Chromatin immunoprecipitation technique (ChIP)

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The basic principle is to immobilize the protein-DNA complex in a living cell state, and randomly cut it into small fragments of chromatin in a certain length range, and then immunoprecipitate the complex to specifically enrich the binding of the target protein. In the DN segment, information on the interaction between proteins and DNA is obtained by purifying and detecting the target fragment. CHIP can not only detect the dynamic effects of trans-factors and DNA in vivo, but also study the relationship between various covalent modifications of histones and gene expression. Moreover, the combination of CHIP and other methods has expanded its range of applications: CH [ChIP chromatin immunoprecipitated cellular protein antibody]

The basic principle is to immobilize the protein-DNA complex in a living cell state, and randomly cut it into small fragments of chromatin in a certain length range, and then immunoprecipitate the complex to specifically enrich the binding of the target protein. In the DN segment, information on the interaction between proteins and DNA is obtained by purifying and detecting the target fragment. CHIP can not only detect the dynamic effects of trans-factors and DNA in vivo, but also study the relationship between various covalent modifications of histones and gene expression. Moreover, the combination of CHIP and other methods has expanded its range of applications: CHIP-on-chip methods based on the combination of CHIP and gene chips have been widely used for high-throughput screening of specific trans-factor target genes; CHIP and in vivo footprint method Binding, used to find in vivo binding sites for trans-factors; RNA-CHIP is used to study the role of RNA in gene expression regulation. It can be seen that with the further improvement of CHIP, it will play an increasingly important role in the study of gene expression regulation.

1. 37% formaldehyde

2. Glycine

3. PBS

4. Protease inhibitor

5. RnaseA

6. 0.5MEDTA

7. 1MTris.HCl (PH6.5)

8. 10 mg/ml proteinase K, etc.

1. 10 cm plate

2. Water bath

3. Cell scraper

4. Ultrasonic breaker

5. 15 ml centrifuge tube

6. High speed centrifuge

7. Cross-linking instrument, etc.

Well cultured cells

1) Remove 1 plate of cells (10 cm plate) and add 243 ul of 37% formaldehyde to a final concentration of 1% formaldehyde (9 ml in medium).

2) Incubate for 10 min at 37 °C.

3) Termination of cross-linking: add glycine to a final concentration of 0.125 M. 450 ul 2.5 M glycine in a dish. After mixing, leave it at room temperature for 5 min.

4) Wash out the medium and wash the cells twice with ice-cold PBS.

5) Cell scraper collect the cells in a 15 ml centrifuge tube (PBS 5 ml, 3 ml and 3 ml). The cells were collected at 2000 rpm for 5 min after pre-cooling.

6) Pour the supernatant. According to the amount of cells, SDS Lysis Buffer was added to give a final concentration of 2 x 106 cells per 200 ul. This contains 1 x 106 cells per 100 ul of solution. A protease inhibitor complex is then added. Assume that MCF7 is 5 x 106 cells in a long plate. This time the cells grow about 80%. That is 4 x 106 cells. Therefore, add 400ul Lysis Buffer to each tube. Mix 2 tubes together for a total of 800ul.

7) Ultrasonic crushing: VCX750, 25% power, 4.5S impact, 9S clearance. 14 times in total.

1) After the end of sonication, centrifuge at 10,000 g for 4 min for 10 min to remove insoluble materials. Take 300 ul for the experiment and the rest at -80 degrees. In 300 ul, 100 ul plus antibody was used as the experimental group; 100 ul was not added as the control group; 100 ul was added with 4 ul of 5 M NaCl (the final concentration of NaCl was 0.2 M), and the cross-linking was performed at 65 degrees for 3 h, and the electrophoresis was performed to detect the effect of ultrasonication.

2) Add 900ul of ChIPDilutionBuffer and 20ul of 50xPIC to 100ul of sonicated product. Add 60ul of ProteinAAgarose/SalmonSpermDNA. 4 degrees and mix for 1 h.

3) After 1 h, the pellet was allowed to stand at 4 ° C for 10 min and centrifuged at 700 rpm for 1 min.

4) Take the supernatant. Each was allowed to take 20 ul as input. 1 ul of antibody was added to one tube, and no antibody was added to the other tube. 4 degrees reversed overnight.

100 ul of ultrasonically disrupted product was added, 4ul of 5M NaCl was added, and treated with 65 degrees for 2 h to decrosslink. Half of the phenol/extraction was used. Electrophoresis detects ultrasound effects.

1) After overnight incubation, add 60 ul of ProteinAAgarose/SalmonSpermDNA to each tube for 4 h.

2) After standing at 4 ° for 10 min, centrifuge at 700 rpm for 1 min to remove the supernatant.

3) The precipitated complex was washed successively with the following solution. Washing step: The solution was added, and the solution was inverted at 4 degrees for 10 minutes, left at 4 degrees for 10 minutes, and centrifuged at 700 rpm for 1 minute to remove the supernatant.

a. Low salt solution once

b. High salt solution once

c. LiCl solution once

d. TE buffer twice

4) After the cleaning is completed, the elution starts. Formulation of eluent: 100 ul 10% SDS, 100 ul 1 M NaHCO 3 , 800 uld d H 2 O, 1 ml total. A 250 ul elution buffer was added to each tube, and the mixture was inverted at room temperature for 15 min. After standing and centrifuging, the supernatant was collected. Repeat the wash once. The final eluent is 500 ul per tube.

5) Decrosslinking: 20ul of 5M NaCl (0.2M final concentration of 0.2M) was added to each tube. Mix and mix at 65 degrees overnight.

1) After the end of the cross-linking, add 1ul of RNaseA (MBI) to each tube and incubate at 37 degrees for 1 h.

2) Add 10ul of 0.5MEDTA, 20ul1MTris.HCl (pH6.5), 2ul10 mg/ml proteinase K to each tube. 45 degrees treatment for 2 h.

3) Recovery of DNA fragments - omega gel recovery kit. The final sample is dissolved in 100 ulddH2O.

ChIP-chip technology has a proven track record of large-scale mining of cis-regulatory information, and it can be used for embryonic stem cells and mechanisms for the development of diseases such as cancer, cardiovascular disease, and central nervous disorders. Researchers can also use this technology to develop treatments. At present, ChIP-chip technology research mainly focuses on two areas: the binding and condition specificity of transcription factors; histone modification, histone modification protein and chromosome reconstruction.

ChIP-chip is also widely used in the study of the dynamics of transcriptional binding factors, the distribution of chromosomal structural components, the modification of histones, histone modification proteins and chromosome reconstruction. The advantage of ChIP-chip technology is that it can be reacted in vivo; a simple image of the DNA relationship is obtained in a given mode of testing the cell environment; and a specific modified antibody is used to identify a protein containing a specific post-transcriptional correction. Related sites; direct or indirect (by protein-protein interactions) identification of genomic-protein related sites. The disadvantage is that a specific protein antibody is required, which is sometimes difficult to obtain; in order to obtain a high-abundance binding fragment, it is necessary to demonstrate the expression of the target protein under intracellular conditions; the acquisition of the gene regulating the protein may need to be restricted to the tissue source.

In summary, the development of ChIP-chip technology provides an extremely powerful tool for analyzing the relationship between DNA and proteins in living cells or tissues. In the future research, the construction of the chip will be improved to improve its practicability. Increase the availability of this method by using readily available antibodies.