Quick detection method of TGFbetaR1(T204D) enzyme activity, and application thereof

Abstract

The invention belongs to the technical field of the biochemistry, and specifically relates to a quick detection method of TGFbetaR1(T204D) enzyme activity, and an application thereof. The detection method comprises the following steps: (1) taking each of a to-be-detected sample, a blank reference substance and a positive reference substance to co-incubate with the TGFbetaR1 (T204D) enzyme, TGFbetaR1 primer / ATP mixed solution, performing kinase reaction to produce ADP; (2) adding ADP-GloTM reaction reagent in each of three groups of kinase reaction systems in the step (1) to perform co-incubation; (3) adding the kinase detection reagent in each of three groups of reaction systems in the step (2) to perform co-incubation, transforming the ADP into ATP, and reading a light unit RLU of the newly synthesized ATP; and (4) computing TGFbetaR1(T204D) enzyme activity according to a condition that the enzyme activity is equal to (RLU(Sample)-RLU(Blank)) / (RLU(Pos.Ctrl)-RLU(Blank))*100%. The method is based on the ADP-GloTM kinase experiment, and the reaction progress is quantified by directly determining the ATP consumed in the reaction; all experiments can be accomplished only in one day, and the detection time is greatly shortened. The method can be applied to the screening of TGFbetaR1(T204D) receptor antagonist and the anti-tumor medicine related to the target spot.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a rapid detection method of TGFβR1 (T204D) enzyme activity and an application thereof. Background technique [0002] TGFβR1, also known as TGF-β receptor, belongs to the TGFβR receptor family and is a serine / threonine protein kinase. When the receptor binds to TGF-β and activates, the downstream will transmit the TGF-β signal from the cell surface to the cytoplasm. TGF-β has multiple functions in the TGF-β signaling pathway, including regulating cell proliferation and differentiation, regulating embryonic development, wound healing, promoting the formation of extracellular matrix, etc., and the completion of the above functions requires the help of TGFβR1 receptor. The TGFβR1 receptor acts as a serine / threonine protein kinase that, by transferring phosphate residues to amino acids of certain intracellular polypeptides, activates these protein substrates and init...

AI Technical Summary

Problems solved by technology

The normally active TGFβR1 receptor is the core of cell signal transduction, but when its activity is abnormal or overexpressed, it will cause abnormal phosphorylation of certain proteins

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