Isolation of nuclei for the co-IP and whole ChIP protocols is based on the
methods of Gendrel et al. (2002, 2005), Johnson et al. (2002), and Nelson et
al. (2006).
METHOD
For extraction and co-IP of nuclear proteins, see Steps 1-39 (Fig. 1). For
the ChIP procedure, see Steps 40-69 (Fig. 2).
Figure 1.
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Figure 1.
Flowchart for the timeline and organization for co-IP of nuclear proteins
from Arabidopsis seedlings.
Figure 2.
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Figure 2.
Flowchart for the timeline and organization for ChIP of DNA from Arabidopsis
seedlings.
Extraction and Co-IP of Nuclear Proteins
Nuclear Extraction for co-IP
For nuclear proteins grow ~50-100 Arabidopsis seedlings per sample on
soil in large trays, with good separation between plants for ~4 wk under
short-day conditions (21°C, 8-h light [150-175 μmol photons/m2/sec)], 16-h
dark).
1. Harvest seedlings. Grind tissue in a mortar with liquid nitrogen to a
fine powder. Transfer the powder to a 250-mL beaker on ice until the
nitrogen has evaporated.
2. Add 30 mL of NEB1 per 5 g of tissue to 5-10 g of plant tissue powder
(depending on the abundance of the protein under investigation). Mix gently
with a spatula; do not vortex.
3. Filter the suspension through a double layer of Miracloth into 50-mL
Falcon tube(s).
Do not squeeze the Miracloth.
4. Centrifuge at 3000g for 10 min at 4°C. Gently decant the supernatant
. Drain the tube upside-down on paper toweling briefly.
5. Resuspend the pellet in 1 mL of NEB2 (containing 1% Triton X-100),
first using an inoculation loop, then by gently pipetting up and down. Avoid
foaming.
6. Centrifuge at 10,000g for 10 min at 4°C. Discard the supernatant.
If necessary, repeat Steps 5 and 6 with NEB2 containing 0.5% TritonX-100
until the pellet looks white or slightly green.
7. Resuspend the pellet in 300 μL of NEB3.
8. Add 500 μL of NEB3 to a new 1.5-mL tube. Carefully overlay the
resuspended pellet on top of the 500 μL NEB3, so that the suspension stays
as a top layer.
9. Centrifuge at 16,000g for 45 min at 4°C. Remove the supernatant.
Store the pellets at -80°C overnight or longer.
The pellets represent enriched, isolated nuclei.
10. Thaw the pellet on ice. Add 300 μL IP-B to the pellet. Resuspend
first with an inoculation loop, then by gently pipetting up and down. Avoid
foaming at all costs.
11. Working in a cold room, sonicate the sample five times, 10 sec each,
with the sonicator set at 40% of full capacity. Keep the samples on ice for
2 min between each sonication.
Sonication disrupts the nuclei. The intensity and duration of sonication
depends upon the sonicator used and is best determined empirically.
12. Add 2 μL of Benzonase to each sample. Incubate on ice for 1 h.
Benzonase removes DNA and RNA, thereby releasing DNA-binding proteins (
Fig. 3). One can infer the relative distribution of proteins between
chromatin-bound and unbound states based on Benzonase treatment. Also, the
resulting nuclear extracts will not be complicated by the presence of
genomic DNA and mRNA.
Figure 3.
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Figure 3.
Benzonase treatment releases DNA-binding proteins. Nuclear extracts
after sonication were treated with or without 20 U of Benzonase on ice for 1
h. 100 μg of the total nuclear extracts were resolved on SDS-PAGE and
immunoblotted with anti-WRKY33 or anti-WRKY25 antibodies. WRKY33 is detected
mainly in samples before Benzonase treatment, whereas the opposite pattern
is detected for WRKY25. This suggests that most WRKY33 might not be bound to
DNA, while most WRKY25 could be.
13. Centrifuge at 12,000g for 30 min at 4°C. Save the supernatant.
Immunoprecipitation for co-IP
14. Prepare protein A or G Sepharose or agarose beads according to the
manufacturer’s instructions. Wash three times with IP-B.
Selection of protein A or G depends on the antibody used for the
immunoprecipitation (for details on selecting an appropriate antibody, see
Harlow and Lane [1999]).
15. Centrifuge at 3000g for 2 min to collect the beads. Resuspend them
in IP-B to make a 50% slurry.
16. Preclear the samples by rotating with 60 μL of the bead slurry for
1 h at 4°C.
17. Collect the beads by centrifugation at 5000g for 2 min at 4°C.
Transfer the supernatant to a new tube.
Avoid transferring beads.
