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Chromosome immunoprecipitation method and kit for DNA binding proteins in eukaryotic cells

A technology of co-immunoprecipitation and eukaryotic cells, which is applied in the field of chromosomal co-immunoprecipitation of DNA-binding proteins in eukaryotic cells and related kits, which can solve the problems affecting analysis and detection, different binding abilities, different target DNA, etc. question

Inactive Publication Date: 2019-02-15
上海微斯生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, protein A or protein G specifically binds to the Fc region of IgG. If the specific binding of protein A or protein G to the Fc region of IgG affects the antigen binding site of the antibody, it will greatly affect the enrichment of the target DNA, thereby affecting the subsequent Analysis and detection; and even protein A and protein G, which also bind to the Fc region of IgG, have different binding abilities. The binding ability of protein G is greater than that of protein A. Therefore, the amount of enriched target DNA is also will be different

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  • Chromosome immunoprecipitation method and kit for DNA binding proteins in eukaryotic cells

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Embodiment 1

[0111] 1. Get 10 grams of neural brain tissue from 8 mice, and wash with PBS (137mM NaCl; 2.7mM KCl; 10mM NaCl 2 HPO 4 12H 2 O; 2 mM KH 2 PO 4 ; 0.1% by weight PMSF) was dispersed into 990ml cell suspension by pipetting and washing.

[0112] 2. Add 10ml formaldehyde to a final concentration of 1% (v / v), add 1ml 2M glycine to neutralize the cross-linking effect after 5 minutes, centrifuge and discard the supernatant, wash with PBS, centrifuge and discard the supernatant, the number of eukaryotic cells is about 10 8 indivual.

[0113] 3. Add 500 μl 1% SDS lysate and place on ice for 10 minutes.

[0114] 4. Ultrasound to disrupt cells, the ultrasonic program is as follows: 30S on, 30S off, 9 ultrasonic cycles, ultrasonic frequency 60kHz. After the sonication is over, centrifuge to take the supernatant and discard the precipitate.

[0115] 5. Pretreat the agarose beads coated with protein A, protein G and protein L (use the first washing solution to wash the agarose beads ...

Embodiment 2

[0122] 1. Take 10 grams of liver tissue from 2 mice, wash with PBS (137mM NaCl; 2.7mM KCl; 10mM NaCl 2 HPO 4 12H 2 O; 2 mM KH 2 PO 4 ; 0.1% by weight PMSF was dispersed into 999ml cell suspension by pipetting and washing.

[0123] 2. Add 1ml of formaldehyde to a final concentration of 0.1% (v / v), add 1ml of 2M glycine to neutralize the cross-linking effect after 10 minutes, centrifuge and discard the supernatant, wash with PBS, centrifuge and discard the supernatant, the number of eukaryotic cells is about 5×10 7 indivual.

[0124] 3. Add 500 μl 1% SDS lysate and place on ice for 15 minutes.

[0125] 4. Ultrasonic disrupts the cells. The ultrasonic program is as follows: 20S on, 45S off, 8 ultrasonic cycles, and the ultrasonic frequency is 40kHz. After the sonication is over, centrifuge to take the supernatant and discard the precipitate.

[0126] 5. Pretreat the agarose beads coated with protein A, protein G and protein L (wash the agarose beads for 1 hour with the fi...

Embodiment 3

[0133] 1. Take the spleen tissues of 30 mice (10 grams in total), wash with PBS (137mM NaCl; 2.7mM KCl; 10mM NaCl 2 HPO 4 12H 2 O; 2 mM KH 2 PO 4 ; 0.1% by weight PMSF) was dispersed into 950ml cell suspension by pipetting and washing.

[0134] 2. Add 50ml formaldehyde to a final concentration of 5% (v / v), add 1ml 2M glycine to neutralize the cross-linking effect after 20 minutes, centrifuge and discard the supernatant, wash with PBS, centrifuge and discard the supernatant, the number of eukaryotic cells is about 15×10 7 indivual.

[0135] 3. Add 500 μl 1% SDS lysate and place on ice for 8 minutes.

[0136] 4. Ultrasound to disrupt cells, the ultrasonic program is as follows: 40S on, 60S off, 15 ultrasonic cycles, ultrasonic frequency 20kHz. After the sonication is over, centrifuge to take the supernatant and discard the precipitate.

[0137] 5. Pretreat the agarose beads coated with protein A, protein G and protein L (use the first washing solution to wash the agarose...

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Abstract

The invention provides a chromosome immunoprecipitation method for DNA binding proteins in eukaryotic cells. The method comprises the following steps: crosslinking proteins and chromatins in the eukaryotic cells of eukaryotic cell suspension; pyrolyzing the eukaryotic cells; shearing a released crosslinked protein-chromatin complex; adding magnetic beads respectively coated with protein A, proteinG and protein L, or adding magnetic beads coated with protein A, protein G and protein L, and adding a desired antibody to the DNA-binding proteins; performing incubation; washing the magnetic beads;eluting the magnetic beads; performing decrosslinking; extracting and purifying DNA. The invention further provides a related kit. The chromosome immunoprecipitation method and kit for the DNA binding proteins in the eukaryotic cells can enrich a large number of target DNAs, and fragments can be well controlled between 100 and 500 bp, which is beneficial to improving the detection effect of scientific research and clinical samples and is favorable for the development of epigenetic research and clinical biomedicines and the detection of results. The method and kit are ingenious in design, operated easily and quickly, low in cost and suitable for large-scale popularization and application.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to the technical field of DNA extraction and purification, and specifically refers to a method for chromosome co-immunoprecipitation of DNA-binding proteins in eukaryotic cells and related kits. Background technique [0002] The genomic DNA of eukaryotes exists in the form of chromatin, so studying the interaction between proteins and DNA in the chromatin environment is the basic way to elucidate the gene expression mechanism of eukaryotes. Chromatin immunoprecipitation (chromatinimmunoprecipitation, CHIP) is currently the only method for studying the interaction between DNA and protein in vivo. Its principle is to cross-link protein-DNA complexes in the state of living cells and cut them into small fragments, and then specifically enrich the DNA fragments by immunological methods, so as to obtain information on the interaction between proteins and DNA. Moreover, the comb...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1013
Inventor 安磊丁炳谦项光刚
Owner 上海微斯生物科技有限公司
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