Ultrasonic crushing method for pig adipose tissue and application of ultrasonic crushing method
What is Al technical title?
Al technical title is built by PatSnap Al team. It summarizes the technical point description of the patent document.
A technology of ultrasonic crushing and porcine fat, which is applied in the field of chromatin immunoprecipitation, can solve the problems of affecting experimental results, protein degradation, dynamic changes of chromatin modification, expression regulation relationship limited to the cell level, etc., to reduce the impact and improve the lysis efficiency Effect
Active Publication Date: 2018-12-07
WUHAN IGENEBOOK BIOTECH CO LTD
View PDF3 Cites 4 Cited by
Summary
Abstract
Description
Claims
Application Information
AI Technical Summary
This helps you quickly interpret patents by identifying the three key elements:
Problems solved by technology
Method used
Benefits of technology
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
Method used
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more
Image
Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
Click on the blue label to locate the original text in one second.
Reading with bidirectional positioning of images and text.
Smart Image
Examples
Experimental program
Comparison scheme
Effect test
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 ...
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more
PUM
Login to view more
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...
Claims
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more
Application Information
Patent Timeline
Application Date:The date an application was filed.
Publication Date:The date a patent or application was officially published.
First Publication Date:The earliest publication date of a patent with the same application number.
Issue Date:Publication date of the patent grant document.
PCT Entry Date:The Entry date of PCT National Phase.
Estimated Expiry Date:The statutory expiry date of a patent right according to the Patent Law, and it is the longest term of protection that the patent right can achieve without the termination of the patent right due to other reasons(Term extension factor has been taken into account ).
Invalid Date:Actual expiry date is based on effective date or publication date of legal transaction data of invalid patent.
Login to view more 
Login to view more