Method for screening kinase inhibitor by utilizing HTRF one-step method

Abstract

The invention discloses a method for screening a kinase inhibitor by utilizing an HTRF one-step method, and belongs to the field of inhibitor screening methods. The method for screening the kinase inhibitor by utilizing the HTRF one-step method comprises the following steps: sequentially adding a compound to be detected, a kinase, a substrate and ATP into a microporous plate for co-incubation; sequentially adding an antibody coupled with a recognition substrate phosphorylation site of Donor and streptavidin coupled with biotin on a recognition substrate of Acceptor into the microporous plate;incubating at room temperature, and detecting fluorescence by using an enzyme marker reading, wherein the ratio of 665nm / 620nm is original data; according to the method, screening the kinase inhibitor at the biochemical level can be achieved, and constructing cells is not needed, and the method is more direct and quicker.

Description

technical field [0001] The invention belongs to the field of inhibitor screening methods, in particular to a method for screening kinase inhibitors using HTRF in one step. Background technique [0002] In biochemistry, protein kinases are a class of enzymes that catalyze the phosphorylation of proteins. It can catalyze the transfer of a phosphate group from adenosine triphosphate (ATP) and covalently bind to a specific substrate, thereby changing the conformation and activity of the substrate. Protein kinases can be divided into two categories: tyrosine kinases and serine / threonine kinases according to their selectivity for substrate phosphorylation sites. Protein kinases play key regulatory roles in nearly every aspect of cell biology. They are involved in the regulation of apoptosis, cell cycle progression, cytoskeletal rearrangement, differentiation, development, immune response, nervous system function and signal transduction. Once the kinase is out of balance, it wil...

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Problems solved by technology

[0005] The radioisotope method has the following disadvantages: 1. It produces a large amount of radioactive waste

It has certain damage to the human body; 2. The half-life of radioactive ATP is very short, only 14 days (32P) or 25 days (33P), so new isotopes need to be ordered frequently; 3. The energy of radioactive isotopes is very high, and there are obvious Cross-interference phenomenon, difficult to apply to high-throughput or ultra-high-throughput screening of compounds

[0006] As a traditional method, ELISA has some shortcomings that are difficult to overcome, such as: 1) There are many experimental steps, it takes a long time, and it needs to go through multiple washings, adding samples, color development, etc., and it takes at least one day; 2) It cannot reach high-pass 3) The coated protein may shield some protein sites, resulting in missing positive results, that is, false negative results; 4) Due to many steps, it is easy to cause experimental errors, leading to poor repeatability, unstable

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