Native Chromatin Immunoprecipitation Protocol

Disclaimer

This procedure should be used as a guideline only. Please keep in mind that Creative Biolabs cannot guarantee specific results for natural chromatin immunoprecipitation.

Chromatin immunoprecipitation (ChIP) is a versatile technique for detecting protein-DNA interactions. Creative Biolabs summarizes the steps and process of natural chromatin immunoprecipitation and provides custom antibody development services for ChIP to aid your research.

Preparation of Native Chromatin in Cultured Human Cells

Preparation

1. Solution formulation

2. Protein A Sepharose

Pre-swell protein A Sepharose in buffer A at 4°C overnight. It is then centrifuged (10,000 g, 10 min), and the protein A Sepharose is finally resuspended in an approximately equal volume (50% v/v) of buffer A.

Note

1. All solutions should be ice cold.
2. Solutions containing sucrose must be prepared fresh on the same day.
3. Add protease inhibitors (0.1 mM PMSAF and complete mini protease inhibitor) to all lysates prior to use.
4. When using antibodies against acetylated histones, 5 mM Na butyrate must be present in all solutions throughout the chromatin separation process to prevent deacetylation.

Procedure

1. Grow cultured cells (e.g., HL-60 or lymphoblastoid cells) to a density of 1 x 10⁶ cells/ml approximately until they are in the log phase.
2. Centrifuge samples (1,000 g, 10 min, 4°C) and wash cell pellet 3 times with ice-cold PBS (phosphate-buffered saline).
3. Resuspend the cell pellet in TBS (Tris-buffered saline) at 2 x 10⁷ cells/ml and add an equal volume of 1.0% v/v Tween 40 to TBS. Add PMSF to a final concentration of 0.5 mM. Stir gently on ice for 1 hr (transfer the suspension to a 50 ml tube with a small magnetic bar or flea; place the tube on ice (of a magnetic stirrer).
4. Transfer the cell lysate to an all-glass homogenizer and homogenize a 7 ml aliquot with an 'A' or 'tight' mortar and pestle. Check by phase contrast microscopy whether the nucleus has been released; intact cells should have a central dark area of the nucleus surrounded by a halo, which is the less dense cytoplasm. (You may need to increase or decrease this homogenization step to maximize the yield of nuclei, depending on the cell line. Keep the cells on ice between homogenization rounds.)
5. Centrifuge samples (8,000 g, 20 min, 4°C).
6. Nuclei are resuspended in 25% [w/v] sucrose/TBS at a concentration of 4 x 10⁶ nuclei/ml and primed with 0.5 volume of 50% [w/v] sucrose/TBS; samples were centrifuged (10,000 g, 15 min, 4°C).
7. The supernatant is discarded, and the nucleolus is washed with 5 ml of 25% [w/v] sucrose/TBS; samples are centrifuged (10,000 x g, 15 min, 4°C).
8. Resuspend the nucleolus pellet in 5 ml of digestion buffer and check the absorbance ratio of the nucleolus suspension diluted sample at 260 nm and 280 nm; calculate the approximate DNA concentration from the A260 readings (the ratio of A260/A280 should be approximately 1:1).

The yield of chromatin (in µg) is obtained from the following formula: A260 x dilution factor x volume x 50.

Centrifuge the sample (10,000 rpm, 10 min, 4°C) and resuspend the nuclei pellet at 0.5 mg/ml in 1.7 ml Eppendorf tube (s). If necessary, divide into 1 ml aliquots.

Micrococcal Nuclease Digestion

Note

1. Typically, we add 5 U of micrococcal nuclease per 50.0 mg of DNA in a reaction volume of 1.0 ml. This is usually provided in powder form; dissolve the micrococcal nuclease in dH20 to the desired concentration and store as small aliquots at -20°C. Aliquots can be refrozen and reused once. This step needs to be carefully controlled, especially during the initial preparation phase.
2. High concentrations of micrococcal ribozyme may over-digest chromatin, resulting in subribosomal particles. Your goal should be to obtain a long/medium oligoribosomal ladder. If pure monoribosomal preparation is required, perform a linear sucrose gradient (5–20%), which will improve resolution.

Procedure

1. Perform a Chlorella nuclease digestion at 37°C for 5 minutes.
2. Stop the reaction by adding 0.2 M EDTA to a final concentration of 5 mM.
3. Place all samples on ice for 5 min, and then centrifuge the samples (12,000 g, 5 min).
4. Remove and retain the first S/N (this is referred to as S1 fraction; total volume is 1.0 ml); store overnight at 4°C.
5. The precipitate is resuspended in 1.0 ml of lysis buffer and dialyzed overnight against Sl of the same buffer.
6. After overnight dialysis, centrifuge the sample (1,800 g, 10 min, 4°C).
7. The supernatant was removed and retained (called the S2 fraction; the total volume after dialysis was approximately 1.2 ml); it was stored at 4°C.
8. Resuspend the water-insoluble pellet in 200 μl of lysis buffer (referred to as the P fraction).

