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Ultrasonication method for heterocephalus glaber tissues in ChIP (Chromatin Immunoprecipitation)

A technology of co-immunoprecipitation and ultrasonic fragmentation, applied in the field of immunoprecipitation, can solve problems such as unsatisfactory experimental results

Active Publication Date: 2017-06-13
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the traditional ChIP experimental process directly performs cell cross-linking, fragmentation and precipitation. However, when this method is applied to chromatin immunoprecipitation of animal tissues, the experimental results are not ideal.

Method used

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  • Ultrasonication method for heterocephalus glaber tissues in ChIP (Chromatin Immunoprecipitation)
  • Ultrasonication method for heterocephalus glaber tissues in ChIP (Chromatin Immunoprecipitation)
  • Ultrasonication method for heterocephalus glaber tissues in ChIP (Chromatin Immunoprecipitation)

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1 Ultrasonic disruption method of naked mole rat muscle tissue in chromatin immunoprecipitation

[0032] 1. Sample preparation

[0033] After the naked mole rats were grouped and subjected to the experimental treatment, the animals were sacrificed at a predetermined time point, and the suprafemoral muscle was surgically cut and quickly put into liquid nitrogen for preservation.

[0034] 2. Exploration of sonication conditions and verification of results in the experimental process of chromatin immunoprecipitation

[0035] The kit used in the experiment was ChIPTM G Tissue Kit (Millipore, USA). Cut an appropriate amount (50-80 mg) of frozen naked mole rat muscle tissue, put it on ice to thaw slowly and chop it into pieces → add 1 ml at a volume ratio of 1:1: 1 Mix collagenase Ⅱ (Sigma, USA), hyaluronidase (Sigma, USA), DPBS (Gibco, USA) solution, digest naked mole rat muscle tissue with shaking in a 37°C water bath for 3h→4°C, centrifuge at 1000rpm for 5min, remov...

Embodiment 2

[0039] In order to test the effect of digesting naked mole rat muscle tissue with a mixed solution of collagenase Ⅱ and hyaluronidase on the final chromatin immunoprecipitation experiment results, the subsequent steps of ChIP experiments were performed on the above two differently processed sonicated products:

[0040] Add the target gene and negative control protein antibody (IgG) to bind to the DNA-protein complex → use magnetic beads to collect the precipitated antibody-protein-DNA complex → wash and elute the complex adsorbed on the magnetic beads → Resolve cross-linking of the complex→purify the precipitated DNA fragments→PCR detection. The ChIP kit used in the experiment is ChIP from Millipore TM G Tissue Kit (Millipore, USA). figure 2 Shown is the comparison of the final ChIP experiment results of naked mole rat muscle tissue digested with collagenase II and hyaluronidase mixed solution and without mixed enzyme digestion under the same experimental conditions.

[00...

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Abstract

The invention relates to the field of immunoprcipitation, and in particular to an ultrasonication method for heterocephalus glaber tissues in ChIP (Chromatin Immunoprecipitation). The ultrasonication method comprises the following steps: firstly adding a mixed solution of a collagenase II solution, a hyaluronidase solution and a DPBS (Dulbecco's Phosphate Buffered Saline) buffer solution which are 1 to 1 to 1 in a volume ratio in the heterocephalus glaber tissues, oscillating under 37 DEG C for digesting tissues, centrifuging and removing supernatant, and then carrying out the next conventional ultrasonication step. By using the ultrasonication method disclosed by the invention, a DNA (Deoxyribonucleic Acid)-protein complex which is proper in fragment size can be well obtained, and a good foundation is laid for success of a whole follow-up ChIP experiment.

Description

technical field [0001] The invention relates to the field of immunoprecipitation, in particular to an ultrasonic disruption method of naked mole rat tissue in chromatin immunoprecipitation. Background technique [0002] In the complex biological process of living organisms, gene expression is carried out in an orderly manner, which depends on the precise regulation of gene expression. In cells, genomic DNA is combined with histone to exist in the form of chromatin, and the interaction between protein (transcription factor) and DNA is an important condition for the expression of genes to exert their corresponding functions. Therefore, studying the interaction between protein and DNA in the form of chromatin can well clarify the mechanism of gene expression regulation. At present, the commonly used methods for studying DNA-protein interaction include gel retardation experiment, methylation interference experiment, electrophoretic mobility shift analysis, footprinting method, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N1/06C12N13/00
CPCC12N1/066C12N5/0602C12N13/00C12N2509/00
Inventor 崔淑芳肖邦程继帅赵善民林丽芳杨文静余琛琳丛薇徐晨蔡丽萍孙伟
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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