Formulation Supplied in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.25 mM DTT, 10 mM glutathione, 0.1 mM EDTA, 0.1 mM PMSF, and 25% glycerol.
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & 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.
1. Thaw the [33P]-ATP Assay Cocktail in a shielded container in a designated radioactive work area.
2. Thaw the Active ROCK1, Kinase Assay Buffer, Substrate, and Kinase Dilution Buffer on ice.
3. In a pre-cooled microfuge tube, add the following reaction components bringing the initial reaction volume up to 20 μL.
a. Diluted Active ROCK1: 10 μL
b. Substrate (1 mg/mL; on ice): 5 μL
c. Distilled or deionized water (on ice): 5 μL
4. Set up the blank control as outlined in step 3, excluding the addition of the substrate. Replace the substrate with an equal volume of distilled or deionized water.
5. Initiate the reaction with the addition of 5 μL [33P]-ATP Assay Cocktail, bringing the final volume up to 25 μL. Incubate the mixture in a water bath at 30 °C for 15 minutes.
6. After the 15 minute incubation, terminate the reaction by spotting 20 μL of the reaction mixture onto individual pre-cut strips of phosphocellulose P81 paper.
7. Air dry the pre-cut P81 strip and sequentially wash in a 1% phosphoric acid solution (add 10 mL of phosphoric acid to 990 mL of distilled or deionized water) with constant gentle stirring. It is recommended that the strips be washed a total of three times for approximately 10 minutes each.
8. Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter.
9. Determine the corrected cpm by subtracting the blank control value (see step 4) for each sample and calculate the kinase specific activity as outlined below:
Calculation of [33P]-ATP Specific Activity (SA) (cpm/pmol)
Specific Activity (SA) = cpm for 5 μL [33P]-ATP/pmole of ATP (in 5 μL of a 250 μM ATP stock solution' i.e. 1250 pmol)
Calculation of Kinase Specific Activity (SA) (pmol/minutes/μg or nmol/minutes/mg)
Corrected cpm from reaction / [(SA of 33P-ATP in cpm/pmol) x (Reaction time in minutes) x (Enzyme amount in μg or mg)] x [(Reaction volume) / (Spot Volume)]
|The approximate molecular weight is 85 kDa and the average purity is 90%.|
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).
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