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

A detection method and enzyme activity technology, applied in the field of biochemistry, can solve problems such as abnormal protein phosphorylation, and achieve the effects of simple steps, shortened detection time, and low sample consumption.

Pending Publication Date: 2019-09-03
武汉合研生物医药科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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

Method used

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

Examples

Experimental program
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Embodiment 1

[0036] Example 1: Rapid detection method of TGFβR1 (T204D) enzyme activity

[0037] In this example, TGFβR1 (T204D) recombinant protein, TGFβR1 substrate, 5X polypeptide buffer and DTT were all purchased from Signalchem ​​Company, ADP-Glo TM Kinase reagents were purchased from Promega, and Staurosprine was purchased from MCE.

[0038] 1. A rapid detection method for TGFβR1 (T204D) enzyme activity, comprising the following steps:

[0039] (1) Take the sample to be tested, the blank control substance, and the positive control substance and incubate with the TGFβR1 (T204D) enzyme reagent and the TGFβR1 substrate / ATP mixture respectively to perform the kinase reaction to generate ADP; the sample to be tested, the positive control substance, and the TGFβR1 (T204D) ) Enzyme reagent, TGFβR1 substrate / ATP mixture all use buffer as solvent, wherein the positive control substance is a solution formed by dissolving staurosporine, a universal kinase inhibitor, in buffer, with a concentrati...

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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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/573
CPCG01N33/573G01N2333/90
Inventor 石榴徐燕华
Owner 武汉合研生物医药科技有限公司
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