Principle and Protocol of ChIP
Chromatin immunoprecipitation assay (ChIP) is an analytical method to study the DNA-protein interaction in vivo, which can truly and completely reflect the regulatory protein binding on DNA sequence. Using formaldehyde to immobilize DNA and protein in living cells, the interaction between trans-acting factors and DNA in vivo, the relationship between histone modification and gene expression, and the analysis of protein and its DNA binding sequence in vivo can be studied by immunoprecipitation separation of complex. At present, the technology of ChIP is applied to the study of the dynamics of chromatin structure, the regulation of transcription factors, co-regulatory factors and other epigenetic changes. It can not only detect the dynamic interaction between trans-acting factors and DNA in vivo, but also study the relationship between various covalent modifications of histones and gene expression. The analysis of immunoprecipitated DNA can verify the specific binding of target protein and DNA target sequence by hybridization of target sequence-specific probe and chromatin immunoprecipitated DNA, and of course, DNA fragments of known or newly discovered unknown regulatory elements can also be separated through ChIP. Compared with the traditional EMSA technology, the chromatin immunoprecipitation analysis technology developed based on in vivo analysis can more truly and completely reflect the regulatory proteins bound to the DNA sequence.
ChIP can be divided into three main steps: fixation, precipitation and detection:
(1) After formaldehyde fixation, chromatin is separated and broken, that is, in the living cell state, the protein-DNA complex is fixed by formaldehyde, and then it is randomly cut into small chromatin fragments within a certain length range by means of chemical microsphere enzyme or mechanical ultrasound;
(2) Immunoprecipitation refers to the formation of protein-antibody complex through antigen-antibody reaction with specific antibody of target protein to complete the immunoprecipitation of specific chromatin fragments;
(3) Analyze the immunoprecipitation fragments to determine whether the target DNA binds to the protein of interest.
The purpose of this lab manual is to help researchers master the screening of DNA sequences interacting with target proteins (regulators), to obtain information on protein-DNA interactions, and to master the principles, applications, and major steps of ChIP.
Chromatin immunoprecipitation analysis is a method developed based on in vivo analysis, using the specific binding of antigenic proteins and antibodies, and the specific binding of bacterial proteins to FC fragments of immunoglobulins. Its basic principle is to fix the protein-DNA complex in the living cell state and randomly cut it into small fragments of chromatin in a certain length range, then precipitate this complex by immunological methods, specifically enrich the target protein-bound DNA fragments, and obtain information on protein-DNA interactions by purification and detection of the target fragments. chIP combines antigen-antibody reaction and polymerase chain reaction (PCR) and is mainly used to identify regulatory proteins on the promoter of target genes that bind directly or indirectly to DNA. The process can be summarized as follows.
(1) Immobilization of protein-DNA complexes by formaldehyde cross-linking, preventing the separation of DNA-binding proteins from the target DNA sequence.
(2) Randomly severing DNA with high-frequency acoustic waves to break it into small fragments within a certain length range.
(3) Incubating solutions containing DNA-protein complexes with antibodies to the target protein to capture antigen-antibody complexes by agarose containing natural antibodies to protein A or G.
(4) washing the agarose with various concentrations of salt and detergent to remove proteins from the immune complexes and precipitating the target DNA with ethanol.
(5) Analysis of target DNA by PCR or southern blot transfer techniques.
Formaldehyde can prevent the separation of protein from protein, protein from DNA and protein from RNA, and the chromatin structure of cells can be effectively preserved after formaldehyde treatment. Moreover, chromatin treated with formaldehyde can be restored to its original state using sodium chloride.
1. Main Instruments and Equipment
Vacuum apparatus, desiccator, vortex oscillator, frozen centrifuge, ultrasonic apparatus.
