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Native ChIP
10 x TBS
- 0.1 M Tris-HCl (pH 7.5)
- 1.5 M NaCl
- 30 mM CaCl2
- 20 mM MgCl2
- 50 mM Na butyrate (pH 8.0)
Digestion buffer
- 0.32 M sucrose
- 50 mM Tris-HCl (pH 7.5)
- 4 mM MgCl2
- 1 mM CaCl2
- 0.1 mM PMSF
- 5 mM Na butyrate
Lysis buffer
- 1.0 mM Tris-HCl (pH7.4)
- 0.2 mM Na2EDTA
- 0.2 mM PMSF
- 5 mM Na butyrate
Incubation buffer
- 50 mM NaCl
- 20 mM Tris-HCL (pH 7.5)
- 20 mM Na butyrate
- 5 mM Na2EDTA
- 0.1 mM PMSF
Buffer A
- 50 mM Tris-HCl, (pH 7.5)
- 10 mM EDTA
- 5 mM Na butyrate
- 50 mM NaCl
Buffer B
- 50 mM Tris-HCL (pH 7.5)
- 10 mM EDTA
- 5 mM Na butyrate
- 100 mM NaCl
Buffer C
- 50 mM Tris-HCL (pH 7.5)
- 10 mM EDTA
- 5 mM Na butyrate
- 150 mM NaCl
Protein A Sepharose
- Pre-swell protein A Sepharose overnight in buffer A at 4°C.
- Centrifuge (10,000 x g, 10 min) and resuspend pellet in approximately an equal volume (50% v/v) of buffer A.
Stage 1 - Preparation of native chromatin from cultured human cells.
All solutions used in this stage need to be ice-cold.
Solutions containing sucrose must be made fresh on the day you're using them and protease inhibitors should be added to all lysis solutions before use (0.1mM PMSAF and complete mini protease inhibitors; commercially available).
Steps
Grow cultured cells (eg HL-60 or lymphoblastoids) to a density of approximately 1 x 106 cells/ml, until they are in log phase.
Harvest cells: centrifuge samples (1,000 g, 10 min, 4°C) and wash the cell pellet 3 x with ice-cold PBS.
It is essential that 5 mM Na butyrate is present in all solutions throughout chromatin isolation when using antibodies to acetylated histones to prevent deacetylation.
Resuspend cell pellet in TBS at 2 x 107 cells/ml and add an equal volume of 1.0% v/v Tween 40 in TBS.
- Add PMSF to a final concentration of 0.5 mM.
- Leave stirring gently on ice for 1 hr:
- Transfer the suspension into a 50 ml tube with a small magnetic bar or flea.
- Place the tube in ice on top of a magnetic stirrer.
Transfer cell lysate to an all-glass homogenizer and homogenize 7 ml aliquots with seven strokes using an ‘A’ or ‘tight’ pestle.
- Check that nuclei have been released by phase-contrast microscopy; intact cells should have the central dark region of the nucleus surrounded by a halo, which is the less dense cytoplasm.
You may have to increase or decrease this homogenization step to maximize the yield of nuclei depending on the cell line.
Centrifuge samples (8,000 g, 20 min, 4°C).
Resuspend nuclei pellet in 25% [w/v] sucrose/TBS at 4 x 106 nuclei/ml.
- Underlay with 0.5 vol of 50% [w/v] sucrose/TBS.
- Centrifuge the samples (10,000 g, 15 min, 4°C).
Discard the supernatant and wash nuclei pellet in 5 ml 25% [w/v] sucrose/TBS.
- Centrifuge samples (10,000 x g, 15 min, 4°C).
Resuspend nuclei pellet in 5 ml digestion buffer and check absorbance ratios at 260 nm and 280 nm for a diluted sample of the nuclei suspension.
- Calculate the approximate DNA concentration from the A260 reading (the ratio of A260/A280 should be about 1.1).
- The yield of chromatin (in µg) is given by: A260 x dilution factor x volume x 50.
- Centrifuge samples (10,000 rpm, 10 min, 4°C) and resuspend the nuclei pellet at 0.5 mg/ml in 1.7 ml Eppendorf tube(s).
- Divide into 1 ml aliquots, if necessary.
Stage 2 - Micrococcal nuclease digestion
We usually add 50 U micrococcal nuclease per 0.5 mg DNA, in a reaction volume of 1.0 ml. This is usually provided as a powder; dissolve the micrococcal nuclease in dH20 to the required concentration and store as small aliquots at -20°C. Aliquots may be re-frozen and reused once. This step needs to be carefully controlled, especially in the initial preparations.
High concentrations of micrococcal nuclease may over-digest the chromatin, leading to sub-nucleosomal particles. You should aim to obtain a long/medium oligonucleosome ladder. If pure mononucleosome preparations are required to carry out a linear sucrose gradient (5-20%), this will increase resolution.
Steps
Perform microccal nuclease digestions at 37°C for 5 min.
Stop reaction by addition of 0.2 M EDTA to a final concentration of 5 mM.
Place all samples on ice for 5 min; centrifuge samples (12,000 g, 5 min).
Remove and keep the first S/N
- Store overnight at 4°C.
S/N is called the S1 fraction; total vol 1.0 ml.
