Sample Preparation

For optimal ChIP results, use approximately 4 x 10⁶ cells per immunoprecipitation (at least 12 x 10⁶ cells including positive and negative controls).

1. To crosslink proteins with DNA, add 540 µl of 37% formaldehyde to a 15 cm culture dish (20 mL medium). For suspension cells, add 540 µl of 37% formaldehyde to cells suspended in 20 mL medium (for optimal fixation of suspension cells, the cell density should be less than 0.5 x 10⁶ cells/ml during fixation). Mix gently and incubate on a shaker at room temperature for 10 minutes. The final concentration of formaldehyde is 1%. A color change in the medium will be observed after adding formaldehyde.

2. Add 2 ml of 10x glycine (to a final concentration of 0.125 M) to terminate the reaction, and incubate at room temperature for 5 minutes. A color change in the medium will occur upon adding glycine.

3. For adherent cells, discard the medium and wash the cells twice with ice-cold 1x PBS, completely removing the wash solution from the dish each time. Add 5 mL ice-cold 1x PBS (containing protease inhibitor cocktail), scrape the cells with a cell scraper, and transfer to a 15 mL tube. Centrifuge at 1000g for 5 minutes at 4°C, discard the supernatant.

4. For suspension cells, transfer the cells to a 50 mL centrifuge tube, centrifuge at 500g for 5 minutes at 4°C, and wash the pellet twice with ice-cold 1x PBS, discard the supernatant.

5. Add ChIP lysis buffer (1 mL per 1x10⁷ cells) and incubate on ice for 10 minutes. Alternatively, samples can be temporarily stored at -80°C for up to 3 months.

Chromatin Sonication

1. Sonicate to shear genomic DNA, resulting in most DNA fragments between 200-1000bp, preferably with most controlled between 400-800bp. This step requires optimization, and different cell lines require different sonication times.

Note：

1) Exploration conditions for contact probe Xinzhi sonicator: 2mm diameter micro-probe, amplitude 20% (power 130W), sonicate for 5s, pause for 15s, set sonication time gradient of 2, 4, 8, 12, 16 minutes. For HeLa cells, 2 minutes can yield good chromatin fragmentation samples.

2) Sonication conditions may vary for different cells, requiring pre-experiments. Note that the sonication volume and cell dosage should be fixed each time, otherwise a relatively fixed sonication condition cannot be used for subsequent experiments. During sonication, please ensure the sample is in an ice bath and at a low temperature. Do not let the probe touch the bottom or wall of the sonication tube. If foam is generated during sonication, pause sonication and adjust the position of the sonication tube.

3) For sonication buffer: higher SDS concentration makes DNA easier to break; higher salt ion or detergent concentration makes DNA easier to break; higher cell density makes DNA harder to break; compared to commonly used tumor cell lines, chromatin sonication of many primary cells and tissue cells is more difficult, such as T cells, which are often difficult to sonicate because they are very small and compact cells. In this case, different lysis and sonication conditions are needed to achieve the best results, and crosslinking reaction time can be reduced to 5 minutes.

2. Centrifuge at 8,000g for 10 minutes at 4°C to pellet cell debris, transfer the supernatant to a new EP tube.

DNA Concentration and Fragment Size Determination

1. Add 8 µL 5 M NaCl to 200 µL chromatin and incubate overnight at 65°C with shaking.

2. Add 1 µL Proteinase K (20 mg/mL), incubate at 42°C with shaking for 1h.

3. Purify DNA using a PCR purification kit.

4. Determine DNA fragment size by 1.5% agarose gel electrophoresis.

Immunoprecipitation

1. After optimizing sonication conditions, sonicate the samples.

2. Prepare appropriate amount of ChIP Dilution Buffer containing protease inhibitors (protease inhibitors: ChIP Dilution Buffer=1:100). Take 100µl sonicated sample, add 900µl ChIP Dilution Buffer containing protease inhibitors, make the final volume 1mL.

3. Take out 20µL (2%) sample as Input and store at -20°C for subsequent detection. Set positive control and isotype control (negative control) simultaneously.

4. Add 2—5µg primary antibody to the sample, rotate slowly at 4°C overnight or for at least 4h.

5. Add 40µL Protein A/G Magnetic Beads/Salmon Sperm DNA, rotate slowly at 4°C for 60 minutes to precipitate proteins recognized by the primary antibody or corresponding complexes.

Note: Protein A/G magnetic beads can be used directly. For applications highly sensitive to non-specific binding, blocking reagents can be added to the ChIP reaction. Blocking reagents: 2.5 µg/µL BSA, 2.5µg/µL Salmon Sperm DNA (final concentration).

6. Place on a magnetic rack for 1 minute to separate, remove the supernatant, do not touch the magnetic beads. Wash the magnetic beads with the following solutions in sequence, each wash solution volume is 1mL, each wash is rotated or shaken slowly at 4°C for 3—5 minutes, then placed on a magnetic rack for 1 minute, carefully remove the supernatant, do not touch the magnetic beads.

1) Wash once with Low Salt Immune Complex Wash Buffer.

2) Wash once with High Salt Immune Complex Wash Buffer.

3) Wash once with LiCl Immune Complex Wash Buffer.

4) Wash twice with TE Buffer.

Elution and Decrosslinking

1. Freshly prepare appropriate amount of Elution buffer (1% SDS, 0.1M NaHCO3).

2. After completing all washing steps, add 150µL Elution buffer.

3. Place the sample in a metal bath with oscillation function, incubate at 65°C for 30 minutes (amplitude 300 rpm) to elute chromatin from the magnetic beads.

4. Centrifuge at 10,000g for 10 seconds to collect residual sample on the centrifuge tube cap.

5. Place the centrifuge tube on a magnetic rack for 60 seconds, transfer the supernatant to a new centrifuge tube and label them respectively.

6. Add 6µL 5M NaCl and 2µL Proteinase K (20mg/mL) to all tubes, including input tubes (add 150µL Elution buffer to each corresponding input sample), heat at 65°C for 2h or overnight to remove crosslinks between protein and genomic DNA.

DNA Purification and Analysis

1. Process 150µL samples according to the DNA purification kit instructions.

2. PCR detection and analysis.

Protocol Download

https://huabio.cn/resources?category=Protocol

Product Recommendation

染色质免疫沉淀检测试剂盒ChIP Assay Kit (Protein A/G磁珠)