2. Material
Arabidopsis seedlings of 3-4 weeks old
3. Main Reagents
(1) Extraction buffer 1
| 0.4 mol/L | Sucrose |
| 10 mmol/L | Tris-HCl (pH8.0) |
| 10 mmol/L | MgCl2 |
| 5 mmol/L | β-mercaptoethanol, protease inhibitors |
(2) Extraction buffer 2
| 0.25 mol/L | Sucrose |
| 10 mmol/L | Tris-HCl (pH8.0) |
| 10 mmol/L | MgCl2 |
| 1% | Triton X-100 |
| 5 mmol/L | β-mercaptoethanol, protease inhibitors |
(3) Extraction buffer 3
| 1.7 mol/L | Sucrose |
| 10 mmol/L | Tris-HCl (pH8.0) |
| 2 mmol/L | MgCl2 |
| 0.15% | Triton X-100 |
| 5 mmol/L | β-mercaptoethanol, protease inhibitors |
(4) Nuclei lysis buffer
| 50 mmol/L | Tris-HCl (pH8.0) |
| 10 mmol/L | EDTA |
| 2 mmol/L | MgCl2 |
| 1% | SDS, with protease inhibitors |
(5) CHIP dilution buffer
| 1.1% | Triton X-100 |
| 1.2 mmol/L | EDTA |
| 16.7 mmol/L | Tris-HCl (pH8.0) |
| 16.7 mmol/L | NaCl |
(6) Elution buffer
| 1% | SDS |
| 0.1 mol/L | NaHCO3 |
(7) Low salt wash buffer
| 0.1% | SDS |
| 1% | Triton X-100 |
| 2 mmol/L | EDTA |
| 150 mmol/L | NaCl |
| 20 mmol/L | Tris-HCl (pH 8.0) |
(8) High salt wash buffer
| 0.1% | SDS |
| 1% | Triton X-100 |
| 2 mmol/L | EDTA |
| 500 mmol/L | NaCl |
| 20 mmol/L | Tris-HCl (pH 8.0) |
(9) LiCl wash buffer
| 0.25 mol/L | LiCl |
| 1% | Nonidet P-40 |
| 1% | Sodium deoxycholate |
| 1 mmol/L | EDTA |
| 10 mmol/L | Tris-HCl (pH 8.0) |
(10) TE buffer
| 10 mmol/L | Tris-HCl (pH 8.0) |
| 1 mmol/L | EDTA |
(11) Protease inhibitors
| 100 mmol/L | PMSF |
1. Chromatin Cross-linking
(1) Place 1.5 g of Arabidopsis seedling material into a 50 mL falcon tube and wash the material twice with 40 mL of double-distilled water to remove residual soil*1 and remove as much excess water as possible.
(2) Add 37mL of 1% formaldehyde solution to submerge Arabidopsis seedlings. After screwing on the lid and making small holes with a needle, place in a vacuum desiccator and evacuate for 10 min, slowly deflate and gently remove the air bubbles in the tube, at which time the Arabidopsis seedlings will appear transparent.
(3) Add 2.5 mL of 2 mol/L glycine to terminate the cross-linking, evacuate for 5 min, and gently remove the air bubbles in the tube by slow deflation.
(4) The supernatant waste solution was recovered and the seedlings were washed twice with 40 mL of double-distilled water to remove as much excess water as possible, and the seedlings were placed between two layers of blotting paper to absorb the residual liquid, leaving as little liquid as possible.
2. Chromatin Preparation
(1) Pre-chill the mortar with liquid nitrogen, add 2 tsp of silica and plant material and grind to a powder.
(2) Add the powder with a pre-cooled spoon to 30 mL of extraction buffer 1 pre-cooled on ice, vortex to mix and keep at 4°C.
(3) Filter the homogenate with a miracloth into a new 50 mL falcon tube pre-chilled on ice and repeat the filtration once.
(4) Centrifuge at 4000r/min for 20min at 4°C. Carefully decant the supernatant, add 1mLextraction buffer 2, resuspend the precipitate by pipetting and transfer to an eppendorf tube.
(5) Centrifuge at 13,000 r/min for 10 min at 4°C. Carefully decant the supernatant, add 300μL of extraction buffer 2, and pipette the resuspended precipitate.
