Chromatin immunoprecipitation of in vivo protein-DNA complexes:
(modified 5/2/04)
7/12/2000
Once you have your nuclei: (see nuclear prep)
- if they are from "fresh" (i.e. non-fixed) tissue you can treat the isolated nuclei with 1%
formaldehyde in M1 buffer for 15 to 30 minutes on ice (typically resuspend the nuclear pellet after
M2 washes in 1 ml M1 buffer, add 28 microliters of 37% Formaldehyde). After the 30 minutes,
stop the crosslinking by addition of glycine to 0.125 M final (from a 2.5 M stock). Pellet the nuclei
and wash with M3 buffer. Proceed to the sonication step.
- if you made your nuclei from fixed tissue, after washing with M3, proceed to the
sonication step.
Sonication:
Resuspend the nuclear (and starch) pellet in 1 ml of sonication buffer and glass beads (note: the
glass beads are supposed to help sonicate chromatin to a reasonable size. However, we have
recently found that sonication works fine without the glass beads). The sonication buffer is 10 mM
potassium phosphate, pH 7, 0.1 M NaCl, 0.5% Sarkosyl, and 10 mM EDTA. Two volumes of
sonication buffer are mixed with one volume of glass microbeads. The glass microbeads are
supposed to help shear the chromatin. They are treated as described below. Add PMSF (200 mM
stock in isopropanol) to 1 mM final.
Sonicate with a probe sonicator. We use 4 pulses of 10 to 15 seconds each, setting 50 (on a
Fisher brand sonicator. 50 is one-half the maximum). Cool on ice in between pulses. You want to
shear the chromatin to reasonable-sized pieces (0.6 to 1 kb about).
Spin in cold microfuge, 12,500 rpm, 5 minutes. Remove the supernatant – this is the solubilized
chromatin. Save 50 microliters to check on a gel for solubilization of your protein. Also save the
pellet (mostly glass if you use the beads) to check on the gel to check efficiency of solubilization.
An aliquot of the super (20 microliters) can be analyzed for size of the DNA as well and as a total
(input) sample for PCR tests of enrichment of specific targets (you will need to RNase treat,
proteinase K treat, extract, precipitate and run on a gel).
To preadsorb:
To the rest of the solubilized chromatin: add 5 to 7.5 microliters of preimmune serum (typically 7.5
microliters). Incubate on the rotator for one hour at 4 degrees.
Spin at top speed, microfuge for 2 minutes 4 degrees.
Remove supernatant to a new tube (avoid any pellet). Add 40 microliters of protein A-Sepharose
(50% slurry). Incubate on the rotator for 1 hour at 4 degrees.
*** it is possible to let either of these preadsorption steps incubate overnight; this step may also
be omitted if simply performing enrichments tests of known targets, but is suggested if cloning
and analyzing new targets ***
Spin at top speed for 2 minutes, 4 degrees. Carefully remove the supernatant.
To immunoprecipitate:
Divide the supernatant between the samples that you want to do. Typically we do the immune
precipitation, and preimmune and “no sera” controls, so we would make three equal aliquots. Add
an equal amount of immunoprecipitation buffer (see below). Typically we add 2.5 microliters of
immune or preimmune sera. No serum is added to the "no sera" control, which gives an idea of
general stickiness.
Incubate for one to two hours at 4 degrees on the rotator.
Spin top speed, 2 minutes, in the cold microfuge.
Remove the top 85% of the volume to a new tube. Add 20 microliters of 50% slurry of protein ASepharose and incubate for one hour at 4 degrees with rotation. You need to mix the slurry well,
and pipette using a "cut-off" yellow tip. Aliquot the protein A-Sepharose into the tube first and
check to verify equal amounts in all tubes. Then add the chromatin. The other 15% of the sample
can be analyzed by Western blot as a “binding” reaction to verify that your DNA-binding protein is
present and undegraded to this point.
Pellet the beads by spinning 1-2 minutes at 4 degrees, top speed. Remove and save the
supernatant – this is the “post-bind” sample for the Western blot. If all works well, your protein
should be immunodepleted specifically from the immune precipitation.
Wash the beads with immunoprecipitation buffer (1 ml each tube). The wash is added cold, but
then the wash is incubated on the rotator for 10 minutes at room temperature. Beads are pelleted
by microfuging for 1 minute, top speed, R.T. The wash is repeated for a total of 3 to 5 times
(generally 5). When the last wash is added to the beads, the wash and beads are moved to a
new tube (helps eliminate anything just sticking to the tube). The beads are pelleted, wash
removed and the tube spun again to remove the remainder of the wash. A drawn out Pasteur
pipette works well for this step.
To elute:
Now the beads are ready for elution. Add 100 microliters of cold glycine elution buffer to the
beads, vortex (typically to the count of 15), and pellet in the R.T. microfuge (1 min., top speed).
Remove the super and add to a tube that has 50 microliters of 1 M Tris, pH 9 to neutralize.
Repeat the elution and neutralization twice (so a total of three times, giving a total of 450
microliters of eluted sample).
Spin the eluted sample top speed for 2 minutes in a R.T. microfuge. Remove the top 300
microliters to a new tube – this is the sample for DNA workup. The remaining approximately 150
microliters in the original tube may be used as a sample for Western blot to verify recovery of
your protein. The beads may also be analyzed by Western blot (resuspend in about 40 microliters
of water, neutralize with about 10 microliters of 1 M Tris, pH 9, add SDS-Sample buffer, etc).
DNA workup:
Add 1 microliter of Rnase A (1 mg/ml stock in TE - boiled to inactivate any DNases). Incubate at
37 degrees for 15-30 minutes.
