Chromatin immunoprecipitation (ChIP) is a technique for studying protein-DNA interactions in vivo.
The genomic DNA of eukaryotes exists in the form of chromatin, and the study of protein-DNA interactions in the chromatin environment is a fundamental way to elucidate the gene expression mechanism of eukaryotes.
ChIP utilizes the specificity of antigen-antibody reaction, which can sensitively detect the binding of target proteins to specific DNA fragments, and is currently the best method to identify the genomic regions that bind to specific proteins, and the proteins that bind to specific genomic regions.
ChIP is commonly used to study the binding of transcription factor (TF) to promoter, and to study the relationship between various covalent modifications of histones and gene expression.
Chromatin immunoprecipitation techniques generally include cell fixation, chromatin breakage, chromatin immunoprecipitation, reversal of cross-linking reaction, purification of DNA, and DNA identification.
Principle
The basic principle of chromatin immunoprecipitation is to cross-link and immobilize intracellular DNA with proteins under physiological state, and randomly cut chromatin into small chromatin fragments within a certain length range by ultrasound or enzyme treatment.
This cross-linked complex is precipitated with an antibody specific for the target protein to be studied, and the DNA fragment bound by the target protein is specifically enriched, so that information on the interaction between the protein and DNA can be obtained through purification and detection of the target fragment.
Appliance
Chromatin immunoprecipitation is commonly used to study the binding of transcription factor (TF) and promoter, and to study the relationship between various covalent modifications of histones and gene expression.
Operation method
chromatin immunoprecipitation
Principle
The basic principle of chromatin immunoprecipitation is to cross-link and immobilize intracellular DNA with proteins under physiological state, and randomly cut chromatin into small chromatin fragments within a certain length range by ultrasound or enzyme treatment. This cross-linked complex is precipitated with an antibody specific for the target protein to be studied, and the DNA fragment bound by the target protein is specifically enriched, so that information on the interaction between the protein and DNA can be obtained through purification and detection of the target fragment.
Materials and Instruments
Equipment: Move The basic process can be divided into the following steps: (1) Lysis (FA Lysis) buffer 50 mmol/L HEPES-KOH, pH 7.5; 140 mmol/L NaCl; 1 mmol/L EDTA, pH 8.0; 1% Triton X-100; 0.1% sodium deoxycholate; 0.1% SDS; protease inhibitor (added before use). (2) RIPA buffer 50 mmol/L Tris-HCI, pH 8.0; 150 mmol/L NaCl; 2 mmol/L EDTA, pH 8.0; 1% NP- 40; 0.5% sodium deoxycholate; 0.1% SDS; protease inhibitor (added before use). (3) Wash buffer 0.1% SDS; 1% TritonX-100; 2 mmol/L EDTA, pH 8.0; 150 mmol/L NaCl; 20 mmol/L Tris-HCI, pH 8.0. (4) Final Wash buffer 0.1% SDS; 1% Triton X-100; 2 mmol/L EDTA, pH 8.0; 500 mmol/L NaCl; 20 mmol/L Tris-HCl, pH 8.0. (1) Take a flat dish of cells (150 mm dish), the cell confluence is about 80% to 90% (HeLa cells are about 1x107 ), the dish contains 20 ml of medium, add 550 μl of 37% formaldehyde (or 1,100 μl of 18.5% formaldehyde) to make the final concentration of formaldehyde is 1%.
①Mute mixer
② High-speed low-temperature centrifuge
③Ultrasonic crusher
④Electrophoresis instrument, horizontal electrophoresis tank
⑤PCR instrument
⑥ Real-time PCR instrument
Reagents:
①Materials: HeLa cells
②HEPES
③NaCl, KOH, HCl, Triton X-100, NP-40, SDS, NaHCO
3
④Sodium deoxycholate, EDTA, Tris
⑤ Protease inhibitors, Protease K, Protein A/Gbeads (salmon sperm DNA)
⑥Control primer (GAPDH)
(vii) Positive control antibody (Anti-Acetyl Histone H3), negative control antibody (Normal IgG)
⑧Agarose
⑨ PCR reaction Mix, SYBR Green qPCR Mix
(2) Mix gently and incubate for 10 min at room temperature.
(3) Terminate cross-linking: add glycine to a final concentration of 125 mmol/L, mix gently and incubate for 5 min at room temperature.
(4) Place the dish on ice.
(5) Aspirate the medium and wash the cells with 20 ml of ice-cold PBS.
(6) Aspirate the PBS and repeat washing the cells 1 time.
(7) Add 2 ml ice-cold PBS (containing 1x protease inhibitor complex) and collect the cells with a cell scraper in a 1.5 ml centrifuge tube.
(8) Centrifuge at 1000 g, 4 ℃ for 5 min.
(9) Aspirate off the supernatant, add 750 μl FA Lysis buffer and resuspend the cells to obtain cell lysate.
