CN104651506A - Histone-modified chromosome co-immunoprecipitation method applicable in tissue sample - Google Patents
Histone-modified chromosome co-immunoprecipitation method applicable in tissue sample Download PDFInfo
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- CN104651506A CN104651506A CN201510073437.2A CN201510073437A CN104651506A CN 104651506 A CN104651506 A CN 104651506A CN 201510073437 A CN201510073437 A CN 201510073437A CN 104651506 A CN104651506 A CN 104651506A
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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
The invention belongs to technical field of immunoassay, especially relate to one and be applied to histone modification karyomit(e) co-immunoprecipitation method in tissue samples.
Background technology
Histone modification is the important epigenetics mechanism of regulate gene expression, plays an important role in the maintenance totipotency of cell and the pathologic process of cancer.Histone modification karyomit(e) co-immunoprecipitation (Chromatin Immunoprecipitation, ChIP) method is research body internal protein and the interactional powerful of DNA, is generally used for the research of Binding site for transcription factor or histone-specific decorating site.By the ChIP-Seq technology that ChIP combines with s-generation sequencing technologies, the region of DNA section done mutually with histone, transcription factor etc. can be detected efficiently within the scope of full-length genome.Current the method is generally suitable for cell sample, and not yet stable in clinical tissue sample application, and does not reach the object of widespread use, causes to study for domestic important diseases (lung cancer, cancer of the stomach, liver cancer etc.).
In current ChIP-Seq technology, cross-linking method ChIP (Cross-linked ChIP is all adopted to karyomit(e) pretreatment process, hereinafter referred to as XChIP), namely with formaldehyde, reversible crosslinking reaction is carried out to DNA-protein, by ultrasonication (sonication) by chromosome segment.But the effect of XChIP is subject to the many factors such as crosslinking time, concentration of formaldehyde, ultrasonic treatment time and intensity, the experiment condition of crosslinked three steps of crosslinked-ultrasonic wave-solution needs collaborative discussion.Because tissue samples and cell sample also exist very large difference, on early stage handling tissue samples, need to carry out condition optimizing, isolate cell individual, reach the optimum efficiency of antibodies.In addition due to the specificity of tissue, cross-linking efficiency and process may cause some antigenic determinants to be closed, and the good antibody of specificity specificity after crosslinking Treatment is originally reduced, and false positive raises.
Summary of the invention
Object of the present invention be exactly in order to overcome above-mentioned prior art exist defect and provide one to be applied to histone modification karyomit(e) co-immunoprecipitation method in tissue samples.
Method of the present invention takes to use separation, micrococcal nuclease (the Micrococcal Nuclease of tissue samples individual cells, hereinafter referred to as MNase) combine with each node optimization technology such as ultrasonic, cut the karyomit(e) ChIP (being called for short NXChIP) under state of nature.ChIP early stage time, optimize and is separated the not damaged formula cell individual of tissue samples, NXChIP takes enzyme incision technology process, thus minimizing ChIP rate of loss; Simultaneously in view of histone H 3 and H4 combine closely with DNA, the early stage of therefore not carrying out formaldehyde is in the method fixed.In addition, with the lingker district between Mnase enzyme cutting nucleosome, single nucleosome can be obtained.Therefore, this technology is also well suited for the research of applicable single core corpusculum (i.e. nucleosome resolving power, Nucleosoome resolution).Simultaneously also because this characteristic of Mnase makes NChIP not be suitable for nonhistones research, at this moment just need to solve with NXChIP.
Object of the present invention can be achieved through the following technical solutions:
One is applied to histone modification karyomit(e) co-immunoprecipitation method in tissue samples, comprises the following steps:
(1) tissue samples pre-treatment:
Get tissue samples and cut into 1-3mm
3bulk, the PBS washing composition (containing 1 × protein inhibitor protein inhibitor, being namely diluted to a times) containing protein inhibitor adding precooling washs 3 times; Described protein inhibitor is specifically made up of AEBSF, EDTA, Leupeptin and Pepstatin A.
