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DNA binding protein magnetic nanoparticle separation system and preparation and application thereof

A magnetic nanoparticle and protein binding technology, applied in the biological field, can solve the problems of high cost, antibodies affecting the binding of DNA and protein, and health effects, and achieve the effects of mild conditions, good separation platform, and simple operation.

Inactive Publication Date: 2007-10-03
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a DNA-binding protein magnetic nanoparticle separation system, to overcome the existing gel hysteresis experiment can only verify the interaction between known proteins and DNA, and isotope labeling is used in the experiment, It has an impact on the health of the experimenter; the chromatin immunoprecipitation method needs to guess the protein factor that interacts with DNA in advance, and the monoclonal antibody of the protein factor is used, which is costly. In addition, the addition of the antibody may affect the DNA and protein. combined defects

Method used

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  • DNA binding protein magnetic nanoparticle separation system and preparation and application thereof
  • DNA binding protein magnetic nanoparticle separation system and preparation and application thereof
  • DNA binding protein magnetic nanoparticle separation system and preparation and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Preparation of Magnetic Nanoparticles with Amino Surface Modification

[0045] Methanol, glycerol and ddH 2 O is mixed in a ratio of 100:60:1, and the surface is coated with SiO 2 Add 20mg of magnetic nanoparticles to the above solution, disperse evenly by ultrasonication for 20-50min, add 1-5mL AEAPS dropwise into the mixture, mix by ultrasonication for 5-10min, and stir in a water bath at 50-80°C for more than 5hr. The magnetic nanoparticles are separated by paramagnetism by applying an external magnetic field, washed with methanol, and vacuum-dried overnight at 40-90° C. to collect the magnetic nanoparticles.

Embodiment 2

[0047] Surface modification of magnetic nanoparticles with Strepavidin

[0048] Add 3 mg of amino-modified MNP to 500 μL of phosphate buffer (such as 0.1 mol / L, pH 7.0) around pH 7.0, disperse evenly by ultrasonication, add 75 ug of Strepavidin, and shake the solution at room temperature for 24 hours. Add 0.5 mL of glutaraldehyde (25% aqueous solution) and react at room temperature for 2-4 hrs. Wash with phosphate buffer, redisperse the particles in phosphate buffer, and store at 4°C for later use.

Embodiment 3

[0050] Capture of Biotin-modified double-stranded DNA by magnetic nanoparticles modified with Strepavidin on the surface

[0051] 20 μL of the above-mentioned Strepavidin-modified magnetic nanoparticles (containing about 0.15 mg particles) was equilibrated with 10 times the volume of buffer, mixed with 10 pmol 5'-biotin-labeled hCMV MIEP fragment (588 bp), and shaken at room temperature for 30 min. Wash the particles with 10 volumes of buffer.

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Abstract

This invention relates to a separation system of DNA binding protein magnetism nanometer particle, composed by follows component: magnetism nanometer particle with strepto avidin surface finish; double strand DNA noted biotin; Nucleoprotein bonded with double strand DNA; and external magnetic field. The linkage of magnetic nanometer particle and long chain dsDNA is high efficiency and singleness. The protein and DNA keep each other idiosyncratic combine at optimal identify system in the separation process. The magnetic separation elution method has gentle condition, not create damage to sample, and opreation simple. You can use this nanometer separation system to obtain DNA binding protein, supply reliable technology platform for DNA and protein interaction research.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a system for separating DNA-binding proteins by using nanoparticles. Background technique [0002] The interaction between DNA and protein contains a large amount of life activity information, which is an important content of research in the post-genome era. In particular, the research on the recognition of DNA and protein is related to the resolution of the conformation-specific recognition of biological macromolecules and the mechanism of action of protein factors in the process of gene transcription regulation. There are some difficulties in the traditional method of studying the interaction between DNA and protein: gel hysteresis experiment (EMSA) can only verify the interaction between known protein and DNA, and isotope labeling is used in the experiment, which has an impact on the health of the experimenter; staining Co-immunoprecipitation method (CHIP) needs to pre-guess the ...

Claims

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Application Information

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IPC IPC(8): C07K1/14G01N33/68C12Q1/68
Inventor 沈鹤柏王皓月周海清费俭陈伟
Owner SHANGHAI NORMAL UNIVERSITY
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