自然染色质免疫沉淀实验设计
1. The preparation of native chromatin from cultured human cells
1.1.Cultured cells (e.g. HL-60 or lymphoblastoids) are grown to a density of approximately 1 x 106 cells/ml until they are inlog phase.
1.2.Harvest
cells: centrifuge samples (7,000 g, 10 min, 4°C) and wash the cell
pellet 3 x ice cold PBS (Phosphate BufferedSaline).
It is
essential that 5 mM Na butyrate is present in all solutions throughout
chromatin isolationwhen using antibodies to acetylated histones to
prevent deacetylation.
1.3.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 inTBS. Add PMSF
to a final concentration of 0.5 mM. Leave stirring gently on ice for 1hr
(Transfer the suspension into a50ml tube with a small magnetic bar or
flea; place the tube in ice on top of a magnetic stirrer).
1.4.Transfer cell lysate to an all-glass homogeniser and homogenise 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 darkregion of the nucleus
surrounded by a halo, which is the less dense cytoplasm.
You may have to increase or decrease this homogenisation step to maximise the yield of nucleidepending on cell line.
1.5.Centrifuge samples (10,000 g, 20 min, 4°C).
1.6.Resuspend
nuclei pellet in 25% [w/v] sucrose/TBS at 4x106 nuclei / ml and
underlay with 0.5 vol of 50% [w/v] sucrose /TBS; centrifuge the samples
(14,000 g, 25 min, 4°C).
1.7.Discard supernatant and wash nuclei pellet in 5 ml 25% [w/v] sucrose/TBS; centrifuge samples (14,000 x g, 25 min,4°C).
1.8.Resuspend
nuclei pellet in 5ml digestion buffer and check absorbance ratios at
260 nm and 280 nm for a dilutedsample of the nuclei suspension;
calculate the approximate DNA
concentration from the A260 reading (the ratio ofA260/A280 should be
about 1.1). Centrifuge samples (10,000 rpm, 10 min, 4°C) and resuspend
the nuclei pellet at0.5mg/ml in 1.7 ml Eppendorf tube(s)
2. 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 apowder; 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 tosub-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.
2.1. Perform microccal nuclease digestions at 37°C for 5 min.
2.2. Stop reaction by addition of 0.2 M EDTA to a final concentration of 5 mM.
2.3. Place all samples on ice for 5 min; centrifuge samples (8,000 g, 5 min).
2.4. Remove and keep the first S/N (this is called the S1 fraction; total vol 1.0 ml); store overnight at 4°C.
2.5. Resuspend the pellet in 1.0 ml Lysis buffer and dialyse overnight against 2 litres of the same buffer.
2.6. After overnight dialysis centrifuge samples (500 g, 10 min, 4°C).
2.7. Remove and keep the supernatant (called the S2 fraction; total vol about 1.2 ml after dialysis); store at 4°C.
2.8. Resuspend insoluble pelleted material in 200 ul lysis buffer (called the P fraction).
3. Analysis of soluble chromatin fractions
3.1. Check A260/A280 in all samples; the ratios for S1, S2 and P fractions are approximately 1.7, 1.5 and 1.3 respectively.
3.2. Analyze all samples by 1.2% agarose gel electrophoresis.
Do not place ethidium bromide in the agarose gel or the electrophoresis buffer, because of thepresence of SDS (see below).
3.3.
Preparation of samples: xul (total of 5ug) chromatin fraction (S1, S2
and P) yul dH2O (x+y = 25ul) 3ul 1% [w/v] SDS(final conc 0.1%) 2 ul gel
loading buffer, containing bromophenol blue3.4. Stain the gel with
0.5ug/ml ethidium bromide after the run has finished.
4. Immunoprecipitation
4.1.100-200ug
unfixed chromatin + 100-200ul affinity purified antibody (50-100ug Ig)
and the final volume made up to 1.0ml with incubation buffer. A negative
control, with no added antibody, also needs to be set up to test for
any nonspecificbinding of the chromatin to the protein A Sepharose.
4.2.Incubate
overnight at 4°C on a slow rotating turntable. Add 200ul 50% v/v
protein A Sepharose; use a siliconizedpipette 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).
4.3.Centrifuge samples (3,000 g, 10 min, 4°C), remove and keep the S/N; this is the unbound (or “U”) fraction.
4.4.Resuspend
the Sepharose pellet in 1ml buffer A and layer onto 9ml of the same
buffer using a siliconised pasteurpipette and siliconized 15 ml tube.
4.5.Centrifuge
samples (10,000 g, 10 min, 4°C), discard the S/N and wash the Sepharose
sequentially in 10 ml buffer Band buffer C.
4.6.Finally, resuspend the Sepharose in 1 ml buffer C and transfer back to siliconized Eppendorfs.
4.7.Centrifuge
samples (3,000 g, 10 min, 4°C) and resuspend the sepharose pellet in
250ul 1.0% SDS / incubation bufferand incubate for 15 min at RT on a
fast turntable. (Ensure that the Sepharose is thoroughly resuspended at
all times).
4.8.Centrifuge the samples (3,000 g, 10 min, 4°C) and remove and keep S/N; this is the bound (or “B”) fraction.
4.9.Wash the sepharose in 250ul 1.0% SDS / incubation buffer and centrifuge immediately (3,000 g, 10 min, 4°C).
Remove the S/N and pool with the previous bound fraction from the previous step.
5. DNA Isolation
Add 500ul incubation buffer to each bound fraction, to reduce the SDS concentration to 0.5% SDS.
Unbound and bound fractions then treated as follows:
5.1.Add
0.33 vol (330ul) phenol/chloroform; vortex and spin (13,000 rpm, 10
min, microcentrifuge). Keep the organicphase and interface; this is used
to isolate immunoprecipitated proteins (see below).
5.2.Transfer
the aqueous supernatant to an equal volume (1.0 ml) of
phenol/chloroform; vortex and spin (13,000 rpm, 10min,
microcentrifuge)5.3.Transfer supernatant to an equal volume (1.0 ml) of
chloroform; vortex and spin (13,000 rpm, 10 min,
microcentrifuge)5.4.Transfer S/N to a clean centrifuge tube and add 0.1
vol (100ul) 4 M LiCl, 50ug 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.
5.5.Centrifuge samples (13,000 g, 15 min) to precipitate the DNA.
5.6.Wash the pellet with 70% ethanol and redissolve the DNA in 250ul TE buffer.
5.7.Store samples at -20°C or proceed with detection method (PCR, microarray, etc).
5.8.PCR
is used to quantify DNA levels of specific loci. This is analyzed
semi-quantitatively (analyses of PCR end-productby agarose gel) using
primers which can be designed using the URL below.
http://biotools.umassmed.edu/bioapps/primer3_www.cgiAlternatively,
DNA levels are quantitatively measured by real-time PCR. Primers and
probes are often designed usingsoftware provided with the real-time PCR
apparatus.
6. Protein Isolation
6.1.To the first
phenol/chloroform phase (see DNA isolation; step1) add 5ul of a 1 mg/ml
solution of BSA (to be used as acarrier), 0.01 vol (4ul) 10 M H2SO4 and
12 vol of acetone.
6.2.After precipitation at -20°C wash the
protein pellets once in acidified acetone (1:6 100 mM H2SO4:acetone) and
3 timesin 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 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 resuspendpellet 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)