(2) tissue monocytes is separated:
With tissue grinder, in ice bath after grinding, filter with sterile gauze, collect filtrate, examine under a microscope, viable cell is counted, collect viable cell (1000rpm, 10min);
(3) lysis and enzymic digestion:
Collect the cell in step (2) filtrate, be suspended in digestions damping fluid (containing 50mM Tris-HCl, 1mM CaCl
2, 0.2v/v%Triton X-100 or NP-40, pH be 7.6) in, 37 DEG C of water-baths 2 minutes;
(4) micrococcal nuclease endonuclease reaction:
To the cell of digestions damping fluid be added, add the micrococcal nuclease that enzyme activity is 0.1-0.8U, after 37 DEG C of enzymes cut 5 minutes, add and to cut stop buffer (be 7.6 containing 10mM Tris and 5mM EDTA, pH) with the enzyme of micrococcal nuclease equivalent;
(5) ultrasonication:
Under ice bath, use Bioruptor ultrasonic apparatus, 9000g high frequency ultrasound carried out 5 minutes to step (4) gained liquid, then 4 DEG C centrifugal 5 minutes, obtain broken chromatin solution;
(6) antibodies:
Get Protein A/G, with 500ml containing after the PBS buffer solution three times of 5mg/ml bovine serum albumin, add the PBS damping fluid of 250ul containing 5mg/ml bovine serum albumin, add 2-5ug antibody again, 4 DEG C hatch 2-4 hour after, add broken chromatin solution, 4 DEG C of night incubation;
(7) DNA extraction after ChIP:
Solution after hatching, magnetic frame repeatedly washs with high level salt solution, use TE solution (containing 10mM Tris and 5mM EDTA after washing, pH is 7.6) DNA in wash-out magnetic bead, with refined solution (mixed solution of the phenol of volume ratio 25:24:1, chloroform and primary isoamyl alcohol) purification DNA, use ultrapure water dissolving DNA.
(8) method efficiency assessment:
With the enrichment degree of real time quantitative PCR method inspection ChIP.
Compared with prior art, the present invention adopts tissue samples process in early stage and cellular segregation, effectively can improve the deposition efficiency of antibody; Secondly, adopt enzyme to cut and the ultrasonic method combined, effectively can not only obtain chromatin fragments, and nucleosome on chromatin can be obtained, can on high flux levels the binding site of precise positioning histone or transcription factor, improve the Detection results of clinical sample.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1
Take clinical lung adenocarcinoma tissue sample.Concrete grammar is as follows:
Aseptically, gather about 30-50mg pulmonary adenocarcinoma, tissue is cut into 1-3mm
3block.The 1ml PBS (containing 1 × protein inhibitor protein inhibitor) adding precooling washs 3 times; With tissue grinder, in ice bath after grinding, filter with sterile gauze, collect filtrate, examine under a microscope, viable cell is counted, collect the viable cell (1000rpm, 10min) of 1M (1,000,000).Collecting cell, is suspended in 300ul digestions damping fluid (50mM Tris-HCl, pH7.6; 1mM CaCl
2; 0.2%TritonX-100 (or NP-40)) in, 37 degree of water-baths 2 minutes.Add the MNase enzyme of 0.4U, after 37 degree of enzymes cut 5 minutes, add 300ul enzyme and cut stop buffer (10mM Tris, pH7.6,5mM EDTA).By Bioruptor ultrasonic apparatus high frequency, ice-bath ultrasonic 5 minutes, 9000g, 4 degree centrifugal 5 minutes, takes out 100ul solution as input.Get 10ul Protein A/G, add 500ml PBS (5mg/ml BSA) and wash three times, add 250ul PBS (containing 5mg/ml BSA), add 2-5ug antibody again, 4 degree hatch 2-4 hour after, add broken chromatin solution, 4 degree of night incubation.Solution after hatching, magnetic frame repeatedly washs with high level salt solution, washs the DNA in rear TE (10mM Tris, pH7.6,5mM EDTA) eluant solution magnetic bead.DNA and the input DNA of ChIP uses phenol respectively: chloroform: primary isoamyl alcohol (25:24:1) purification, finally uses ultrapure water dissolving DNA.With the enrichment degree of real time quantitative PCR method inspection ChIP.
