FAQ: Rho Kinase Activity Assay, 96-Well

Q: What is the recommended lysis buffer?

A: Cells and tissues can be lysed in RIPA buffer or with the following lysis buffer we have used: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM 2-glycerophosphate, 1 % Triton X-100 or 1 % Nonidet P-40, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4 and Proteinase inhibitor cocktail.


Q: How much protein should be added to the assay?

A: The amount of protein to add is sample dependent and will depend on the ROCK activity level of the sample.  We recommend adding as much as possible (90 µL) in a preliminary experiment, and then scaling back if necessary with subsequent experiments.


Q: How do I calculate ROCK activity without a standard?

A: The ROCK Activity assay measures kinase activity, which is different from an ELISA that can be compared to a standard curve.  Most researchers instead report their results as relative OD values for different conditions tested.  Another option is to run a ROCK II dose curve with the active ROCK II positive control that is provided with the kit and use for comparison with your samples.