18. Measure the protein concentration. Aliquot equal amounts of protein
for each sample to new tubes. Adjust to equal volumes with IP-B.
A final protein concentration of ~1 μg/μL or higher is desired.
19. Add antibody against the protein of interest. Incubate with rotation
overnight at 4°C.
The amount of antibody depends on its purity and affinity. Affinity-
purified antibodies are preferred, and normally 5 μg of such antibody is
used.
20. Add 60 μL of the bead slurry to the samples. Incubate with rotation
for 2-3 h at 4°C.
Cut the end of the pipette tips to allow easily handling of the beads.
21. Centrifuge at 3000g for 2 min at 4°C to collect the beads. Remove
the supernatant.
The supernatant can be used as a control for checking
immunoprecipitation efficiency.
22. Wash the beads through three changes of 600 μL of IP-B.
23. Use a 50-100 μL Hamilton syringe to remove all the remaining
solution from the beads.
Immunoblotting
24. Add 45 μL of 2.5X SDS loading buffer to each sample. Heat for 10
min at 95°C.
25. Centrifuge the samples at maximum speed for 1 min at room
temperature.
26. Load the samples on an SDS-PAGE gel. Run the gel until the dye front
runs out.
27. Blot the proteins to a membrane by semidry transfer according to the
manufacturer’s instructions.
28. Trim off the membrane outside of the gel. Rinse the membrane twice
in H2O for 2 min.
29. Wash the membrane with PBS for 5 min with gentle shaking.
30. Wash the membrane with PBT for 5 min with gentle shaking.
31. Incubate the membrane with PBTB with gentle shaking for at least 1 h
at room temperature.
32. Discard the solution. Add fresh PBTB. Incubate with gentle shaking
for 10 min at room temperature.
33. Add primary antibody (diluted 1:1000). Incubate with gentle shaking
overnight at 4°C.
Depending on antibody purity and affinity, the amount of antibody added
might need to be optimized.
34. Rinse the membrane with PBT briefly, twice.
35. Wash the membrane with PBT six times, 10 min each, with gentle
shaking.
36. Add PBTB. Incubate with gentle shaking for 30 min at room
temperature.
37. Add secondary antibody. Incubate with gentle shaking for 1-2 h.
The amount of secondary antibody also often needs to be optimized,
depending on the antibody and method for detection (Step 39).
38. Wash membrane as in Steps 34 and 35.
39. Detect secondary antibody with substrates appropriate for the
conjugate on the secondary antibody.
ChIP Procedure
Formaldehyde Cross-linking of Proteins to DNA
Grow plants ~3 wk on either soil or Murashige and Skoog (MS) plates.
40. Harvest seedlings as quickly as possible into precooled 50-mL Falcon
tubes on ice, 1.5 g per tube.
Process all the samples for one experiment at the same time.
41. Dilute formaldehyde in NEB1 to a final concentration of 1% (i.e., 1
mL formaldehyde to 36 mL buffer). Add the formaldehyde/NEB1 to the Falcon
tubes to cover the seedlings.
Pieces of nylon mesh can be used to keep seedlings submerged in buffer.
42. Vacuum-infiltrate for 10-20 min at room temperature. Release the
vacuum.
The seedlings should look translucent. Seedlings grown on plates require
shorter infiltration time than those from soil.
43. Stop cross-linking by adding 2 M glycine to a final concentration of
0.125 M (2.5 mL per 37 mL). Vacuum-infiltrate for 5-10 min.
44. Remove the solution. Rinse the plants three times with 40 mL of
Milli-Q H2O. Remove as much H2O as possible from the seedlings by gently
dabbing with tissue paper.
45. Freeze the seedlings in liquid nitrogen. Store at -80°C.
Nuclear Extraction for ChIP
46. Grind 1.5 g of cross-linked plant seedlings in liquid nitrogen to a
fine powder.
47. Add the powder to 30 mL of NEB1 in a 50-mL Falcon tube. Mix by
inverting the tube gently; do not vortex.
48. Extract nuclei as described in Steps 3-9. Because only 1.5 g of
tissue is required, do not repeat Steps 5 and 6.
Use pipettes with filtered tips to reduce contamination between samples.
49. Resuspend the pellet in 273 μL of nuclei lysing buffer by pipetting
up and down gently, keeping the samples cold. Incubate for 30 min on ice.
50. Add 2 volumes (i.e., 546 μL) of ChIP dilution solution. Aliquot 400
μL each into two reaction tubes.
The optimal sonication volume for this procedure is 400 μL, but the
volume can vary depending on the sonicator.
Sonication
51. Working in a cold room, sonicate the samples six times in a VirSonic
Cup horn at an output power of level 3, for 10 sec each.