Embodiment 2
For murine liver tissue
1. tissue samples pre-treatment:
Aseptically, get about 50mg murine liver tissue, tissue is cut into 1-3mm
3block.The 1ml PBS (containing 1 × protein inhibitor protein inhibitor) adding precooling washs 3 times;
2. tissue monocytes is separated:
With tissue grinder, in ice bath after grinding, filter with sterile gauze, collect filtrate, examine under a microscope, viable cell is counted, collect viable cell (1000rpm, 10min).
3. lysis and enzymic digestion:
Collecting cell, is suspended in digestions damping fluid (50mM Tris-HCl, pH7.6; 1mM CaCl
2; 0.2v/v%Triton X-100 (or NP-40)) in, 37 DEG C of water-baths 2 minutes.
4.MNase endonuclease reaction:
By add Digestive system cell decile after, add MNase enzyme (0.1-0.8U not etc.), after 37 DEG C of enzymes cut 5 minutes, add equivalent amounts of enzyme and cut stop buffer (10mM Tris, pH7.6,5mM EDTA).
5. ultrasonication:
By Bioruptor ultrasonic apparatus high frequency, ice-bath ultrasonic 5 minutes, 9000g, 4 DEG C centrifugal 5 minutes.
6. antibodies:
Get 10ul Protein A/G, add 500ml PBS (5mg/ml BSA) and wash three times, add 250ul PBS (5mg/ml BSA), then add 2-5ug antibody, 4 DEG C hatch 2-4 hour after, add broken chromatin solution, 4 DEG C of night incubation.
DNA extraction after 7.ChIP
Solution after hatching, magnetic frame repeatedly washs with high level salt solution, washs the DNA in rear TE (10mMTris, pH7.6,5mM EDTA) eluant solution magnetic bead.With phenol: chloroform: primary isoamyl alcohol (25:24:1) purification DNA, use ultrapure water dissolving DNA.
8. method efficiency assessment
With the enrichment degree of real time quantitative PCR method inspection ChIP.
Above-mentioned is can understand and use invention for ease of those skilled in the art to the description of embodiment.Person skilled in the art obviously easily can make various amendment to these embodiments, and General Principle described herein is applied in other embodiments and need not through performing creative labour.Therefore, the invention is not restricted to above-described embodiment, those skilled in the art, according to announcement of the present invention, do not depart from improvement that scope makes and amendment all should within protection scope of the present invention.
Claims (8)
1. be applied to a histone modification karyomit(e) co-immunoprecipitation method in tissue samples, it is characterized in that, comprise the following steps:
(1) tissue samples pre-treatment:
Get tissue samples and cut into bulk, add the PBS detergent washing containing protein inhibitor of precooling;
(2) tissue monocytes is separated:
With tissue grinder, in ice bath after grinding, filter with sterile gauze, collect filtrate;
(3) lysis and enzymic digestion:
Collect the cell in step (2) filtrate, be suspended in digestions damping fluid, 37 DEG C of water-baths 2 minutes;
(4) micrococcal nuclease endonuclease reaction:
By adding the cell of digestions damping fluid, adding micrococcal nuclease, after 37 DEG C of enzymes cut 5 minutes, adding and cutting stop buffer with the enzyme of micrococcal nuclease equivalent;
(5) ultrasonication:
Under ice bath, high frequency ultrasound is carried out and centrifugal treating to step (4) gained liquid, obtain broken chromatin solution;
(6) antibodies:
Get Protein A/G, with containing after the PBS buffer solution of bovine serum albumin, add the PBS damping fluid containing bovine serum albumin, then add antibody, 4 DEG C hatch 2-4 hour after, add broken chromatin solution, 4 DEG C of night incubation;
(7) DNA extraction after ChIP:
Solution after hatching, magnetic frame repeatedly washs with high level salt solution, with the DNA in TE eluant solution magnetic bead after washing, with refined solution purification DNA, uses ultrapure water dissolving DNA.
2. one according to claim 1 is applied to histone modification karyomit(e) co-immunoprecipitation method in tissue samples, it is characterized in that, in the digestions damping fluid described in step (3), containing 50mMTris-HCl, 1mM CaCl
2, 0.2v/v%Triton X-100 or NP-40, pH be 7.6.
