Ultrasonic crushing method for pig adipose tissue and application of ultrasonic crushing method

Abstract

The invention belongs to the field of co-immunoprecipitation chromatin of chromatins and particularly relates to an ultrasonic crushing method for a pig adipose tissue and application of the ultrasonic crushing method. The method comprises the following steps: (1) carrying out ice formaldehyde cross-linking reaction on the pig adipose tissue; (2) stopping cross-linking reaction through glycine, and then carrying out centrifugation and ice-bath layering; (3) acquiring a cell deposition layer of the pig adipose tissue; (4) extracting a cell nucleus deposition layer; and (5) ultrasonically breaking chromatins, so as to obtain small fragments of a protein-nucleic acid compound. The ultrasonic crushing method has the beneficial effects that a low-temperature condition is maintained in the wholeexperiment operation, so that a natural structure of a protein is guaranteed to a great extent, and the DNA-protein compound adsorbed with a real antibody is obtained; the obtained small fragments ofthe protein-nucleic acid compound can be directly applied to subsequent ChIP, the concentration of obtained DNA is determined, and the combination condition between DNA and a target protein in a whole genome range is detected by virtue of high-flux sequencing; and DNA obtained through enrichment is used for establishing a bank, is subjected to second-generation sequencing and is applied to the bioinformatics analysis of DNA of the pig adipose tissue.

Description

technical field [0001] The invention belongs to the field of chromatin immunoprecipitation, and in particular relates to a method for ultrasonic fragmentation of porcine adipose tissue and its application. Background technique [0002] The regulation of biological processes in life is very complex and orderly. Most of the genetic information of living organisms exists in cellular chromatin, and in the biological activities of living organisms, the interaction between proteins and DNA is an important basis for precise regulation of cells. At present, the commonly used methods to detect DNA-protein interactions in vitro include footprinting method, electrophoretic mobility shift assay, DNA-Western blotting, yeast one-hybrid assay, phage display technology, etc. (Dey B, Thukral S, Krishnan S, Chakrobarty M , Gupta S, Manghani C, Rani V. DNA-protein interaction: methods for detection and analysis. Molecular and Cellular Biochemistry. 08 Mar 2012, 365(1-2): 279-299), but none of...

AI Technical Summary

Problems solved by technology

[0004] The pretreatment method of tissue samples in ChIP technology is mainly to cut the tissue samples into pieces, mix them with enzymes such as collagenase and hyaluronidase, digest them at 37°C for 3 hours, and centrifuge the filtrate obtained by conventional formaldehyde fixation and ultrasonic crushing. When this method is used to deal with adipose tissue, the experimental results are very unsatisfactory

Adipose tissue contains a large amount of fat, which makes it difficult for small adipose tissue to precipitate during centrifugation, and the standard ChIP operation procedure requires the use of SDS lysis buffer as the buffer for sonication, but the presence of a large amount of fat seriously affects the sonication efficiency, resulting in The results of chromatin fragmentation are not suitable for subsequent co-immunoprecipitation; and during the enzymatic digestion process of co-immunoprecipitation experiments at 37°C, due to the instability of the protein structure, it is easy to cause partial protein degradation and affect the experimental results

[0005] At present, most studies on adipose ChIP-seq are conducted in preadipocytes, and the dynamic changes of chromatin modification and the relationship with gene expression regulation are also limited to the cellular level, and there is no report on porcine adipose tissue. application

Images