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Rapid detection method of TTK enzyme activity and application of rapid detection method

A detection method and enzyme activity technology, applied in the field of biochemistry, can solve the problems of long and unsuitable experimental operation steps, and achieve the effect of low sample consumption, increased throughput, and extremely high sample consumption

Inactive Publication Date: 2019-08-16
武汉合研生物医药科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

During the operation of ELISA method, there are many plate washing steps, which are not suitable for 384-well plate; and the experimental operation steps are long, so it is not suitable for a large number of parallel test plate operations in one day

Method used

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  • Rapid detection method of TTK enzyme activity and application of rapid detection method
  • Rapid detection method of TTK enzyme activity and application of rapid detection method
  • Rapid detection method of TTK enzyme activity and application of rapid detection method

Examples

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Effect test

Embodiment 1

[0038] Embodiment 1: the rapid detection method of TTK enzymatic activity

[0039] In this example, TTK enzyme, MBP protein, 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.

[0040] 1. A rapid detection method for TTK enzyme activity, comprising the following steps:

[0041](1) Take the sample to be tested, blank control substance, and positive control substance and incubate with TTK enzyme reagent and MBP protein / ATP mixture respectively for kinase reaction to generate ADP; the sample to be tested, positive control substance, TTK enzyme reagent, MBP protein / ATP mixture All ATP mixtures use buffer as solvent, and the positive control substance is a solution formed by dissolving staurosporine, a universal kinase inhibitor, in buffer, with a concentration of 50μM-0.64nM; the concentration of TTK enzyme reagent is 1.0-5.0ng / μl , the concentration of the s...

Embodiment 2

[0082] Example 2: The detection method of the present invention screens two compounds, CC-671 and CFI-402257, for TTK target inhibitors

[0083] Get CC-671 and CFI-402257 to experiment according to the method of embodiment 1 respectively, and calculate the enzymatic activity and IC50 value of two kinds of compounds to TTK, such as image 3 shown. The IC50 of CC-671 and CFI-402257 are 4.26nM and 0.18nM, respectively.

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Abstract

The invention relates to a rapid detection method of TTK enzyme activity and an application of the rapid detection method. The detection method includes 1), taking a to-be-detected sample, blank control and positive control to perform co-incubation with TTK enzyme and MBP / ATP (mannose-binding protein / adenosine triphosphate) mixture respectively to produce ADP (adenosine diphosphate); 2), respectively adding ADP-Glo<TM> reaction reagents to three groups of kinase reaction systems for co-incubation, and terminating the kinase reaction and consuming the remaining TAP; 3), respectively adding kinase detection reagents to the reaction systems for co-incubation to convert ADP to ATP, and reading the luminous value RLU of the newly synthesized ATP; 4), calculating the TTK enzyme activity according to the formula: the enzyme activity=(RLU(Sample)-RLU(Blank)) / (RLU(Pos.Ctrl)-RLU(Blank))X100%. On the basis of ADP-Glo<TM> kinase experiment, the reaction process is quantified by direct direction ofATP consumed in the reaction; the reaction is carried out in 384-hole plates, which does not involve repeated suction and washing process, the operation error is small, the flux is increased, and themethod is suitable for a large number of microhole plate operation combined with high-flux liquid transfer workstation equipment; the method is stable in system, high in precision and can be used forscreening TTK target inhibitors and TTK target related drugs.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a rapid detection method for TTK enzyme activity and an application thereof. Background technique [0002] TTK is a protein kinase that phosphorylates tyrosine, serine, and threonine. It is a spindle checkpoint complex, and its main biological function is to participate in the replication of centrosomes and spindle checkpoints. During mitosis, this protein is required for overlapping of the center of chromosome arrangement at the centriole. It was discovered as a mitotic checkpoint protein for precise segregation of chromosomes during mitosis. When this protein fails to degrade and excess centrosomes are produced, abnormal mitotic spindles are produced, which can lead to cancer development. [0003] The study found that the dual-specificity protein kinase TTK is a potential substrate of USP9X. Further experimental evidence confirmed that USP9X stabilizes TTK p...

Claims

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Application Information

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IPC IPC(8): C12Q1/48
CPCC12Q1/485
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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