of ADP in RLU/pmol) x (Reaction time in min) x (Enzyme amount in μg or mg)]

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Active ROCK1 Protein, CF

• EC 2.7.11
• EC 2.7.11.1
• MGC131603
• MGC43611
• p160 ROCK-1
• p160ROCK
• p160-ROCK
• PRO0435
• Renal carcinoma antigen NY-REN-35
• Rho kinase
• rho-associated protein kinase 1
• Rho-associated, coiled-coil containing protein kinase 1
• Rho-associated, coiled-coil-containing protein kinase 1
• ROCK1
• ROK beta

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).

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

Bioinformatics

• Gene Symbol: ROCK1
• Uniprot:
  • NM_005406(Human)