Rho-associated Kinase (ROCK) mediates Rho signaling and reorganizes the actin cytoskeleton by phosphorylation of several substrates that contribute to the assembly of actin filaments and contractility. ROCK inactivates myosin phosphatase through the specific phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at Thr-696, which results in an increase in the phosphorylated content of the 20-kDa myosin light chain (MLC20).
Our 96-Well ROCK Activity Assay Kit uses a safe, non-radioactive format to measure the level of active Rho Kinase in cell or tissue lysates. The kit contains a strip-well plate pre-coated with recombinant MYPT1.
96-Well ROCK Activity Assay Principle
Results from the 96-Well ROCK Activity Assay. Top: Active ROCK-II in 10 µL was incubated with 90 µL of 1X Kinase Reaction Buffer for 60 minutes at 30ºC. Bottom: 2.5 ng of active ROCK-II in 10 µL was incubated with 90 µL of 1X Kinase Reaction Buffer at 30ºC for various incubation times as shown. Phosphorylation of MYPT1 substrate was detected by anti-phospho-MYPT1 (Thr-696) antibody according to the assay protocol.