What kind of kinase is MEK?

Mitogen-activated protein kinase kinase
Mitogen-activated protein kinase kinase (also known as MAP2K, MEK, MAPKK) is a kinase enzyme which phosphorylates mitogen-activated protein kinase (MAPK). MAP2K is classified as EC 2.7.

What does a kinase assay measure?

A kinase assay works by simply measuring the activity of the kinase. A kinase is an enzyme that aids in the transfer of a phosphate from ATP to another specific molecule. They regulate many activities in the human body. The measurement is used to test potential drugs to see how they affect kinase activity.

Is MEK a protein kinase?

MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). Several phosphorylation sites present in MEK-1 immunoprecipitated from 32P-labeled cells after stimulation with epidermal growth factor were common to the in vitro phosphorylated enzyme.

What should the substrate concentration be for a kinase assay?

For initial reactions, a substrate concentration of 0.7-1.5 mM should be used. To determine the kinetic parameters for phosphorylation of the synthetic peptide, a range of peptide concentrations is required

How to conduct a kinase assay in a microcentrifuge?

Conduct the kinase assay as above, this time use different concentrations of the substrates starting from 50nM to 1mM. Stop the reaction after 30 minutes by adding 45 ul ice-cold 10% trichloroacetic acid (TCA) to each reaction. Vortex the reactions. 3. Spin for 2 minutes in microcentrifuge (10K rpm). 4.

How is phosphorylation of a substrate determined by a MAPK?

The kinetic parameters for phosphorylation of a substrate by a MAPK can be determined using a variation on the protocol above. 1. Carry out a reaction at a high concentration of substrate to establish that the protein is a substrate. 2. Vary the enzyme concentration in the assay.

Which is the best solution for protein kinase?

Protein Kinase (stock solutions of 1-10 mg/ml pure kinases) – for these assays, I use purified ERK2 kinase (NEB). For each enzyme, it is important to determine the optimal buffer, ionic strength, and pH for activity.