X-ChIP Protocol Summary

Disclaimer

This procedure should be used as a guideline only. Please keep in mind that Creative Biolabs cannot guarantee specific results for the X-ChIP protocol.

Chromatin immunoprecipitation (ChIP) is a versatile technique for detecting protein-DNA interactions. Creative Biolabs summarizes the specific details of performing ChIP on cells. Also, the DNA obtained based on this protocol can be used for ChIP-qPCR or ChIP-seq analysis. In addition, we offer custom antibody development services for ChIP to help you with your research.

Preparation of Native Chromatin in Cultured Human Cells

Preparation

1. Reagents required

Formaldehyde Glycine, cold PBS, ChIP lysis buffer, elution buffer, sodium chloride (NaCl), RNase, proteinase K, PCR purification kit or materials for phenol: chloroform extraction, Tris-EDTA (TE), 1.5% agarose gel with a 100 bp DNA marker, RIPA buffer, primary antibody, low salt wash buffer, high salt wash buffer, lithium chloride (LiCl) wash buffer

For Protein A/G: Protein A beads, Protein G beads, RIPA buffer, single-stranded herring sperm DNA

2. Solution formulation

Procedure

Cross-Linking Proteins to DNA & Harvesting Cells

Formaldehyde is used to cross-link proteins to DNA. Crosslinking is a time-dependent process that needs to be optimized. (We recommend crosslinking samples for 2–30 minutes. Over-crosslinking can reduce antigen accessibility and sonication efficiency. Epitopes may also be masked. Add glycine to quench formaldehyde and terminate the cross-linking reaction.

1. Start with two confluent 150 cm² plates (1x10⁷–5x10⁷ cells per plate). Add formaldehyde directly dropwise to the medium at a final concentration of 0.75% and rotate gently for 10 min at room temperature (RT) to cross-link the protein to the DNA.
2. Add a final concentration of 125 mM glycine to the medium and incubate for 5 min at room temperature with shaking.
3. Rinse the cells twice with 10 mL of cold PBS.
4. Add 5 mL of cold PBS, scrape the plate thoroughly with a cell scraper, and transfer to a 50 mL tube.
5. Add 3 mL PBS to the dish, scrape again, and transfer the remaining cells to a 50 mL tube.
6. Centrifuge for 5 min at 4°C, 1,000 g.
7. Carefully aspirate the supernatant and resuspend the pellet in ChIP lysis buffer (750 μL per 1x10⁷ cells) and incubate on ice for 10 minutes.

When using suspension cells, start with 1x10⁷–5x10⁷ cells and treat with 0.75% formaldehyde and glycine as described above (step 1). Pellet the cells by centrifugation (5 min, 1,000 g). Wash 3 times with cold PBS and resuspend the cell pellet in ChIP lysis buffer (750 μL per 1x10⁷ cells).

Sonicate the Cell Lysate to Separate Chromatin

1. Sonicate the lysate to shear the DNA to an average fragment size of 200–1,000 bp. This will need to be optimized, as different cell lines require different sonication times. (The cross-linked lysate should be sonicated for a certain period of time to determine the optimal conditions. Samples should be removed during the time course and DNA isolated as described in step 3. Fragment size should be analyzed on a 1.5% agarose gel.)
2. After sonication, cell debris is precipitated by centrifugation for 10 min at 4°C, 8,000 g. The supernatant is transferred to a new tube. This chromatin preparation will be used for immunoprecipitation (IP) in step 4.
3. Remove 50 μL from each sonicated sample to determine DNA concentration and fragment size. (Ultrasonicated chromatin can be snap frozen in liquid nitrogen and stored at -80°C for up to 3 months. Avoid multiple freeze-thaws. In addition, prolonged sonication can disrupt ribosome-DNA interactions, so the band size should not be less than 200 bp.)

Calculate DNA Concentration and Measure DNA Fragmentation Size

Ultrasonicated chromatin samples can be used to calculate DNA concentration for subsequent IP and to measure DNA fragment size.