Analysis of Soluble Chromatin Fractions

1. All samples are examined for A260/A280; the ratios of S1, S2, and P fractions are approximately 1.7, 1.5, and 1.3, respectively. All samples are analyzed by 1.2% agarose gel electrophoresis. (Because of the presence of SDS, do not place ethidium bromide in agarose gels or electrophoresis buffers.)

Sample preparation: x μl (total 5 μg) chromatin graded fractions (S1, S2, and P), y μl dH2O (x + y = 25 μl), 3 μl 1% [w/v] SDS (final concentration 0.1%), and 2 μl gel loading buffer containing bromophenol blue.

1. After electrophoresis, the gels are stained with 0.5 μg/ml ethidium bromide.

Immunoprecipitation

1. 100–200 μg unfixed chromatin + 100–200 μl affinity-purified antibody (50–100 μg Ig) with incubation buffer to bring the final volume to 1.0 ml. A negative control without added antibody also needs to be established to test for non-specific binding of chromatin to protein A Sepharose. (Learn about antibody customization services.)
2. Incubate overnight at 4°C on a slowly rotating turntable. Add 200 μl of 50% v/v Protein A Sepharose; use a siliconized pipette with the tip cut off to make this step easier. Incubate on a rapidly rotating turntable at room temperature for 3 hours. (Make sure Sepharose is in suspension at all times).
3. Centrifuge the sample (2,000 g, 10 min, 4°C), remove and retain the S/N; this is the unbound (or "U") fraction.
4. Resuspend the Sepharose pellet in 1 ml of buffer A and layer it into 9 ml of the same buffer using a siliconized barrel pipette and a siliconized 15 ml tube.
5. Centrifuge the sample (2,000 g, 10 min, 4°C), discard S/N, and wash Sepharose sequentially with 10 ml of buffer B and buffer C.
6. Finally, resuspend sepharose in 1 ml of buffer C and transfer back into in silico Eppendorfs.
7. Centrifuge the sample (2,000 g, 10 min, 4°C) and resuspend the Sepharose pellet in 250 μl of 1.0% SDS/incubation buffer and incubate for 15 min at RT on a fast turntable. (Make sure that Sepharose is thoroughly resuspended at all times).
8. Centrifuge the sample (2,000 g, 10 min, 4°C), remove and retain the S/N; this is the bound (or "B") fraction.
9. Wash Sepharose with 250 μl of 1.0% SDS/incubation buffer and centrifuge immediately (2,000 g, 10 min, 4°C). Remove the S/N and mix with the bound fraction from the previous step.

DNA Isolation

1. Add 500 μl of incubation buffer to each bound fraction to reduce the SDS concentration to 0.5% SDS. Unbound and bound fractions are then processed.
2. Add 0.33 volume (330 µl) of phenol/chloroform; vortex and spin (13,000 rpm, 10 min, microcentrifuge). Retain the organic phase and interface; this will be used to separate the immunoprecipitated proteins (see below).
3. Transfer the aqueous supernatant to an equal volume (1.0 ml) of phenol/chloroform, vortex, and spin (13,000 g, 10 min, microcentrifuge).
4. Transfer the supernatant to an equal volume (1.0 ml) of chloroform, vortex, and spin (13,000 g, 10 min, microcentrifuge).
5. Transfer the S/N to a clean centrifuge tube and add 0.1 volume (100 µl) of 4 M LiCl, 50 µg glycogen (molecular biology grade, dissolved in DH20 at 2 mg/ml) as a carrier, and 4 volumes of ethanol. Vortex thoroughly and leave overnight at -20°C.
6. Centrifuge the sample (13,000 g for 15 min) to precipitate the DNA.
7. Wash the pellet with 70% ethanol (molecular biology grade) and redissolve the DNA in 250 μl TE buffer.
8. Store the samples at -20°C or continue with the assay (PCR, microarray, etc.).
9. PCR is used to quantify DNA levels at specific loci. Semi-quantitative analysis is performed using primers that can be designed with this tool (PCR end products by agarose gel analysis). Quantitative measurements of DNA levels are performed by real-time PCR. Primers and probes are usually designed using the software provided with the real-time PCR instrument.

Protein Isolation

1. In the first phenol/chloroform phase (see DNA Isolation; step 1) add 5 µl of 1 mg/ml BSA solution (as carrier), 0.01 volume (4 µl) of 10 M H₂SO₄, and 12 volumes of acetone.
2. After overnight precipitation at -20°C, the protein pellet is washed once with acidified acetone (1:6 100 mM H₂SO₄:acetone) and 3 times with dry acetone. The proteins can be analyzed by SDS-PAGE.

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