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Method for determination of activity of protein kinase PKA based on colorimetric method or electrochemical method

A protein kinase and colorimetric technology, applied in the field of biosensing, can solve the problems of radioactive contamination, high cost, and low specificity.

Inactive Publication Date: 2016-12-21
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] People have developed a large number of methods for detecting PKA activity, such as electrochemical methods, fluorescence methods, surface plasmon resonance methods, radioactive isotope labeling methods, and immunoassays. These detection methods and methods have promoted the research on protein kinase activity to a certain extent. , but each has certain limitations
Radioactive element labeling 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, low specificity, and high cost

Method used

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  • Method for determination of activity of protein kinase PKA based on colorimetric method or electrochemical method
  • Method for determination of activity of protein kinase PKA based on colorimetric method or electrochemical method
  • Method for determination of activity of protein kinase PKA based on colorimetric method or electrochemical method

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Effect test

Embodiment 1

[0078] With cytosine-rich single-stranded DNA (CCC CCC CCC CCC, dC 12 ) At the same time as a template and stabilizer, NaBH 4 Reduce AgNO 3 Synthesize AgNCs with good water solubility and stable fluorescence intensity. First, centrifuge the DNA for 30-60 min at 4℃ at a speed of 12000r / min, add 127μL of secondary water to dilute it in 1OD DNA to make the concentration reach 100μM, and then use the secondary water with 1mM AgNO 3 , Keep in the refrigerator at 4℃ in the dark. Take 18μL 1mM AgNO 3 Solution and 30μL 100μM DNA (of which Ag + :DNA=6:1) Add to a brown centrifuge tube containing 34μL of secondary water, shake for 30-60min at room temperature, and then add 18μL of freshly prepared 1mM NaBH 4 (Ice secondary water as solvent) quickly add the above DNA and AgNO 3 In the mixture, make NaBH 4 Rapid reduction of Ag + For AgNCs. The final reaction solution is stored in a refrigerator at 4°C and protected from light for 3 to 4 hours to synthesize AgNCs.

Embodiment 2

[0080] Place a 3mm diameter glassy carbon electrode containing 0.05μm Al 2 O 3 Polished on the polishing cloth, the polished electrode is rinsed with secondary water, and then ultrasonically cleaned with absolute ethanol and secondary water for 3 to 5 minutes. The polishing powder attached to the electrode surface can be removed, and then blown with nitrogen. dry. Put the processed glassy carbon electrode, Ag / AgCl reference electrode and platinum wire counter electrode together into a 5mmol·L -1 In the reaction cell of the potassium ferricyanide solution, the cyclic voltammetry scan is performed at a voltage of -0.1~0.6V, and the scan speed is set to 0.1V·s -1 , The potential difference is less than 85mV, indicating that the surface of the glassy carbon electrode has been cleaned. Cover the electrode cap and place it in a refrigerator at 4℃ for later use.

Embodiment 3

[0082] ATP can inhibit the silver staining reaction, and its concentration needs to be optimized. Add 50μL of the above-mentioned synthetic AgNCs to the wells of the 96-well plate, and add 50μL of ATP of different concentrations from left to right to make the final concentration 2.5, 5, 10, 20, 40μM, and finally add 100μL of silver stain solution (silver stain Solution A: Silver stain solution B=1:1). The time for observing the blackening was 5, 15, 20, 30, 40, and 90 min. Taking into account the time limit of the test process, 5 μM was selected as the optimal concentration.

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Abstract

The invention discloses a method for determination of activity of protein kinase PKA based on a colorimetric method or an electrochemical method. The method comprises the steps: with a single stranded DNA (dC12) as a template and a stabilizer, reducing AgNO3 by NaBH4 to synthesize silver nanoclusters (AgNCs); carrying out catalytic hydrolysis of ATP with PKA to transfer a gamma phosphate radical to serine of a polypeptide substrate, and combining phosphorylated polypeptide and an AgNCs template DNA by using Zr<4+>, and constructing a molecular probe. The AgNCs as a seed crystal can promote a silver staining reaction, generated silver nanoparticles make the solution turn from light yellow to black, after the polypeptide is wrapped with the AgNCs, the silver staining reaction is suppressed, and the solution discoloration speed slows down; in the same way, the AgNCs can produce an electrical signal on the surface of an electrode, the electrical signal of the AgNCs is greatly enhanced by the silver staining reaction, protein phosphorylation inhibits the occurrence of silver staining, and the electrical signal can be abated; the activity of the protein kinase PKA can be determined through the colorimetric method or the electrochemical method by using the molecular probe, and the method has the advantages of simple operation, high sensitivity, and short detection used time.

Description

Technical field [0001] The invention relates to a method for measuring the activity of protein kinase PKA, in particular to a method for constructing molecular probes and measuring protein kinase activity by colorimetric method or electrochemical method; it belongs to the technical field of biosensing. Background technique [0002] Protein kinase-catalyzed protein phosphorylation is a very important way of protein post-translational modification, which can regulate the function of most proteins in the cell. Phosphorylated proteins are 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 can transfer the γ-phosphate group on adenosine triphosphate (ATP) to specific tyrosine (Tyr), threonine (Thr) and serine (Ser) residues of the substrate protein. Protein kinases are important members of the enzyme family. Normally active protein k...

Claims

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Application Information

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IPC IPC(8): G01N21/78G01N27/26
CPCG01N21/78G01N27/26
Inventor 阳明辉申聪聪张凯娜
Owner CENT SOUTH UNIV
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