(6) Add 300μL of extraction buffer 3 to a new eppendorf tube, and carefully add the resuspended sample to the upper layer with a pipette.
(7) Centrifuge at 13000r/min for 1h at 4°C. Prepare 10mL of nuclei lysis buffer and 20mL of CHIP dilution buffer for pre-cooling at the same time.
(8) Remove the supernatant, resuspend the precipitate with 500μL of pre-cooled nuclei lysis buffer, and vortex shake (remove 10μL for agarose gel electrophoresis detection).
(9) Sonication was performed for 10 s each time, four times, using 40% pulse duration cycle and 20% power. Each two sonication intervals were placed on ice for 1 min*2.
(10) Centrifuge at 13000r/min, 4°C for 10min, and remove the supernatant into a new eppendorf tube. Repeat once and take 10μL for agarose gel electrophoresis detection.
(11) The removed samples were detected in a 1.5% agarose gel. Most of the samples after sonication were broken into 200-2000 bp size and diffuse, more concentrated compared to before treatment, with the size mainly about 500 bp.
3. Pre-purification and Immunoprecipitation
(1) Separately, 150μLof chromatin solution was added to two separate eppendorf tubes, each with 1350μL of CHIP dilution buffer.
(2) Prepare protein A agarose beads in eppendorf tubes by adding 40μL. sheared salmon sperm DNA at a time and wash 3 times with 1 mL. of CHIP dilution buffer in equilibrium. precipitate the beads by centrifugation at 13,000 r/min for 30 s at 4°C.
(3) Add the diluted chromatin solution to a tube with 40μL of well equilibrated beads. 4°C spin incubate for 1h.
(4) Centrifuge at 13 000 r/min, 4°C for 30 s to precipitate beads. mix 3 mL of the same type of chromatin solution into a 14 mL falcon tube. Be careful not to allow residual beads to contaminate each other (remove 60μL of chromatin solution and store at -20°C as an INPUT control).
(5) Add 600μL of chromatin solution and the appropriate amount of primary antibody*3 to each tube of immunoprecipitation reaction. Mix overnight at 4°C with slow rotation or oscillation.
4. Collect, Wash and Dissolve Immune Complex
(1) Prepare fresh CHIP dilution buffer and store at 4°C.
(2) Prepare protein A agarose beads (as before).
(3) Add immunoprecipitation solutions (IPs) to the tubes with equilibrated beads and mix by slow rotation or oscillation at 4°C for 60 min to precipitate the protein recognized by the primary antibody or the corresponding complex. Meanwhile, prepare the elution buffer and place it at 65°C for preheating.
(4) Carefully remove the supernatant, without touching the precipitate. Subsequently, wash the precipitate sequentially with the following solution, 1 mL of each wash solution, 3-5 min per wash at 4°C with slow rotation or oscillation, followed by centrifugation at 4°C, 5000 r/min for 1 min. remove the liquid carefully, do not touch the precipitate.
a. Wash once with low salt wash buffer.
b. Wash once with high salt wash buffer.
c. Wash once with LiCl wash buffer.
d. Wash twice with TE buffer.
(5) Add 250μL of elution buffer to elute the immunoprecipitate complex, vortex briefly and incubate for 15 min at 65°C. Centrifuge at 13000 r/min for 30 s at 4°C and transfer the supernatant to a new eppendorf tube. Repeat the elution once and mix the product of both elutions.
(6) Add 60μL of input control chromatin solution saved during the pre-purification and immunoprecipitation to 460μL elution buffer.
5. Reversal of the Cross-linking Reaction
Add 20μL of 5 mol/L NaCl to the sample tube and incubate overnight at 65°C.
6. DNA Purification
(1) Add 10μLof 0.5 mol/LEDTA, 20μL of 1 mol/LTris-HCl (pH 6.5) and 1μL of 20 mg/mL proteinase K to each tube. incubate at 45°C for 1h.
