自然染色質免疫沉淀實驗設計

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)