Description
• Safe non-radioactive ELISA format
• Active ROCK-II included as positive control
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.
Introduction: 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: ROCK-I (also known as ROKbeta) and ROCK-II (also known as Rho Kinase and ROKalpha). 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).
MyBioSource's 96-well ROCK Activity Assay Kit is an enzyme immunoassay developed for detection of the specific phosphorylation of MYPT1 at Thr696 by ROCK. A strip well microtiter plate is precoated with a recombinant MYPT1. After incubating the substrate wells with ROCK samples (such as purified kinase, cell or tissue lysate) the phosphorylated MYPT1 is detected by an anti-phospho-MYPT1 (Thr696) antibody (Figure 1).
MyBioSource's 96-well ROCK Activity Assay Kit provides a non-isotopic, sensitive and specific method to monitor ROCK activity using its physiological substrate; it can also be used in screening ROCK inhibitors. The kit has detection sensitivity limit of 200 pg of active ROCK-II. A recombinant active ROCK-II is also provided as a positive control. Each kit provides sufficient quantities to perform up to 96 assays