I have never been taught what a kinase assay is and my online searches only point me int the direction of buying such assays. My question is: what is a kinase assay and how is one performed? Sorry if this is a basic question just have never done one but would like to know more about it.
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Posted 15 March 2009 - 01:58 PM
Its a protein coupled assay. YOu take the protein and its two subtrates, one of them as to be ATP. The other two proteins are pyruvate kinase and lactate dehydrogenase. When ATP goes to ADP, pyruvate kinase takes phosphoenolpyruvate and ADP and makes pyruvate. Then LDH takes NADH and pyruvate and makes NAD and lactate. The trick is montoring this using UV-Vis and monitoring absorbance decrease at 340 (which is abosorbance of NADH). This is all dependent on the rate of the initial enzyme.