(2) Add an equal volume of phenol/chloroform as the sample for extraction, centrifuge at 13 000 r/min, 4°C for 15 min, and transfer the supernatant to a 2 mL reaction tube.
(3) Add 1/10 volume of 3 mol/L NaAC (pH 5.2) and 3 times volume of anhydrous ethanol for precipitation. Add 4μL of heparan glycogen to each tube and place at -20°C for at least 1h.
(4) Centrifuge at 13000 r/min at 4°C for 15 min, discard the supernatant, wash the precipitate with 1 mL of 70% ethanol, re-centrifuge, discard the supernatant, and vacuum dry the precipitate for 10 min.
(5) Solubilize DNA with 50uL of 10 mmol/L Tris-HCl (pH 7.5) and perform PCR reaction*4.
7. Identification of DNA
Depending on the gene promoter sequence to be tested, at least one pair of primers is designed for semi-quantitative PCR and real-time PCR to iteratively validate the results of the ChIP experiment*5.
If the target sequence of the target protein is known or needs to be verified, slit blot is used to verify the specific binding of the target protein to the DNA target sequence by hybridizing a target sequence-specific probe to chromatin immunoprecipitated DNA.
If the target sequence of the target protein is unknown or high-throughput, southern hybridization can be used. The precipitated DNA can also be cloned into a vector and sequenced to look for open reading frames near that sequence and discover new gene regulatory sequences.
1. Before immunoprecipitation, take the broken chromatin for input control. Input is the broken genomic DNA, which needs to be reversely cross-linked with the precipitated sample DNA, purified, and finally detected by PCR or other methods.
2. Input control is an essential step in the ChIP experiment. It can not only verify the effect of chromatin breakage, but also convert the efficiency of ChIP according to the content of target sequence in input and the content of target sequence in chromatin precipitation.
3. The agarose beans can also be replaced by magna beans. The magnetic frame is used for separation. The operation process is simple, and the centrifugal step is eliminated, saving time.
4. The antibody of the target protein selected by chromatin immunoprecipitation is the key to the success of the ChIP experiment. Because the antigenic epitope of the antibody may be too close to the binding site to be recognized by the antibody when the protein is cross-linked with chromatin, so it cannot effectively form an immunoprecipitation complex in the body, which directly affects the result of ChIP.
5. When doing the ChIP test, be sure to do a good job of positive antibody and negative antibody control. Positive antibodies usually select antibodies to relatively conservative proteins that combine with known sequences, and commonly used include histone antibodies or RNA polymerase II antibodies. The negative antibody usually selects the IgG or serum of the target protein antibody host. Objective To draw a correct conclusion by comparing the results of protein antibody with those of positive antibody and negative antibody.
6. In addition, it is necessary to consider the possibility of non-specific binding between the target protein antibody and DNA, and select the DNA sequence that the target protein will not bind to design a pair of primers as the negative control of the antibody. The best negative control primer is a sequence upstream of the target sequence that cannot bind to the target protein.
7. For antibodies to new target proteins, protein immunoprecipitation can be performed first. If the antibody can successfully precipitate the protein, then carry out the chromatin immunoprecipitation test.
*1 The residual soil will be with the precipitation and have an effect on the results.
*2 Ultrasound generates a lot of heat, so be careful to keep it cold.
*3 The amount of primary antibody can be referred to the instructions of the antibody. If the dilution ratio for ChIP is not given in the instructions for the antibody, you can refer to the dilution ratio for normal immunoprecipitation. Usually, the amount of primary antibody is 0.5-1μg, but it can be increased to 10μg. You can use no antibody as a negative control or use an unrelated antibody as a negative control, and you can use a solution without cell samples as a blank control.
*4 The promoter region is mostly CG-rich sequences, and its PCR conditions may need to be adjusted accordingly.
*5 The amount of immunoprecipitated DNA is small, so the DNA probe is usually amplified by PCR method before the whole genome is scanned during the study.