1. Add 70 μL of elution buffer to 50 μL of chromatin.
2. Add 4.8 µL of 5 M NaCl and 2 µL of RNase A (10 mg/mL) and incubate overnight at 65°C while shaking.
3. Add 2 μL of proteinase K (20 mg/mL) and incubate at 60°C for 1 hour while shaking.
4. Purify DNA using a PCR purification kit or the phenol:chloroform extraction method.
5. To determine the concentration of DNA, 5 μL of purified DNA was transferred to a tube containing 995 μL TE, a 200-fold dilution was performed, and OD260 was read. The concentration of DNA in μg/mL is OD260 x 10,000. This was used to calculate the concentration of DNA in the chromatin preparation. Purified DNA was run in a 1.5% agarose gel with a 100 bp DNA marker to determine fragment size.

• In the second step, why use RNase A?

When using the PCR purification kit, samples are treated with RNase A, as high levels of RNA can interfere with DNA purification. The yield may drop severely as the column becomes saturated.

• In the third step, why use proteinase K?

Samples are treated with proteinase K, which cleaves peptide bonds near the carboxyl groups of aliphatic and aromatic amino acids. The cross-link between protein and DNA is disrupted, which helps in the purification of DNA.

Chromatin Immunoprecipitation

1. Use the prepared chromatin (the second step in Sonicate the Cell Lysate to Separate Chromatin). It is recommended to have approximately 25 μg of DNA per IP. Dilute each sample 1:10 with RIPA buffer. You will need one sample for the specific antibody and one sample for the control (beads only). Remove 50 μL of chromatin as your input sample and store at -20°C until further use.
2. Add primary antibodies to all samples, except for the pure bead control, and spin for 1 hour at 4°C. The amount of antibody added should be determined empirically; adding 1–10 μg of antibody per 25 μg of DNA usually works well.
3. Preparation of protein A/G beads: If both protein A and protein G beads are used, mix equal volumes of protein A and protein G beads and wash three times in RIPA buffer. Aspirate the RIPA buffer and add single-stranded herring sperm DNA to a final concentration of 75 ng/μL beads and BSA to a final concentration of 0.1 μg/μL beads. Add RIPA buffer to twice the volume of the beads and incubate for 30 min at RT with rotation. Wash once with RIPA buffer and add RIPA buffer to twice the volume of the beads.
4. Add 60 μL of blocked protein A/G beads to all samples and spin IP overnight at 4°C. (Protein A beads, protein G beads, or a mixture of both should be used. The table in Appendix shows the affinity of protein A and G beads for different immunoglobulin isoforms.)
5. Centrifuge the immunoprecipitated samples at 2,000 x g for 1 min and remove the supernatant.
6. Perform washes: once in low-salt wash buffer, once in high-salt wash buffer, and once in lithium chloride wash buffer. Centrifugation at 2,000 g for 1 minute followed each wash to remove the supernatant. (If a high background is observed, additional washing may be required. Alternatively, sonicated chromatin can be pre-cleared by incubating with protein A/G beads for 1 hour prior to step 2. Any non-specific binding to the beads will be removed in this additional step. Transfer the supernatant (sonicated chromatin) to a new tube and incubate with the antibody and beads as described in step 2.)

Elution and Reversal of DNA cross-links

1. Add 120 μL of elution buffer to the protein A/G beads to elute DNA, and vortex slowly for 15 min at 30°C.
2. Centrifuge at 2,000 g for 1 min and transfer the supernatant to a new tube.
3. Add 4.8 µL of 5 M NaCl and 2 µL of RNase A (10 mg/mL) and incubate overnight at 65°C while shaking.
4. Add 2 μL of proteinase K (20 mg/mL) and incubate at 60°C for 1 hour while shaking.
5. DNA can be purified using PCR purification kits or phenol-chloroform extraction methods.

Analysis of DNA Levels in ChIP-qPCR or ChIP-seq

1. DNA levels are usually measured by real-time quantitative PCR. Primers and probes are usually designed using the software provided with the real-time PCR instrument or using an online design tool. A selection of pre-designed primers and probes are available on our website.
2. In addition, when using the ChIP-seq method, analysis can be performed by sequencing. The DNA generated using this protocol is suitable as input for the pre-processing of sequencing libraries.

Appendix:

Affinity of protein A and G beads for immunoglobulin isotypes of different species

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