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Method for detecting PARP-1 (Poly ADP-Ribose Polymerase-1) activity based on fluorescent dye TOTO-1 analysis

A technology of TOTO-1 and PARP-1 is applied in the application field of TOTO-1 fluorescent dye quantitative detection of biological enzymes, which can solve the problems of complicated synthesis, and achieve the effect of simplifying the detection method, reducing the cost of virus detection and low cost.

Active Publication Date: 2018-07-20
HENAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method requires tedious synthetic
Therefore, this method has many limitations

Method used

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  • Method for detecting PARP-1 (Poly ADP-Ribose Polymerase-1) activity based on fluorescent dye TOTO-1 analysis
  • Method for detecting PARP-1 (Poly ADP-Ribose Polymerase-1) activity based on fluorescent dye TOTO-1 analysis
  • Method for detecting PARP-1 (Poly ADP-Ribose Polymerase-1) activity based on fluorescent dye TOTO-1 analysis

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Experimental program
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Effect test

Embodiment 1

[0039] The analytical method for detecting PARP-1 activity by analyzing fluorescent dyes based on TOTO-1, the detection steps are:

[0040] DNA hybridization step: Add two specific DNA single strands to the DNA hybridization buffer solution (10mM Tris-HCl, pH 7.4, 0.1M NaCl), and slowly cool to room temperature in a 95°C water bath for 5 minutes to form a hybridized double-stranded DNA double strand DNA)

[0041] The steps of PARP-1 catalyzing the synthesis of PAR: PARP-1 is configured into different concentrations with reaction buffer solution, and in the presence of double-stranded DNA, NAD + Reaction buffer solution (50mM Tris-HCl, pH 7.4, 50mM KCl, 2mM MgCl 2 , and 50μM Zn(OAc) 2 ) was added dropwise with 0.1U PARP-1, and reacted at 37°C for 1h.

[0042] ExoⅢ excises the DNA double strand and releases the amplified PAR step: add 2 μL of ExoⅢ solution dissolved in 10X ExoⅢ buffer solution, the final concentration of ExoⅢ is 1.6 U / μL, and react at 37°C for two hours.

[...

Embodiment 2

[0050] The difference between the double-stranded DNA and Example 1 is that PARP-1 (1U=45ng) of different concentrations is selected: (a) 0 (b) 0.02 (c) 0.1 (d) 0.2 (e) 0.5 (f) 0.8 (g )1.1(h)1.5(i)2(j)3, get the fluorescence spectrum as Figure 5 As shown, it can be seen that in the range of 0-3U, PARP-1 has a good linear relationship at 0.02-1.5U, and the detection limit is 0.02U.

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Abstract

The invention discloses a method for detecting PARP-1 (Poly ADP-Ribose Polymerase-1) activity based on fluorescent dye TOTO-1 analysis. The method comprises the following steps: (1) mixing and reacting activated DNA, PARP-1 (Poly ADP-Ribose Polymerase-1) and NAD<+> (Nicotinamide Adenine Dinucleotide), and realizing catalyzed synthesis of a PAR polymer (Poly ADP-Ribose) with lots of negative charges by PARP-1; (2) excising double strands from the obtained product by using ExoIII to release PAR; (3) acting TOTO-1 (Thiazole Orange Dimer-1) and the product PAR polymer, and detecting the product solution by utilizing a fluorescence spectrophotometer. The fluorescence signal intensity change is observed by utilizing enhancement of a fluorescence signal generated by combining the TOTO-1 and the product PAR polymer, so the method can be used for detecting the PARP-1. The method disclosed by the invention has the advantages of being simple, convenient, rapid, high in sensitivity and capable ofavoiding from labeling a DNA probe.

Description

technical field [0001] The invention belongs to a technology for quantitatively detecting the activity of PARP-1 (polyadenosine diphosphate-ribose polymerase-1), through the adsorption of TOTO-1 and PAR, the enhancement of the fluorescence signal of TOTO-1 is caused, and the detection of the fluorescence signal is realized The application in clinical detection specifically relates to the application field of TOTO-1 fluorescent dye quantitative detection of biological enzymes. Background technique [0002] PARP, also known as poly ADP ribose polymerase, is a class of protein post-translational modification enzymes present in most eukaryotic cells. This family contains 18 enzymes, of which PARP-1 has the highest content. It is a protein in normal physiological activities and diseases. Regulators of cell function that play a key role in the occurrence and development of cells. It plays an important regulatory role in gene stability and tumor development, inflammation and stres...

Claims

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Application Information

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IPC IPC(8): C12Q1/48G01N21/64
CPCC12Q1/48G01N21/6428
Inventor 刘勇杨海堂卫伟
Owner HENAN UNIVERSITY
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