Add proteinase K to 0.5 mg/ml (7.5 microliters of 20 mg/ml stock in PBS is fine). Incubate
overnight at 37 degrees.
Next day: add another aliquot of proteinase K – incubate at 65 degrees for at least 6 hours (the
high temp reverses the formaldehyde crosslinks – note: proteinase K is not necessary for this
step and in fact is inactivated at this temperature). Let cool to room temp and then chill on ice.
Phenol:chloroform extract (buffer saturated phenol: chloroform: isoamyl alcohol at 25:24:1).
Precipitate the aqueous with glycogen (one microliter), 1/10 vol of 3M sodium acetate and 2.5 vol
of ethanol. Overnight at –20, or 15 to 30 min at –80. Pellet (top speed cold microfuge 15-30 min),
wash with 70% ethanol, and dry in the Speed-vac.
If you are doing enrichment tests – you can simply resuspend DNA in water or TE (20
microliters works well; 40 microliters for the total DNA that you saved) and perform your
PCR reactions. If you want to clone the entire population, continue as below.
Resuspend in 13 to 20 microliters milliQ sterile water. Work-up entire reaction, or 13 of the 20
microliters. (we have in the past saved 7 of 20 microliters frozen for enrichment tests, more
recently we resuspend in 13 microliters and process the entire reaction).
To 13 microliters of the eluted extracted co-precipitated DNA, add 1.5 microliters of 10X Sau3AI
buffer and 0.5 microliters of Sau3AI. Incubate for 2 hours at 37 degrees.
Heat kill the Sau3AI by incubating for 10 min at 75 degrees or 20 min at 65 degrees.
Cool to R.T.
Do the single “G” fill in reaction by adding 1 microliter of 10 mM dGTP and 0.5 microliter of
Klenow. Incubate at R.T. for 10 min. Heat kill the Klenow by incubating at 75 degrees for 10 min.
Cool to R.T.
Add 50 microliters of water, and phenol:chloroform extract and glycogen/ NaOAc/ EtOH ppt as
usual. Pellet, wash and dry.
Resuspend in 10 microliters of ligation mix (one microliter of “catch linker”, one microliter of 10X
T4 ligase buffer, 0.4 microliter of T4 ligase). It is useful to also "mock ligate" any left over "ligation
mix" as a control. Overnight at 16 degrees in a water bath at 4 degrees (so temp gradually cools
overnight).
Next day: Check specificity of co-precipitation of DNA by PCR amplifying using a linker specific
oligo. Each 20 microliter reaction is 2 µl of 10X PC2 buffer, 2 µl of 2 mM dNTP, 0.4 µl of oligo,
0.04 µl of Klentaq (Ab Peptides), DNA, and water to volume. We usually PCR on 0.5, 1 and
sometimes 2 µl of the ligation reaction. Typically 27 rounds, with a 60 degree annealing step,
works fine. (i.e. 94° for 5 ', 27 X(94°, 30"; 60°, 30"; 72°, 30"), 72°, 5'). After PCR, 5 µl of the
reaction is run on an agarose gel to check specificity. Hopefully you will see product in the
immune precipitation, but not the controls. Occasionally, we have some yet undetermined
problem with the linkers by themselves causing a smear of PCR product (you can check this by
doing a "mock linker" ligation). If you obtain smears in the immune precipitation and the controls
(and the mock linker control), you can check by digesting the PCR product with EcoRI. Smears
caused by linkers alone will be digested to "linker-size". Any real DNA that was precipitated will
be more EcoRI-resistant and give a higher band. The remainder may be used for cloning, or
subsequent in vitro selection.
Some recipes:
Sonication buffer1:
10 mM potassium phosphate, pH 7
0.1 M NaCl
0.5 % sarkosyl
10 mM EDTA
Store frozen
Glass beads in sonication buffer:
75 to 105 µm glass beads (Sigma G-3753) are rinsed in 1 N HCl, and then 0.1 N HCl for
30 minutes or more. The beads are washed extensively with water, then with buffer. The pH of
the buffer is checked to make sure it is correct. The beads are resuspended 2:1 in the buffer (2
vol buffer to one vol. beads).
Immunoprecipitation buffer2: (IP bf) - Store frozen
50 mM Hepes, pH 7.5
150 mM KCl
5 mM MgCl2
10 µM ZnSO4
1% Triton X-100
0.05% SDS
Glycine elution buffer, pH 2.8 3
0.1 M glycine
0.5 M NaCl
0.05% Tween-20
pH 2.8
Store at 4° C
1M Tris, pH 9
Protein A-Sepharose (Sigma P-9424)
this is packed in 20% ethanol, so before use, wash throughly with TN buffer
(10 mM Tris, 7.5, 150 mM NaCl, 0.05 % azide) and resuspend as a 50% slurry in the TN buffer.
1
From Ito et al., (1997). Plant Cell Physiol. 38(3), p. 248-258.
From Kinzler and Vogelstein (1989). Nucl. Acids Res. 17(10), p. 3645-3652.
3 From Tang (1993). Methods in Cell Biology 37, p. 95-104.
2
Catch linkers: These are based on a design by Kinzler and Vogelstein (1989). Nucl. Acids Res.
17(10), p. 3645-3653.
We had two oligos made;
Oligo 1: 5'- ATC GAG ATA TTA GAA TTC TAC TCA -3'
Oligo 2: 5'- GAG TAG AAT TCT AAT ATC TC -3'
We phosphorylate oligo 1 using standard protocols and T4 polynucleotide kinase. Stop the
reaction with EDTA. Add an equal amount (we generally work on a scale of 0.0022 µmol) of oligo
2. Place at 95°C water in a beaker and let cool on the benchtop to room temp.
The oligos have an EcoRI site.