Rho-associated Kinase (ROCK) Activity Assay

Introduction:

Small GTPase proteins of the Rho family are pivotal regulators of several aspects of cell behavior, such as cell motility, cell proliferation and apoptosis. They have a central role in many motile responses that involve the actin cytoskeleton and/or microtubule network, from neurite extension to phagocytosis and cancer-cell invasion. In addition, they contribute to changes in gene expression that lead to cell-cycle progression or differentiated cell responses. 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 protein kinase (ROCK). Two ROCK isoforms have been identified: ROCK-I (also known as ROKβ) and ROCK-II (also known as Rho Kinase and ROKα). ROCK mediates Rho signaling and reorganizes the actin cytoskeleton through 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 threonine residue 696, which results in an increase in the phosphorylated form of the 20 kDa myosin light chain (MLC20). This threonine residue may also be phosphorylated by myotonic dystrophy protein kinase (DMPK) family kinases.

Test Principle:
Millipore’s Rho-associated Protein Kinase (ROCK) Activity Assay Kit is an enzyme immunoassay for detection of the active ROCK and DMPK family kinases. Plates are pre-coated with recombinant MYPT1, which contains a Thr696 residue that may be phosphorylated upon addition of ROCK-I, ROCK-II, or DMPK. A detection antibody that specifically detects only MYPT1 phosphorylated at Thr696 is then applied. Subsequently, an HRP-conjugated secondary detection antibody is added. The amount of phoshorylated substrate is measured by addition of the chromogenic substrate tetra-methylbenzidine (TMB), which bound HRP converts from a colorless solution to a blue solution (or yellow after the addition of stop solution). The absorbance signal at 450 nm reflects the relative amount of ROCK activity in the sample.

The ROCK assay kit is designed to determine the presence and relative amount of purified ROCK activity as well as for screening inhibitors or activators of ROCK or DMPK family kinases.

Millipore’s Rho-associated Protein Kinase (ROCK) Activity Assay Kit is an enzyme immunoassay for detection of the active ROCK and DMPK family kinases. Plates are pre-coated with recombinant MYPT1, which contains a Thr696 residue that may be phosphorylated upon addition of ROCK-I, ROCK-II, or DMPK. A detection antibody that specifically detects only MYPT1 phosphorylated at Thr696 is then applied. Subsequently, an HRP-conjugated secondary detection antibody is added. The amount of phoshorylated substrate is measured by addition of the chromogenic substrate tetra-methylbenzidine (TMB), which bound HRP converts from a colorless solution to a blue solution (or yellow after the addition of stop solution). The absorbance signal at 450 nm reflects the relative amount of ROCK activity in the sample.

The ROCK assay kit is designed to determine the presence and relative amount of purified ROCK activity as well as for screening inhibitors or activators of ROCK or DMPK family kinases. It can also be used to assess the potency of pharmacological agents on ROCK or DMPK family kinases.

MYPT1 Pre-coated 96-well Multistrip Plate: One 96-well multistrip plate pre-coated with MYPT1

Anti-phospho-MYPT1(Thr696) Antibody: One vial containing 10 µg of anti-phospho-MYPT1(Thr696) antibody

TMB/E Substrate: One bottle containing 12 mL

Stop Solution: One bottle containing 12 mL

TBS, 20X: One bottle containing 50 mL 20X TBS

20% Tween^® 20 (v/v): One bottle containing 3 mL

Plate Sealers: Two plate covers

Assay Dilution Buffer I: One vial containing 1 mL

ATP (10mM): One vial containing 50 µL

MgCl2 (1M): One vial containing 100 µL

30% BSA: One vial containing 1 mL

Goat Anti-Rabbit IgG HRP Secondary Antibody: One vial containing 30 µL

ROCK Inhibitor Y-27632: One vial containing 150 µL

Active Rho-associated Kinase II (ROCK-II): One vial containing 10 µg

Store kit materials at specified temperatures on the label and manual; use within 4 months from date of receipt.

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