Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP): Chromatin immunoprecipitation technology is a technique used in epigenetic research, which can quickly reflect protein-DNA interactions. This technology enriches DNA fragments that bind to the target protein through specific antibodies, thereby revealing the localization and function of nuclear proteins such as transcription factors and histone modifications on the genome^[1]

Technical principle: Its basic principle is to fix the protein-DNA complex in the living cell state and randomly cut it into chromatin fragments of a certain length range through ultrasound or enzymatic treatment. Then, this complex is precipitated through specific antigen-antibody recognition reactions, specifically enriching the DNA fragments bound to the target protein. Through the purification and detection of the target fragments, and ultimately through PCR, Q-PCR, sequencing and other methods, the information of protein-DNA interaction is obtained.

Development and application: Chromatin immunoprecipitation technology is developing towards higher sensitivity (at the single-cell level), higher throughput (multi-omics integration), and wider applications (clinical diagnosis, precision agriculture). With the progress of microfluidic technology and single-cell sequencing, ChIP technology will play a greater role in analyzing the spatiotemporal dynamics of gene regulatory networks^[2][3].

Note: ChIP experiments require high-quality antibodies and skilled operation. MCE provides high-quality ChIP-specific reagents and professional technical support. https://www.medchemexpress.cn/

MCE has not independently verified the accuracy of these methods; they are for reference only.

The following steps describe ChIP using cell samples as the starting material^[1][4].

2.1. Materials Preparation

Reagents: Proteinase inhibitor (100 mM PMSF), Nuclei lysis buffer (containing SDS), CHIP dilution buffer, low salt, high salt, LiCl, TE solution. The preparation scheme is shown in the appendix.

2.2. Methods

2.2.1. Optimization of Ultrasonic Treatment Conditions for Samples
(1) Prepare an appropriate amount of pre-cooled PBS and 100 mM PMSF. Properly warm-wash the SDS Lysis Buffer to fully dissolve the SDS and mix well.
(2) Cultivate the cells in a 10 cm cell culture dish. After each group of treatment, directly add an appropriate amount of formaldehyde to the cell culture medium and gently mix to reach a final concentration of 1%. Incubate at 37°C for 10 minutes to cross-link the target proteins and the corresponding genomic DNA.
(3) Add 1.1 mL of Glycine Solution (10X), gently mix. Place at room temperature for 5 minutes.
(4) Place the cell culture dish with the cell samples on an ice bath. Remove the culture medium containing formaldehyde and Glycine (HY-Y0966), and try to keep no liquid residue.
(5) During the 5 minutes of room temperature placement, dilute 100 mM PMSF with ice-bathed PBS to 1 mM, that is, prepare ice-bathed PBS containing 1 mM PMSF.
(6) Add 5-10 mL of ice-bathed PBS containing 1 mM PMSF, wash the cells, and remove the liquid, trying to keep no liquid residue.
(7) Add 5-10 mL of ice-bathed 1 mM PMSF PBS, further wash the cells, and remove the liquid, trying to keep no liquid residue.
(8) Add 1 mL of ice-bathed 1 mM PMSF PBS, scrape the cells with a cell scraper, and collect them in a centrifuge tube. Count the cells and aliquot them into tubes containing approximately 1 million cells each.
(9) At 4°C, centrifuge at 800-1000g for 1-2 minutes to fully precipitate the cells. If the precipitation is not sufficient, you can appropriately extend the centrifugation time. Remove the supernatant and try to minimize liquid residue.
(10) Prepare an appropriate amount of SDS Lysis Buffer containing 1 mM PMSF. Resuspend the 1 million cell precipitate with 0.2 mL of the SDS Lysis Buffer containing 1 mM PMSF.
(11) Incubate at the ice bath for 10 minutes to fully lyse the cells.
(12) Perform ultrasonic treatment to shear the genomic DNA until a suitable number of ultrasonic cycles can be found to break most of the genomic DNA into 200-1000bp fragments (ultrasonic conditions: 3 seconds on, 3 seconds off, 20% power, treatment times: 2, 4, 6, 8, 10, 12 min).
(13) Add 8 μL of 5 M NaCl to the ultrasonicated sample, mix well. Heat at 65°C for 4 hours to remove the cross-linking between proteins and genomic DNA.
(14) Use a DNA purification kit to recover the DNA.
(15) Take 5 μL of the recovered product and perform agarose gel electrophoresis to observe the shearing effect of the ultrasonic treatment on the genomic DNA.