This step requires optimization (Fig. 4). Sonication should shear DNA to
~500 bp. Both the duration and intensity of the sonication should be
adjusted depending on the apparatus. Flat-bottom reaction tubes provide more
efficient sonication when used in the cup horn.
Figure 4.
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Figure 4.
Time series performed using a Virsonic 600 sonicator at output 3 to
estimate duration of sonication. Ten-second intervals of sonication (with 10
-sec breaks between each sonication) for 00:30, 01:00, 01:30, and 2:00 min
are shown. DNA shearing leading to a smear peaking at about 500 bp is
preferred for ChIP. Based on these observations, 1-min sonications were used
for further experiments.
52. Recombine the two 400-μL samples into a 1.5-mL tube. Centrifuge at
maximum speed for 10 min at 4°C. Transfer supernatant to a new tube.
Immunoprecipitation for ChIP
53. Wash ChIP grade (i.e., DNA-blocked) Protein A-agarose/salmon sperm
DNA beads three times with ChIP dilution solution. Centrifuge at 2000g for 1
min to collect beads.
54. Preclear each sample with 40 μL of the beads for 1 h at 4°C with
gentle rotation.
This reduces nonspecific binding to the beads. Cutting the end of a
pipette tip facilitates bead transfer.
55. Centrifuge at 2000g for 1 min. Transfer the supernatant to a fresh
tube. Discard the beads.
56. Reserve 1% of the total volume as an input control. Aliquot the
remaining solution into the tubes needed for the experiment: one tube per
antibody and another one either with preimmune serum or without antibody
added (as a mock control).
57. Add antibody to the appropriate tubes. Incubate with rotation
overnight at 4°C.
The amount of antibody added depends on purity and affinity, but is
usually 5-20 μL.
58. Wash the beads three times with ChIP dilution solution.
59. Add 40 μL of beads to each sample. Incubate with rotation for 3 h
at 4°C.
60. Pellet the beads by centrifuging at 2000g for 1 min. Remove the
supernatant.
The supernatant can be reserved as an additional input control and for
performing primer standard curves for real-time PCR quantification.
61. Wash the beads with 1 mL of each of the following buffers for 10 min
at 4°C, with gentle agitation. Between washes, collect the beads by
centrifugation at 2000g for 1 min. Discard the supernatant:
i. Low-salt wash buffer
ii. High-salt wash buffer
iii. LiCl wash buffer
iv. TE buffer, twice
Elution and Purification of DNA
62. Add 250 μL freshly prepared bead eluting buffer to the beads.
Vortex briefly. Incubate with agitation for 15 min at 65°C.
63. Centrifuge at maximum speed for 1 min. Carefully transfer the
supernatant to another tube. Repeat Step 62. Combine the eluates (for a
total volume of ~500 μL).
64. Add 20 μL of 5 M NaCl to each eluate. Wrap the tubes with Parafilm.
Reverse the cross-linking with shaking for at least 6 h (or overnight) at
65°C.
65. Add 10 μL of 0.5 M EDTA, 20 μL of 1 M Tris-Cl (pH 6.5), and 1.5 μ
L of 14 μg/μL (~20 μg) proteinase K to each eluate. Incubate for 1-3 h at
45°C with shaking.
66. Use a PCR purification kit or phenol:chloroform extraction to
isolate the DNA.
67. Elute or dissolve DNA with 40 μL of TE buffer containing 10 μg/mL
RNase.
68. Dilute a 10-μL aliquot of the DNA solution 1:10 with 90 μL of H2O.
The dilution factor will depend on overall DNA yield.
69. Quantify the DNA by real-time PCR.
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DISCUSSION
Standard procedures for plant nuclear protein extraction generally include
isolating protoplasts, disrupting the plasma membrane, and releasing
cytoplasmic proteins by Triton X-100 in the presence of Mg++, followed by
collection and lysis of the nuclei (Schäffner and Sheen 1991; Sheen
1993). Triton X-100 solubilizes most organellar proteins, and Mg++ helps to
maintain nuclear integrity (Cho et al. 2006). However, most nuclear lysis
buffers are incompatible with co-IP. The nuclear extraction protocol
described here is compatible with co-IP and includes sonication in co-IP
buffer to disrupt nuclear membranes and treatment with Benzonase. This
method has allowed us to successfully coimmunoprecipitate several proteins
on nuclear extracts from Arabidopsis seedlings. Although the protocol
described here uses IP followed by confirmatory immunoblotting, other
applications can be used, such as mass spectrometry to identify novel in
vivo interacting proteins or components of protein complexes (Cho et al.
2006).
