A method for analyzing protein kinase activity based on fluorescence quenching

Abstract

The invention discloses a fluorescence quenching-based protein kinase activity analysis method. Firstly target protein kinase and a corresponding fluorescence labeling polypeptide substrate are mixed, and phosphorylation reaction is performed on a specific amino acid site in polypeptide of the substrate; then the generated phosphorylated fluorescence labeling polypeptide substrate is quickly and highly selectively adsorbed by using nanometer cerium oxide, so as to cause fluorescence quenching of the substrate, wherein interaction between the non-phosphorylated fluorescence labeling polypeptide substrate and the cerium oxide is very weak and cannot cause quenching of fluorescence signals, and thus accurate analysis on protein kinase activity can be realized by monitoring the quenching situation of the fluorescence signals in a system. By only mixing the protein kinase reaction system and the nanometer cerium oxide, the fluorescence signals can be directly measured, and immediate-mixing immediate-detecting quick analysis of the protein kinase activity is realized by simple and convenient operation steps. Meanwhile, the method can be applied to screening of protein kinase inhibitor.

Description

technical field [0001] The invention belongs to the technical field of biomolecular detection, and specifically relates to a method for detecting protein kinase activity of a mix-and-measure protein kinase with simple operation and low cost, using NanoCeria as a phosphorylated polypeptide-specific recognition and fluorescence quenching element . Background technique [0002] Cell signal transduction refers to the combination of extracellular signal molecules with cell surface or intracellular receptors, triggering intracellular cascade reactions, and then regulating the activity of specific intracellular proteases or inducing the expression of specific genes, so that cells respond the process of. In this process, protein phosphorylation plays a vital role. Most of the life processes in cells, such as metabolism, material movement, growth, development, apoptosis, and neural activity, are closely related to protein phosphorylation. [0003] Protein kinases are the most impor...

AI Technical Summary

Problems solved by technology

Although this type of method has made good progress, it still faces the following challenges: First, the preparation of phosphorylated recognition antibodies is complicated, different antibodies are required to detect different kinases, the cost is high, and its activity will be affected by storage environment, reaction The influence of various factors such as medium and personal operation has a great impact on the stability of the test results

Secondly, protein kinase activity detection methods based on antibody recognition often require pre-connected fluorescent or electrochemically active markers on the antibody, which greatly increases the complexity of the operation and limits its application range.

In addition, most phosphorylated recognition antibodies are active proteins. When using this kind of kinase activity detection method to screen and develop enzyme inhibitor drugs, it is sometimes difficult to distinguish whether the inhibitory effect is derived from the inhibition of the kinase by the molecule to be screened or the inhibition of antibody activity. , not suitable for large-scale inhibitor drug screening

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