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Histone-modified chromosome co-immunoprecipitation method applicable in tissue sample

A technology of co-immunoprecipitation and tissue samples, which is applied in the field of immunoassay, can solve the problems of increased false positives and reduced specificity, and achieves the effect of improving precipitation efficiency and detection effect

Inactive Publication Date: 2015-05-27
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, due to tissue specificity, cross-linking efficiency and process, some antigenic determinants may be blocked, so that the specificity of antibodies with good specificity will be reduced after cross-linking treatment, and false positives will increase.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Take clinical lung adenocarcinoma tissue samples. The specific method is as follows:

[0028] Under sterile conditions, collect about 30-50mg of lung adenocarcinoma tissue, and cut the tissue into 1-3mm 3 Lumpy. Add pre-cooled 1ml PBS (containing 1× protein inhibitor) and wash 3 times; use a tissue grinder, grind in an ice bath, filter with sterile gauze, collect the filtrate, observe under a microscope, and perform a test on the living cells Count and collect 1M (million) viable cells (1000rpm, 10min). Collect the cells, suspend in 300ul digestion buffer (50mM Tris-HCl, pH7.6; 1mM CaCl 2 ; 0.2% Triton X-100 (or NP-40)), 37 degrees water bath for 2 minutes. Add 0.4U of MNase enzyme, digest at 37°C for 5 minutes, and then add 300ul of enzyme digestion stop solution (10mM Tris, pH7.6, 5mM EDTA). Use a Bioruptor ultrasonic instrument to sonicate at high frequency and ice bath for 5 minutes, centrifuge at 9000g, 4 degrees for 5 minutes, and take out 100ul of the soluti...

Embodiment 2

[0030] Taking mouse liver tissue as an example

[0031] 1. Pretreatment of tissue samples:

[0032] Under sterile conditions, take about 50mg of mouse liver tissue, and cut the tissue into 1-3mm 3 Lumpy. Add pre-cooled 1ml PBS (containing 1× protein inhibitor) and wash 3 times;

[0033] 2. Tissue single cell isolation:

[0034] After grinding in an ice bath with a tissue grinder, filter with sterile gauze, collect the filtrate, observe under a microscope, count the viable cells, and collect the viable cells (1000 rpm, 10 min).

[0035] 3. Cell Lysis and Enzyme Digestion:

[0036] Cells were collected and suspended in enzyme digestion buffer (50mM Tris-HCl, pH7.6; 1mM CaCl 2 ; 0.2v / v% Triton X-100 (or NP-40)), 37 ° C water bath for 2 minutes.

[0037] 4. MNase digestion reaction:

[0038] After aliquoting the cells added with the digestion solution, add MNase enzyme (ranging from 0.1-0.8 U), digest at 37°C for 5 minutes, and then add an equal amount of digestion stop sol...

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Abstract

The invention relates to a histone-modified chromosome co-immunoprecipitation method applicable in a tissue sample, which comprises the following steps: carrying out tissue sample pretreatment; grinding in an ice bath, filtering with sterile gauze, collecting cells in the filtrate, and treating the suspended cells in an enzyme digestion buffer solution under water-bath conditions; adding micrococcus nuclease into the cells with the enzyme digestion buffer solution, carrying out enzyme digestion, and adding an enzyme digestion termination solution with the same amount as the micrococcus nuclease; carrying out high-frequency ultrasonic treatment and centrifugation in an ice bath to obtain a pulverized chromatin solution; taking ProteinA / G, adding a BSA (bovine serum albumin)-containing PBS (phosphate buffer solution), adding an antibody, incubating, adding the pulverized chromatin solution, and incubating over night; and washing with a high salt solution many times on a magnetic rack, eluting DNA (deoxyribonucleic acid) on the magnetic bead with a TE solution, and purifying with a purification solution, extracting the DNA, and dissolving the DNA in ultrapure water. Compared with the prior art, the method can accurately position the combination site of the histone or transcription factor on the high flux level, thereby enhancing the detection effect of the clinic sample.

Description

technical field [0001] The invention belongs to the technical field of immunodetection, and in particular relates to a co-immunoprecipitation method for histone modified chromosomes in tissue samples. Background technique [0002] Histone modification is an important epigenetic mechanism that regulates gene expression, and plays an important role in maintaining cell pluripotency and the pathological process of cancer. Chromatin Immunoprecipitation (ChIP) method is a powerful tool for studying the interaction between proteins and DNA in vivo, and is usually used for the study of transcription factor binding sites or histone-specific modification sites. ChIP-Seq technology, which combines ChIP with second-generation sequencing technology, can efficiently detect DNA segments that interact with histones and transcription factors on a genome-wide scale. At present, this method is generally applicable to cell samples, but it is not stable in the application of clinical tissue sam...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 李旦刘小乐
Owner TONGJI UNIV
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