Rapid detection method for RSK4 enzyme activity and application thereof

Abstract

The invention relates to a rapid detection method for RSK4 enzyme activity and application thereof. The detection method comprises the follows steps: (1) taking a sample to be tested, a blank controland a positive control, and respectively co-incubating with RSK4 enzyme and an RSK polypeptide substrate / ATP mixed liquor to carry out a kinase reaction, thus producing ADP; (2) respectively adding anADP-Glo<TM> reaction reagent into three groups of kinase reaction systems in step (1) to co-incubate, terminating the kinase reaction and exhausting the remaining ATP; (3) respectively adding a kinase detection reagent to the three reaction systems of step (2), co-incubating to convert ADP into ATP, and reading a luminescence value RLU of newly synthesized ATP; (4) calculating the enzyme activityaccording to the following formula: enzyme activity=(RLU(Sample)-RLU(Blank)) / (RLU(Pos.Ctrl)-RLU(Blank)) is multiplied by 100%. The method is based on an ADP-Glo<TM> kinase assay to directly measure the ATP consumed in the reaction to quantify the progress of the reaction. The method is operated under ordinary laboratory conditions to avoid radiation injury to operators and the environment; the system is stable, measured data has high accuracy, and the method can be applied to screening of RSK4 target inhibitors and screening of RSK4 target anti-tumor drugs.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a rapid detection method of RSK4 enzyme activity and its application. Background technique [0002] The protein size of RSK4 is 90-kDa. It is an important member of the ribosomal S6 kinase family and belongs to the growth factor that regulates the proliferation, survival and differentiation of cell growth. RSK4 is activated after phosphorylation by ERK and PDK1. RSK4 is often associated with consecutive genetic syndromes including X-linked deafness (dfn3), mental retardation (mrx), and choroideremia (chm). RSK4 is normally highly expressed in the brain and kidney, and in patients with colon cancer, the expression of RSK4 is significantly increased. In patients with non-small cell lung cancer, the content of RSK4 in cancerous tissues was significantly lower than that in normal tissues, and RSK4 constituted a putative NSCLC tumor suppressor gene in preliminary cl...

AI Technical Summary

Problems solved by technology

The use of isotope methods to evaluate the activity of RSK4 compounds requires laboratory hardware and qualifications, which limits the development of experiments

Moreover, isotope reagents are expensive, and the cost of reagent waste treatment is high, so it is not suitable for large-scale screening experiments to evaluate the activity of compound libraries.

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