3. one according to claim 1 is applied to histone modification karyomit(e) co-immunoprecipitation method in tissue samples, it is characterized in that, the enzyme activity of the micrococcal nuclease described in step (4) is 0.1-0.8U.
4. one according to claim 1 is applied to histone modification karyomit(e) co-immunoprecipitation method in tissue samples, it is characterized in that, the enzyme described in step (4) is cut stop buffer and contained 10mM Tris and 5mMEDTA, and pH is 7.6.
5. one according to claim 1 is applied to histone modification karyomit(e) co-immunoprecipitation method in tissue samples, it is characterized in that, in step (5), the condition of carrying out ultrasonication is: 9000g high frequency ultrasound 5 minutes, then 4 DEG C centrifugal 5 minutes.
6. one according to claim 1 is applied to histone modification karyomit(e) co-immunoprecipitation method in tissue samples, it is characterized in that, in antibodies described in step (6), get Protein A/G, with 500ml containing after the PBS buffer solution three times of 5mg/ml bovine serum albumin, add the PBS damping fluid of 250ul containing 5mg/ml bovine serum albumin, add 2-5ug antibody again, 4 DEG C hatch 2-4 hour after, add broken chromatin solution, 4 DEG C of night incubation.
7. one according to claim 1 is applied to histone modification karyomit(e) co-immunoprecipitation method in tissue samples, it is characterized in that, containing 10mM Tris and 5mMEDTA, pH in the TE solution described in step (7) is 7.6.
8. one according to claim 1 is applied to histone modification karyomit(e) co-immunoprecipitation method in tissue samples, it is characterized in that, the refined solution described in step (7) is the mixed solution of the phenol of volume ratio 25:24:1, chloroform and primary isoamyl alcohol.
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| CN107841533A (en) * | 2017-11-13 | 2018-03-27 | 深圳先进技术研究院 | A kind of chromatin breaking method and its application |
| CN108048543A (en) * | 2017-12-25 | 2018-05-18 | 上海嘉因生物科技有限公司 | Applied to MNase digestion optimization methods in nucleosome site in tissue samples |
| CN108179179A (en) * | 2017-12-28 | 2018-06-19 | 上海嘉因生物科技有限公司 | Applied to MNase digestion optimization methods in nucleosome site in tissue samples |
| CN108181461A (en) * | 2017-12-29 | 2018-06-19 | 上海嘉因生物科技有限公司 | Applied to histone modification chromosome co-immunoprecipitation optimization method in tissue samples |
| CN108753939A (en) * | 2018-06-01 | 2018-11-06 | 华侨大学 | A method of the single stranded DNA damage of detection full-length genome |
| CN110320357A (en) * | 2019-06-28 | 2019-10-11 | 华南农业大学 | A kind of natural dyeing matter immunoprecipitation processing method for tissue |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107841533A (en) * | 2017-11-13 | 2018-03-27 | 深圳先进技术研究院 | A kind of chromatin breaking method and its application |
| CN107841533B (en) * | 2017-11-13 | 2021-11-05 | 深圳先进技术研究院 | Chromatin fragmentation method and application thereof |
| CN108048543A (en) * | 2017-12-25 | 2018-05-18 | 上海嘉因生物科技有限公司 | Applied to MNase digestion optimization methods in nucleosome site in tissue samples |
| CN108179179A (en) * | 2017-12-28 | 2018-06-19 | 上海嘉因生物科技有限公司 | Applied to MNase digestion optimization methods in nucleosome site in tissue samples |
| CN108181461A (en) * | 2017-12-29 | 2018-06-19 | 上海嘉因生物科技有限公司 | Applied to histone modification chromosome co-immunoprecipitation optimization method in tissue samples |
| CN108753939A (en) * | 2018-06-01 | 2018-11-06 | 华侨大学 | A method of the single stranded DNA damage of detection full-length genome |
| CN108753939B (en) * | 2018-06-01 | 2021-08-03 | 华侨大学 | Method for detecting single-stranded DNA damage of whole genome |
| CN110320357A (en) * | 2019-06-28 | 2019-10-11 | 华南农业大学 | A kind of natural dyeing matter immunoprecipitation processing method for tissue |
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