Native chromatin

immunoprecipitation The preparation of native chromatin from cultured human cells.

protocol
All solutions need to be ice cold.

Sucrose containing solutions must be made up fresh on the day.

Add protease inhibitors to all lysis solutions before use (0.1mM PMSAF and complete
mini protease inhibitors; commercially available).

1. Cultured cells (e.g. HL-60 or lymphoblastoids) are grown to a density of

approximately 1 x 106 cells/ml, until they are in log phase.

2. Harvest cells: centrifuge samples (1,000 g, 10 min, 4°C) and wash the cell pellet 3 x
with ice cold PBS (Phosphate buffered saline).

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.

3. Resuspend 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 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).

4. 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 cell line. Keep cells on ice between the rounds of
homogenisations.

5. Centrifuge samples (8,000 g, 20 min, 4°C).

6. Resuspend nuclei pellet in 25% [w/v] sucrose/TBS at 4 x 106 nuclei/ml and underlay
with 0.5 vol of 50% [w/v] sucrose/ TBS; centrifuge the samples (10,000 g, 15 min,
4°C).

7. Discard supernatant and wash nuclei pellet in 5 ml 25% [w/v] sucrose/TBS;

centrifuge samples (10,000 x g, 15 min, 4°C).

8. 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.

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 Micrococcal nuclease digestion

Normally we 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 re-used 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 carry
out a linear sucrose gradient (5-20%), this will increase resolution.

1. Perform microccal nuclease digestions at 37°C for 5 min.

2. Stop reaction by addition of 0.2 M EDTA to a final concentration of 5 mM.

3. Place all samples on ice for 5 min; centrifuge samples (12,000 g, 5 min).

4. Remove and keep the first S/N (this is called the S1 fraction; total vol 1.0 ml); store
overnight at 4°C.

5. Resuspend the pellet in 1.0 ml Lysis buffer and dialyze overnight against 2 l of the
same buffer.

6. After overnight dialysis centrifuge samples (1,800 g, 10 min, 4°C).

7. Remove and keep the supernatant (called the S2 fraction; total vol about 1.2 ml
after dialysis); store at 4°C.

8. Resuspend insoluble pelleted material in 200 μl lysis buffer (called the P fraction).

Analysis of soluble chromatin fractions

1. 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.

Do not place ethidium bromide in the agarose gel or the electrophoresis buffer,
because of the presence of SDS (see below).

Preparation of 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.

2. Stain the gel with 0.5 μg/ml ethidium bromide after the run has finished.

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 Immunoprecipitation

1. 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.

2. Incubate overnight at 4°C on a slow rotating turntable. Add 200 μl 50% v/v protein
A Sepharose; use a siliconized pipette with the tip cut off to make this step easier.
Incubate for 3 hr at room temperature on a fast rotating turntable. (Make sure
that the Sepharose is in a suspension at all times).

3. Centrifuge samples (2,000 g, 10 min, 4°C), remove and keep the S/N; this is the
unbound (or “U”) fraction.

4. Resuspend the Sepharose pellet in 1 ml buffer A and layer onto 9 ml of the same
buffer using a siliconized pasteur pipette and siliconized 15 ml tube.

5. Centrifuge samples (2,000 g, 10 min, 4°C), discard the S/N and wash the
Sepharose sequentially in 10 ml buffer B and buffer C.

6. Finally, resuspend the Sepharose in 1 ml buffer C and transfer back to siliconized

Eppendorfs.

7. Centrifuge samples (2,000 g, 10 min, 4°C) and resuspend the sepharose pellet in
250 μl 1.0% SDS / incubation buffer and incubate for 15 min at RT on a fast
turntable. (Ensure that the Sepharose is thoroughly resuspended at all times).

8. Centrifuge the samples (2,000 g, 10 min, 4°C) and remove and keep S/N; this is the
bound (or “B”) fraction.

9. Wash the Sepharose in 250 μl 1.0% SDS / incubation buffer and centrifuge
immediately (2,000 g, 10 min, 4°C). Remove the S/N and pool with the previous
bound fraction from the previous step.

DNA Isolation

Add 500 μl incubation buffer to each bound fraction, to reduce the SDS concentration
to 0.5% SDS. Unbound and bound fractions then treated as follows:

1. Add 0.33 vol (330 μl) phenol/chloroform; vortex and spin (13,000 rpm, 10 min,
microcentrifuge). Keep the organic phase and interface; this is used to isolate
immunoprecipitated proteins (see below).

2. Transfer the aqueous supernatant to an equal volume (1.0 ml) of

phenol/chloroform; vortex and spin (13,000 g, 10 min, microcentrifuge).

3. Transfer supernatant to an equal volume (1.0 ml) of chloroform; vortex and spin
(13,000 g, 10 min, microcentrifuge).

4. Transfer S/N to a clean centrifuge tube and add 0.1 vol (100 μl) 4 M LiCl, 50 μg
glycogen (Molecular biology grade, dissolved in dH20 at 2 mg/ml) as a carrier
and 4 vol of ethanol. Vortex thoroughly and leave at -20°C overnight.

5. Centrifuge samples (13,000 g, 15 min) to precipitate the DNA.

6. Wash the pellet with 70% ethanol (molecular biology grade) and redissolve the
DNA in 250 μl TE buffer.

7. Store samples at -20°C or proceed with detection method (PCR, microarray, etc).
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 8. 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.

Alternatively, DNA levels are quantitatively measured by real-time PCR. Primers and
probes are often designed using software provided with the real-time PCR apparatus.

Protein Isolation

1. 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.

2. After overnight precipitation at -20°C, wash the protein pellets once in acidified
acetone (1:6 100 mM H2SO4:acetone) and 3 times in dry acetone. Proteins can
be analyzed by SDS-PAGE.

Solutions

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 Na22EDTA
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

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 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.

(Adapted from protocols used by Laura O’Neill and Prof. Bryan Turner. University of Birmingham)