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Method for measuring activity of protein kinase and method for measuring activity of protein kinase inhibitor

A protein kinase inhibitor and protein kinase technology, applied in the field of biosensing, can solve the problems of inability to carry out tissue labeling, radioactive contamination, and low specificity, and achieve the effects of low investment, short detection time, and convenient operation.

Inactive Publication Date: 2016-05-04
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The traditional method of detecting protein kinase is generally through the degree of protein phosphorylation, using radioactive isotope labeling method, surface plasmon resonance method, mass spectrometry, fluorescence method and immunoassay to detect, these detection methods and methods promote to a certain extent have been used to study the activity of protein kinases, but each has certain limitations
Radioactive element-labeled ATP technology has a direct detection method and high sensitivity, but there is radioactive contamination, and tissue labeling cannot be performed; immunoassays have high requirements for phosphorylated recognition antibodies, and the specificity is not strong and the cost is high

Method used

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  • Method for measuring activity of protein kinase and method for measuring activity of protein kinase inhibitor

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Embodiment 1

[0035]A method for measuring protein kinase activity of the present invention, comprising the following steps:

[0036] (1) Treatment of gold electrodes: A plurality of gold electrodes with a diameter of 2 mm are placed in a layer containing 0.05 μm Al 2 o 3 Polish on a polishing cloth, rinse the polished gold electrode with secondary water, and then ultrasonically clean it with absolute ethanol and secondary water for 5 minutes, remove the polishing powder attached to the surface of the gold electrode, and then dry it with nitrogen. Put the cleaned gold electrode, Ag / AgCl reference electrode and platinum wire counter electrode together into a 5mmol·L -1 In the reaction cell of potassium ferricyanide solution, under the voltage of -0.1-0.6V, perform cyclic voltammetry scanning, and set the scanning speed to 0.1V s -1 , the potential difference reached 85mV, indicating that the surface of the gold electrode was cleaned and could be used for electrode modification. Take 5 μL ...

Embodiment 2

[0041] A method for measuring protein kinase inhibitor activity of the present invention, comprising the following steps:

[0042] (1) Treatment of gold electrodes: the method is the same as in Example 1.

[0043] (2) Optimization of ATP concentration: same as Example 1.

[0044] (3) Determination of the activity of protein kinase inhibitors: PKA inhibitor H-89 with concentrations of 0, 0.01, 0.1, 0.5, 1, 5, 10, and 25 μM was mixed with 50 U / mL of PKA and 250 μM ATP at 37° C. React in a water bath for 1 hour to obtain a group of reaction solutions with different concentrations of PKA inhibitors, and then take 7.5 μL of the above-mentioned reaction solutions with different concentrations of PKA inhibitors and 7.5 μL of 6 mM Na 2 MoO 4 Add it dropwise on the surface of the gold electrode treated in step (1), react for 20min, then dry it in an oven at 60°C, and wash it with 0.5M H 2 SO 4 It is an electrolyte, within the range of 0.1 ~ 0.5V, the frequency sweep square wave vol...

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Abstract

The invention discloses a method for measuring activity of protein kinase. The method mainly comprises the following steps of selecting a group of different concentrations of PKA (protein kinase A) and same concentration of ATP (adenosine triphosphate) to react, so as to obtain a group of PKA reaction solution; respectively dripping the PKA reaction solution and a Na2MoO4 (sodium molybdate) solution to the clean surface of a gold electrode, drying, respectively measuring a square wave voltammetry curve of the gold electrode, obtaining peak current according to the square wave voltammetry curve, and forming a standard curve by the obtained peak current and the PKA concentration corresponding to each gold electrode; finally, calculating the activity of PKA under different conditions according to the standard curve. A method for measuring the activity of a protein kinase inhibitor adopts the same principle as the method of measuring the activity of the protein kinase. The method has the advantages that the operation is simple, the sensitivity is high, the detection range is wide, the detection time is short, the method can be applied to measure the activity of other phosphohydrolase, and a new concept is provided for the efficient, quick and low-cost sieving of multiple types of effective PKA inhibitors.

Description

technical field [0001] The invention relates to the technical field of biosensing, in particular to a method for measuring protein kinase activity and a method for protein kinase inhibitor activity using an electrochemical method. Background technique [0002] Protein kinase-catalyzed protein phosphorylation is a very important post-translational modification of proteins, which can regulate the function of most proteins in cells. Protein phosphorylation is closely related to many physiological processes, such as signal transduction, metabolic regulation, DNA damage repair, gene transcription and cell apoptosis. Protein kinase is a phosphotransferase that transfers the γ-phosphate group on adenosine triphosphate (ATP) to specific tyrosine (Tyr), threonine (Thr) and serine (Ser) residues in substrate proteins. Protein kinase is an important member of the enzyme family. The protein kinase system with normal activity is the core of cell signal transduction, but when its activit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/26
CPCG01N27/26
Inventor 阳明辉申聪聪
Owner CENT SOUTH UNIV
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