A Nonradioactive Assay for the Activity of the Stress Activated Protein Kinase

ObjectiveTo establish a nonradio active assay for determining activity of stress-activated protein kinase.MethodsA new non radioactive assay for measuring SAPK activity was established as follow:SPAK was pulled down from cell lysates using c-Jun fusion protein beads.The kinase reaction was carried out in the presence of cold ATP.The level of c-Jun phosphorylation was specifically determined with western immunoblotting of phosphoc-Jun antibody and chemiluminescent detection system.In this detect ion system,the activity of SAPK was indirectly reflected by the level of c-Jun phosphory lation which was the substract of phosphorylation.ResultsAs the primarily cultured fasciculataglomerulosa(FG)cells of males guinea pig were incubated with 25μmol/L~100μmol/L cadmium chloride for two hours,the activity of SAPK was showed to have a mildly elevation as the dosage increased.ConclusionsThe protocol for activity assay is an effective method with the features of highly sensitivity and specificity.