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...

Examples

Embodiment 1

[0028] Example 1: Ultrasonic disruption of porcine sub-abdominal fat in chromatin co-immunoprecipitation

[0029] 1. Ultrasonic fragmentation of pig sub-abdominal fat

[0030] (1) Take 50 ml of PBS, 100 μl of PIS, and 200 μl of PMSF to pre-cool on ice.

[0031] (2) Slowly thaw 1 g of frozen porcine adipose tissue on ice and cut it into pieces with a volume of 1-3 cubic centimeters. The thawed pieces are dispersed with 30 ml of PBS, and then added with a final concentration of 1% formaldehyde, and shake gently on ice on a shaker. 10min; then add glycine to stop the cross-linking reaction, make the final concentration of glycine 0.125M, shake gently on ice for 10min.

[0032] (3) Centrifuge at 2500 rpm for 5 min and place it on ice immediately. When the solution is divided into three layers, take the upper layer and wash it with PBS, centrifuge at 2500 rpm for 5 min at 4°C, repeat the PBS washing and centrifugation operations; discard the supernatant and lyse the sediment with ...

Embodiment 2

[0044] Example 2: Ultrasonic disruption of lard suet fat in chromatin co-immunoprecipitation

[0045] 1. Ultrasonic crushing of lard suet fat

[0046] (1) Take 50ml of PBS, 100ul of PIS, and 200ul of PMSF to pre-cool on ice.

[0047] (2) Slowly thaw 1 g of frozen lard suet adipose tissue on ice and cut it into pieces with a volume of 1-3 cubic centimeters. The thawed pieces are dispersed with 30 ml of PBS, and then added with a final concentration of 1% formaldehyde. Shake for 10min; then add glycine to stop the cross-linking reaction, make the final concentration of glycine 0.125M, shake gently on ice for 10min.

[0048] (3) Centrifuge at 2500 rpm for 5 min and place it on ice immediately. When the solution is divided into three layers, take the upper layer and wash it with PBS, centrifuge at 2500 rpm for 5 min at 4°C, repeat the PBS washing and centrifugation operations; discard the supernatant and lyse the sediment with 3 ml of adipocytes The filtrate (10 mM HEPES, 80 mM ...

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...

Examples

Embodiment 1

[0028] Example 1: Ultrasonic disruption of porcine sub-abdominal fat in chromatin co-immunoprecipitation

[0029] 1. Ultrasonic fragmentation of pig sub-abdominal fat

[0030] (1) Take 50 ml of PBS, 100 μl of PIS, and 200 μl of PMSF to pre-cool on ice.

[0031] (2) Slowly thaw 1 g of frozen porcine adipose tissue on ice and cut it into pieces with a volume of 1-3 cubic centimeters. The thawed pieces are dispersed with 30 ml of PBS, and then added with a final concentration of 1% formaldehyde, and shake gently on ice on a shaker. 10min; then add glycine to stop the cross-linking reaction, make the final concentration of glycine 0.125M, shake gently on ice for 10min.

[0032] (3) Centrifuge at 2500 rpm for 5 min and place it on ice immediately. When the solution is divided into three layers, take the upper layer and wash it with PBS, centrifuge at 2500 rpm for 5 min at 4°C, repeat the PBS washing and centrifugation operations; discard the supernatant and lyse the sediment with ...

Embodiment 2

[0044] Example 2: Ultrasonic disruption of lard suet fat in chromatin co-immunoprecipitation

[0045] 1. Ultrasonic crushing of lard suet fat

[0046] (1) Take 50ml of PBS, 100ul of PIS, and 200ul of PMSF to pre-cool on ice.

[0047] (2) Slowly thaw 1 g of frozen lard suet adipose tissue on ice and cut it into pieces with a volume of 1-3 cubic centimeters. The thawed pieces are dispersed with 30 ml of PBS, and then added with a final concentration of 1% formaldehyde. Shake for 10min; then add glycine to stop the cross-linking reaction, make the final concentration of glycine 0.125M, shake gently on ice for 10min.

[0048] (3) Centrifuge at 2500 rpm for 5 min and place it on ice immediately. When the solution is divided into three layers, take the upper layer and wash it with PBS, centrifuge at 2500 rpm for 5 min at 4°C, repeat the PBS washing and centrifugation operations; discard the supernatant and lyse the sediment with 3 ml of adipocytes The filtrate (10 mM HEPES, 80 mM ...

Images