(iii) Chromatin breakage(1) Place the centrifuge tube containing cell lysate on ice, and ultrasonically break it, with ultrasonic power of 310 W, 20 s impact, 2 s gap, and ultrasonication 3 times (the conditions of ultrasonication are different according to different cell strains and cell numbers, and need to be experimented to find out), and ultrasonication probe should be deep into the tube as much as possible, but not touching the bottom or side wall of the tube, so as not to produce foam.
(2) After ultrasonic crushing, 10000 g, 4 ℃, centrifugation for 10 min, to remove cell debris and other insoluble substances.
(3) Aspirate the supernatant into new 1.5 ml centrifuge tubes, 50 μl per tube (can be stored at -80 ℃ for 3 months).
(4) Take 100 μl of ultrasonically broken product, add 5 μl of proteinase K (20 mg/ml), 65 ℃, rotary mixing, decrosslinking for 4-5 h (or overnight), half of the fraction was extracted with phenol-chloroform, and the effect of ultrasound was detected by 1.5% agarose electrophoresis.
(D) Chromatin immunoprecipitation(1) Add 450 μl of RIPA buffer to 50 μl of ultrasonically broken product in each tube, dilute 1:10, set aside 5 μl (1% by volume) as the Input control, and store it at 4 ℃ until the operation of step 4 (1).
(2) Add antibodies: Add positive control antibody, negative control antibody (normal IgG) and target protein antibody to each tube of sample, the amount of antibody is 1-10 μg. According to the potency, purity and specificity of the antibody, dilute the antibody in a gradient, and determine the amount of antibody experimentally.
(3) Add 20 μl of well-mixed Protein A/G beads (Salmon Sperm DNA).
(4) Spin the mixture at 4 ℃ for 1 h or overnight.
(5) Centrifuge at 2000 g for 1 min and discard the supernatant.
(6) Wash beads 3 times, each time with 1 ml Wash buffer, centrifuge at 2000 g for 1 min, discard supernatant.
(7) Wash beads 1 time with 1 ml Final Wash buffer, centrifuge at 2000 g for 1 min, discard supernatant.
(E) Decrosslinking reaction and DNA purificationDecrosslinking was performed with proteinase K at 65 ℃ for 4-5 h. DNA was recovered by DNA purification column or purified by phenol-chloroform extraction and ethanol precipitation.
(1) Add 120 μl Elution buffer (including Input control) to each sample, 30 ℃, spin mixing, 15 min.
(2) Centrifuge at 2000 g for 1 min.
(3) Pipette the supernatant into a new centrifuge tube, which can be stored at -20 ℃.
(4) Add 5 μl of proteinase K (20 mg/ml), 65 ℃, rotate and mix, and de-crosslink for 4-5 h (or overnight).
(5) DNA was extracted with phenol-chloroform, precipitated by ethanol, and 100 μl of water was added to dissolve DNA, which could be stored at -20 ℃.
(F) Identification of DNAThe most commonly used DNA identification methods are semi-quantitative PCR and Real-time PCR.
(VII) Experimental results and analysis(1) The chromatin after cross-linking was broken by ultrasonic waves, and the results of 2% agarose gel electrophoresis showed that the average length of the interrupted chromatin fragments should be around 200-1000 bp.
(2) The DNA fragment obtained after purification was analyzed by PCR, the amplified fragment was the promoter region of the housekeeping gene GAPDH, and the fragment size was 165 bp, the amplification product was visible in the positive control and Input control, the PCR control without DNA template did not have the amplification product, and the negative control could see the weakly amplified bands but the difference was obvious comparing with the positive control.Caveat
(1) The formaldehyde cross-linking reaction time varies according to the cell type and the number of cells, and needs to be determined by pre-experiment.If the cross-linking time is too long, the experimental material is easy to lose, and the DNA-protein complex after cross-linking is difficult to be interrupted by ultrasound; if the cross-linking time is too short, the cross-linking is incomplete.(2) Ultrasound is the use of mechanical force to break chromatin, easy to cause warming or foam, so that the protein denaturation, affecting the efficiency of ChIP, in the ultrasonic break chromatin, should be carried out on ice, and should be designed to intermittent ultrasound program, the ultrasound time should not be too long, so as not to avoid degradation of proteins.(3) When doing ChIP experiments, it is necessary to do a good experimental control, if there is no control, it is difficult to assess the reliability of the experimental results. Positive antibody and negative antibody control is the most basic experimental control.At the same time, we should do a good job of Input control, Input control can not only verify the effect of chromatin breakage, but also can be based on the content of target sequences in the Input and the content of target sequences in the chromatin precipitation, in accordance with the sampling ratio to convert the efficiency of ChIP.(4) The selection of antibodies to the target proteins used for chromatin immunoprecipitation is the key to the success of ChIP experiments. Because when the protein is cross-linked and bound to chromatin, the antigenic epitope of the antibody may not be recognized by the antibody because it is too close to the binding site.Therefore, it cannot effectively form immunoprecipitation complexes in vivo, which directly affects the results of ChIP. Therefore, not all antibodies can do ChIP experiments, and only the antibodies that have been verified by ChIP experiments can ensure the reliability of the experimental results.
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