Resuspend the pellet in 1.0 ml Lysis buffer and dialyze overnight against 2 l of the same buffer.
After overnight dialysis, centrifuge samples.
- We recommended centrifuging at 1,800 g for 10 mins at 4°C.
Remove and keep the supernatant at store at 4°C.
The supernatant can be called the S2 fraction; total vol about 1.2 ml after dialysis.
Resuspend insoluble pelleted material in 200 μl lysis buffer.
Pelleted material may also be called the P fraction.
Stage 3 - Analysis of soluble chromatin fractions
Steps
Check A260/A280 in all samples.
- The ratios for S1, S2, and P fractions are approximately 1.7, 1.5 and 1.3, respectively.
- Analyze all samples by 1.2% agarose gel electrophoresis.
- Prepare samples: x μl (total of 5 μg) chromatin fraction (S1, S2, and P), y μl dH2O (x+y = 25 μl), 3 μl 1% [w/v] SDS (final conc 0.1%), 2 μl gel loading buffer, containing bromophenol blue.
Stain the gel with 0.5 μg/ml ethidium bromide after the run has finished.
Stage 4 - Immunoprecipitation
Steps
100-200 μg unfixed chromatin + 100-200 μl affinity purified antibody (50-100 μg Ig) and the final volume made up to 1.0 ml with incubation buffer.
- A negative control, with no added antibody, also needs to be set up to test for any nonspecific binding of the chromatin to the protein A Sepharose.
Incubate overnight at 4°C on a slow rotating turntable.
- Add 200 μl 50% v/v protein A Sepharose.
- Incubate for 3 hr at room temperature on a fast-rotating turntable.
Use a siliconized pipette with the tip cut off to add protein A Sepharose.
Centrifuge samples, remove and keep the S/N.
- We recommend centrifuging at 2,000 g for 10 mins at 4°C.
- This is the unbound (or “U”) fraction.
Resuspend the Sepharose pellet in 1 ml buffer A.
- Layer onto 9 ml of the same buffer using a siliconized pasteur pipette and a siliconized 15 ml tube.
Centrifuge samples, discard the S/N and wash the Sepharose sequentially in 10 ml buffer B and buffer C.
- We recommended centrifuging at 2,000 g for 10 mins at 4°C.
Resuspend the Sepharose in 1 ml buffer C and transfer back to siliconized Eppendorfs.
Centrifuge samples and resuspend the sepharose pellet
- We recommend centrifuging at 2,000 g for 10 mins at 4°C.
- Resuspend in 250 μl 1.0% SDS/incubation buffer and incubate for 15 min at RT on a fast turntable.
Centrifuge the samples and remove and keep S/N.
We recommend centrifuging at 2,000 g for 10 mins at 4°C.
Here, the S/N is the bound (or “B”) fraction.
Wash the Sepharose in 250 μl 1.0% SDS/incubation buffer and centrifuge immediately
- We recommend centrifuging at 2,000 g for 10 mins at 4°C.
- Remove the S/N and pool with the previous bound fraction from the previous step.
Stage 5 - DNA isolation
Add 500 μl incubation buffer to each bound fraction, to reduce the SDS concentration to 0.5% SDS. Unbound and bound fractions can be then treated as follows.
Steps
Add 0.33 vol (330 μl) phenol/chloroform; vortex and spin.
- We recommend spinning at 13,000 rpm for 10 mins in a microcentrifuge.
- Keep the organic phase and interface; this is used to isolate immunoprecipitated proteins (see below).
Transfer the aqueous supernatant to an equal volume (1.0 ml) of phenol/chloroform; vortex and spin (13,000 g, 10 min, microcentrifuge).
- We recommend spinning at 13,000 g for 10 mins in a microcentrifuge.
Transfer supernatant to an equal volume (1.0 ml) of chloroform; vortex and spin.
- We recommend spinning at 13,000 g for 10 mins in a microcentrifuge.
Transfer S/N to a clean centrifuge tube and add 0.1 vol (100 μl) 4 M LiCl, 50 μg glycogen as a carrier and 4 vol of ethanol.
- Glycogen should be molecular biology grade, dissolved in dH20 at 2 mg/ml.
- Vortex thoroughly and leave at -20°C overnight.
Centrifuge samples to precipitate the DNA.
- We recommend centrifuging at 13,000 g for 15 mins.
Wash the pellet with 70% ethanol and redissolve the DNA in 250 μl TE buffer.
Ethanol should be molecular biology grade.
Store samples at -20°C or proceed with detection method.
- Detection method can vary based on your experimental design eg PCR, microarray
PCR is used to quantify DNA levels of specific loci. This is analyzed semi-quantitatively (analyses of PCR end-product by agarose gel) using primers which can be designed using this tool.
Stage 6 - Protein isolation
Steps
To the first phenol/chloroform phase (see DNA isolation, step 1) add 5 μl of a 1 mg/ml solution of BSA (to be used as a carrier), 0.01 vol (4 μl) 10 M H2SO4 and 12 vol of acetone.
After overnight precipitation at -20°C, wash the protein pellets once in acidified acetone and 3 times in dry acetone.
- Acidified acetone: 1:6 100 mM H2SO4:acetone.
- Proteins can be analyzed by SDS-PAGE.