2.2.2. Chromatin immunoprecipitation
(1) After optimizing the ultrasonic treatment conditions, for the samples to be tested, perform cell cross-linking, lysis, and chromosome extraction, then conduct ultrasonic treatment according to step (12), and retain a portion of the samples for denaturation and electrophoretic detection of the ultrasonic effect.
(2) Subsequently, centrifuge the ultrasonicated samples at 4°C, 12000-14000g for 5 minutes. Take the supernatant (about 0.2 mL) and transfer it to a 2 mL centrifuge tube, and place it on an ice bath.
(3) Prepare an appropriate amount of ChIP Dilution Buffer containing 1 mM PMSF. Add 1.8 mL of ChIP Dilution Buffer containing 1 mM PMSF to dilute the ultrasonicated samples, making the final volume 2 mL.
(4) Take 100 μL of the sample as Input for subsequent detection. The remaining nearly 2 mL of the sample is added to the solid-phase adsorption matrix. For example, 70 μL Protein A+G Agarose (HY-K0230) or Protein A+G Magnetic Beads (HY-K0202)/ DNA (where approximately 35 μL is the precipitate and 35 μL is the liquid), rotate or shake slowly for 30 minutes at 4°C. The purpose of this step is to reduce non-specific binding of Protein A+G Agarose/Magnetic Beads DNA and target protein or target DNA sequences.
(5) Centrifuge at 4°C, 1000g for about 1 minute or collect the magnetic beads using a magnetic rack and transfer the supernatant to a new 2 mL centrifuge tube.
(6) Add an appropriate amount of primary antibody, rotate or shake slowly at 4°C for overnight. At the same time, take another tube and add normal mouse IgG as a negative control.
(7) Add 60 μL of Protein A+G Agarose/Magnetic Beads/DNA (where approximately 30 μL is the precipitate and 30 μL is the liquid), rotate or shake slowly for 60 minutes at 4°C to precipitate the antibody-recognized protein or the corresponding complex.
(8) Centrifuge at 4°C, 1000g for about 1 minute. Remove the liquid very carefully, do not touch the precipitate or remove the supernatant using a magnetic rack. Then, wash the precipitate with the following solutions, each with a volume of 1 mL, rotate or shake slowly at 4°C for 3-5 minutes each time, and then centrifuge at 4°C, 1000g for about 1 minute or using a magnetic rack, very carefully remove the liquid, do not touch the precipitate.
a. Low Salt Immune Complex Wash Buffer once.
b. High Salt Immune Complex Wash Buffer once.
c. LiCl Immune Complex Wash Buffer once.
d. TE Buffer twice.
The precipitate obtained after all the washing steps can be used for qPCR amplification of the target gene sequence or for Western detection, ChIP-seq analysis, etc.
Note: In the ChIP experiment, performing qPCR verification is to confirm whether the target protein is enriched in specific DNA regions, which is the basis for subsequent analysis (such as ChIP-seq). Select the known or predicted binding regions of the target protein (such as promoters, enhancers) and design primers. If the target region is significantly enriched in the ChIP sample, determine that the target protein can bind to the specific DNA region.

(1) The use of high-quality and specific antibodies is crucial. The efficacy of the antibodies can be confirmed through literature research or experimental verification.
(2) Adequate washing is a key step to remove non-specific binding and ensure the specificity of the experiment.
(3) The ultrasonic disruption step needs to be optimized according to the actual situation (ultrasonic conditions or enzyme digestion concentration and time) to obtain chromatin fragments of appropriate size (usually 200-1000 bp).
(4) The excessive duration of cross-linking may lead to excessive fixation of chromatin, affecting the subsequent DNA extraction.
(5) Quality control of the experiment is carried out, including negative controls and positive controls, to verify the validity and specificity of the experiment.
Group 1: input: 10 μL, directly stored at -20℃
Group 2: add antibody: 140 μL supernatant + 1.4 mL Chip dilution buffer
Group 3: no addition of antibody: 140 μL supernatant + 1.4 mL Chip dilution buffer + 1 μL antibody (not diluted)
Note: The volume ratio of the antibody to 1 mL Chip dilution buffer is 1:1000 - 1:2000. Groups 2 and 3 can use 2 mL centrifuge tubes.
(6) Experimental repetition: To ensure the reliability of the results, it is recommended to conduct multiple independent experiments.