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A method for measuring the activity of protein kinase PKA based on colorimetry or electrochemical method

A technology of protein kinase and colorimetry, applied in the field of biosensing, can solve the problems of radioactive pollution, high requirements and high cost

Inactive Publication Date: 2019-07-16
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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  • A method for measuring the activity of protein kinase PKA based on colorimetry or electrochemical method
  • A method for measuring the activity of protein kinase PKA based on colorimetry or electrochemical method
  • A method for measuring the activity of protein kinase PKA based on colorimetry or electrochemical method

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Experimental program
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Effect test

Embodiment 1

[0078] Cytosine-rich single-stranded DNA (CCC CCC CCC CCC, dC 12 ) as both template and stabilizer, NaBH 4 reduced AgNO 3 Synthesis of AgNCs with good water solubility and stable fluorescence intensity. First, centrifuge DNA at 4°C for 30-60 min at a speed of 12,000 r / min, add 127 μL of secondary water to dilute 1OD of DNA to a concentration of 100 μM, and then mix 1 mM AgNO with secondary water. 3 , protected from light and stored in a refrigerator at 4°C for later use. Take 18 μL of 1mM AgNO 3 solution and 30 μL 100 μM DNA (where Ag + :DNA=6:1) was added to a brown centrifuge tube filled with 34 μL of secondary water, shaken at room temperature for 30-60 min, and then 18 μL of newly prepared 1 mM NaBH 4 (ice secondary water as solvent) quickly add the above DNA and AgNO 3 mixed solution, making NaBH 4 Rapid reduction of Ag + for AgNCs. The final reaction solution was stored in a refrigerator at 4°C in the dark for 3 to 4 hours to synthesize AgNCs.

Embodiment 2

[0080] A glassy carbon electrode with a diameter of 3mm is contained in 0.05μm Al 2 o 3 Polish on a polishing cloth, rinse the polished electrode with secondary water, and then ultrasonically clean it with absolute ethanol and secondary water for 3 to 5 minutes to remove the polishing powder attached to the surface of the electrode, and then blow it with nitrogen Dry. Put the treated glassy carbon electrode, Ag / AgCl reference electrode and platinum wire counter electrode into a 5mmol·L -1 In the reaction cell of the potassium ferricyanide solution, the cyclic voltammetry scan was performed at a voltage of -0.1 to 0.6V, and the scan speed was set to 0.1V s -1 , the potential difference is less than 85mV, indicating that the surface of the glassy carbon electrode is cleaned, and the electrode cap is covered and placed in a refrigerator at 4°C for use.

Embodiment 3

[0082] ATP can inhibit the occurrence of silver staining reaction, and its concentration needs to be optimized. First add 50 μL of the above-synthesized AgNCs to the wells of a 96-well plate, then add 50 μL of ATP with different concentrations from left to right to make the final concentrations 2.5, 5, 10, 20, and 40 μM, and finally add 100 μL of silver staining solution (silver staining solution). Solution A: silver staining solution B=1:1). The time to observe the blackening was 5, 15, 20, 30, 40, and 90 min, respectively, and 5 μM was selected as the optimal concentration considering the time limit of the test process.

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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 a molecular probe and measuring the activity of protein kinase by using a colorimetric method or an electrochemical method; it belongs to the technical field of biosensing. 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. 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 transfers the γ-phosphate group on adenosine triphosphate (ATP) to specific tyrosine (Tyr), threonine (Thr) and serine (Ser) residues in substrate proteins. Protein kinases are important members of the enzyme family. The protein ki...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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