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Method for detection of parp-1 activity based on fluorescent dye toto-1 assay

A technology of TOTO-1 and PARP-1, which is applied in the field of quantitative detection of biological enzymes with TOTO-1 fluorescent dyes, can solve the problems of cumbersome synthesis and achieve the effects of simplifying detection methods, reducing virus detection costs, and avoiding high detection costs

Active Publication Date: 2021-03-30
HENAN UNIVERSITY
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  • Summary
  • 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 detection of parp-1 activity based on fluorescent dye toto-1 assay
  • Method for detection of parp-1 activity based on fluorescent dye toto-1 assay
  • Method for detection of parp-1 activity based on fluorescent dye toto-1 assay

Examples

Experimental program
Comparison scheme
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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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/48G01N21/64
CPCC12Q1/48G01N21/6428
Inventor 刘勇杨海堂卫伟
Owner HENAN UNIVERSITY
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