ROCK Activity Assay Kit

ROCK Activity Assay Kit (ab211175) provides a non-isotopic, sensitive and specific method to monitor ROCK1 (ROKβ) and ROCK2 (Rho Kinase) using its physiological substrate MYPT1 (myosin phosphate target subunit 1). This kit can also be used in the screening of Rho kinase inhibitor molecules in purified protein samples.

This product detects the specific phosphorylation of MYPT1 by ROCK1 and ROCK2 at threonine 696 (T696) using an enzyme-based immunoassay. This substrate is initially incubated with the sample containing ROCK1 and ROCK2 (such as purified kinase or cell lysate). Following the kinase reaction, the sample solution is transferred to a substrate capture plate where phosphorylated MYPT1 is detected by anti-phopsho-MYPT1-Thr696 antibody. A recombinant active ROCK2 is provided as a positive control. The sensitivity limit of this kit is 200 pg of active ROCK2.

Members of the Rho family are essential regulatory components of the signaling pathway that direct cell motility, adhesion, and cytokinesis through reorganization of actin cytoskeleton. Rho is activated by extracellular signals such as lysophosphatidic acid (LPA). The actions of Rho are mediated by downstream Rho effectors. One of these effectors is Rho-associated kinase (ROCK). Two ROCK isoforms have been identified: ROCK1 (also known as ROKβ) and ROCK2 (also known as Rho Kinase and ROKα). ROCK mediates Rho signaling and reorganizes actin cytoskeleton through phosphorylation of several substrates that contribute to the assembly of actin filaments and contractility. For example, ROCK inactivates myosin phosphatase through the specific phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at Thr696, which results in an increase in the phosphorylated content of the 20-kDa myosin light chain (MLC20).