Hello everyone
I am having the question for kinase assay. Exactly, I want to measure the kinetic activity of enzymes, can anyone suggest me some protocol or paper about measuring the kinetic activity?
I have previously been asked from a Hospital that an assay method of protein kinase by using HPLC method, but research fund has been not available at that time. An HPLC method is a uniquely quantitative one to precisely determine the enzyme activity and the enzyme kinetic-parameters (please see file; J Chrom B rat BIN LIP Km).
I have planned to use RP-HPLC method (please see file; Lysozyme by RP-HPLC) and/or
PDMD (Protein-Direct-Microsequencing-Deciphering) method (please see file ; HepG2 Fucoidan), both of these methods are quantitative.
Can you give some more details of your system? Which kinase is it? Do you have the purified enzyme? Do you know what the substrate is? Do you have access to the substrate? How much of the substrate is available?
Thank you for all of the recommendations.
Adam B Shapiro: I had to purify kinase enzyme (in my case, CK1 and GSK3b). I got the really good concentration of these proteins and also detected by Coomassie Brilliant Blue. However, I need to know my purified proteins which have kinase activity or not Until now, I think that I can use MBP as the substrate for kinase enzyme. However, I can not check any protocol for this experiments, design them, and expect how much of the substrate should be used and enough for kinase activity.
If you use a protein, such as MBP, as a substrate, then you will probably have to use gamma-phosphate-labeled radioactive ATP as the other substrate. The assay will consist of measuring the incorporation of radioactive phosphate into MBP. This could be done by SDS-PAGE and autoradiography, for example.
A nonradioactive method for detecting protein phosphorylation is intact protein mass spectrometry, which obviously requires specialized equipment and expertise.
There are commercial kits for measuring kinase activity by various methods, if you can afford them, and if you have the equipment (i.e. plate readers) needed to use them.
- Badges
- Science topic
- Similar topics
- Papers