The principle of Chromatin Immunoprecipitation (ChIP) is to fix protein-DNA complexes in living cells and randomly fragment them into chromatin fragments within a certain length range. Subsequently, the complexes are precipitated using immunological methods to specifically enrich DNA fragments bound to the target protein. By purifying and detecting the target fragments, information on protein-DNA interactions can be obtained.

 

I. Crosslinking and Lysis of Cells

 

Adherent cells are cultured in a 150mm culture dish containing 20ml of growth medium until they reach 80%-90% confluence, approximately 10⁷ cells. If necessary, stimulation or treatment can be performed. It is recommended to use 1×10⁶ cells per ChIP experiment.

 

1) Add formaldehyde to a final concentration of 1% in 20ml of medium, gently vortex the culture dish to mix, and fix the cells in formaldehyde at room temperature for 10 minutes (for transcription factors and co-factors, crosslinking time can be extended appropriately, but not exceeding 30 minutes). The color of the medium will change after adding formaldehyde;

2) Add glycine to a concentration of 0.125M in the culture dish, gently vortex to mix, and react at room temperature for 5 minutes to quench unreacted formaldehyde and terminate crosslinking. The color of the medium will change after adding glycine;

3) Discard the medium and wash the cells with 20ml of pre-chilled 1×PBS, repeating the wash once;

4) Add 2ml of pre-chilled 1×PBS containing a protease inhibitor cocktail to the culture dish. Collect the cells using a cell scraper, and centrifuge at 4°C, 800g, for 5 minutes;

5) Remove the supernatant (the cells can be quickly frozen in liquid nitrogen and stored at -80°C for several months). Resuspend the cells in 0.5ml of lysis buffer containing a protease inhibitor cocktail, and incubate on ice for 15 minutes, vortexing every 5 minutes;

6) Centrifuge at 4°C, 800g, for 5 minutes. Carefully remove the supernatant and resuspend in 0.5ml of nuclear lysis buffer containing a protease inhibitor cocktail.

II. Sonication to Fragment DNA

 

The efficiency of sonication depends on the cell type, cell concentration, and instrument. Optimal sonication conditions must be determined through preliminary experiments to shear crosslinked DNA to approximately 200-1000bp in length, as shown in the figure above. Chromatin fragmentation is crucial for the success of ChIP experiments. Insufficient fragmentation can lead to sample loss, while excessive fragmentation can destroy epitopes of target proteins and reduce ChIP efficiency. During sonication, keep the samples on ice at low temperature. Do not allow the sonication probe to contact the bottom or walls of the tube. If foaming occurs during sonication, pause and adjust the position of the tube.

 

After sonication, centrifuge at 4°C, 12000g for 10 minutes. After centrifugation, take 5μl of the chromatin solution for agarose gel analysis to assess sonication efficiency. Compare 5μl samples taken before and after sonication.

Figure 1 Before and After Sonication

 

III. Immunoprecipitation of Target Protein and DNA

 

Keep the following buffers on ice: low-salt wash buffer, high-salt wash buffer, LiCl wash buffer, and TE wash buffer. Prepare 450μl of dilution buffer (containing protease inhibitor cocktail) and keep it on ice. If multiple samples are used, the buffer can be prepared in bulk.

 

1) Take 50μl of the supernatant from the lysate after centrifugation and transfer it to a new centrifuge tube as the DNA mixture sample for one immunoprecipitation experiment. Each 50μl of lysate contains the lysate products of 1×10⁶ cells. The ChIP reaction includes a positive control antibody tube, a negative control IgG tube, and a target protein antibody tube. It is recommended to use the same species for the negative control IgG and target antibody.

2) Add 450μl of the prepared dilution buffer to the centrifuge tube containing the fragmented chromatin sample (50μl), and mix thoroughly. Take 5μl of the sample from each tube and transfer it to a new centrifuge tube as the 1% “input” for the experiment, which is used for optimization and data processing. Store at 4°C until the protein-DNA complex elution and de-crosslinking step.

3) Add 1-10μg of immunoprecipitation antibody to the centrifuge tube containing the input sample, and incubate on a rotator at 4°C for more than 4 hours or overnight.

4) Add 20μl of protein A/G magnetic beads to each tube (it is recommended to cut off a small part of the pipette tip before aspirating the beads), and incubate on a rotator at 4°C for 2 hours.

5) Place the tubes on a magnetic rack and let them stand for 1-2 minutes to remove the supernatant. Wash the protein A/G magnetic bead-antibody-protein/DNA complex as follows:

Low-salt wash buffer, incubate on a rotator at 4°C for 3-5 minutes, wash once;

High-salt wash buffer, incubate on a rotator at 4°C for 3-5 minutes, wash once;

LiCl wash buffer, incubate on a rotator at 4°C for 3-5 minutes, wash once;

 

Note: Mix equal volumes of solutions A and B just before use. Do not mix in advance, as this will cause precipitation.TE buffer, incubate on a rotator at 4°C for 3-5 minutes, wash once.

Figure 2 Schematic of Target Protein and DNA Immunoprecipitation

IV. Elution and Decrosslinking

 

Preparation Before Experiment: Thaw proteinase K; allow ChIP elution buffer to reach room temperature to ensure SDS dissolution; prepare elution buffer; mix 100μl of ChIP elution buffer with 1μl of proteinase K (can be prepared in bulk for multiple samples).

1) Add ChIP elution buffer (containing proteinase K) to the sample and input tubes;

2) Incubate at 62°C for 2 hours;

3) Incubate at 95°C for 10 minutes;

4) Allow the samples to cool to room temperature;

5) Centrifuge at 10000g for 10 seconds to collect residual samples from the tube caps and walls. Use a magnetic rack to separate the magnetic beads. Carefully transfer the supernatant to a new tube.

 

V. DNA Purification

Purify DNA using a purification column (for both immunoprecipitated and input samples).

 

VI. Analysis

Detect the immunoprecipitated DNA using qPCR or high-throughput sequencing methods.

 

VII. Common Issues

Common Issues	Common Causes/Solutions
High background in negative control (IgG IP) samples	1. Optimize antibody concentration; excess antibody can lead to non-specific binding; 2. Non-specific binding to beads: Use a pre-clearing step to exclude non-targets, or add a blocking agent to the beads; 3. Incomplete chromatin fragmentation: Optimize the fragmentation process to obtain chromatin fragments of 200-1000bp; 4. Contamination of reagents; 5. Increase the number of washes.
Low DNA recovery	1. Ineffective or low-affinity ChIP antibody; use antibodies validated for ChIP; 2. Insufficient antibody amount; 3. Insufficient starting material; increase the amount of starting material; 4. Over-crosslinking or under-crosslinking; 5. Low affinity between magnetic beads and antibody.
How to perform ChIP if a suitable ChIP antibody is not available?	1. Using a tagged antibody is a solution for unavailable antibodies, antibody variability, and epitope masking in crosslinked chromatin. However, tags may interfere with the function of transcription factors; 2. Try antibodies used for IP/IHC/IF, but the effectiveness needs to be determined experimentally.
How to select a negative control antibody?	Use normal IgG from the same species as the ChIP antibody. Therefore, if using a mouse monoclonal antibody, normal mouse IgG is recommended.

 

Catalog Number	Product Name	Specifications
abs50034	Chromatin Immunoprecipitation (ChIP) Kit	22T

Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us

Absin Bioscience Inc. Email: worldwide@absin.cn

Follow us on Facebook: Absin Bio