Bridges Lab Protocols

Chromatin Immunoprecipitation: Difference between revisions

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==== Couple the primary antibody for each transcription factor or chromatin protein to magnetic beads====
==== Couple the primary antibody for each transcription factor or chromatin protein to magnetic beads====
# Add 20 μl re-suspended magnetic bead slurry (for each sample you plan to use with this antibody) to a 1.5 ml microfuge tube on ice containing 1 ml cold PBS/BSA. Mix well, '''do not vortex'''. ''Note: For each wash, you will want to invert the tube while on the magnet rack to collect the beads that remain in the liquid at the top of the tube. This will allow you aspirate the liquid from the top of the tube between washes without sucking up any of the beads.''
1. Prepare enough Dilution Buffer containing protease inhibitors for the number of desired immunoprecipitations and store on ice.
# Place the microfuge tubes on the magnet rack and remove supernatants.
• Each IP requires the addition of 900 μL of Dilution Buffer and 4.5 μL of Protease Inhibitor Cocktail II.
# Resuspend the beads in 1 ml cold PBS/BSA.
• Immunoprecipitations should include the positive control (Anti-RNA Polymerase II), and the negative control, (Normal Mouse IgG), and the antibody of interest (user supplied). It is recommended that the user include a negative control IgG of the same species as the antibody of interest.
# Repeat Steps b and c 3 times.
2. Prepare one microfuge tube containing 100 μL of sheared crosslinked chromatin (Section B, step 5) for the number of desired immunoprecipitations and put on ice. If chromatin has been previously frozen, thaw on ice.
# Add 200 μl PBS/BSA (for each sample you plan to use this antibody with) to beads.
• Alternatively, if multiple immunoprecipitations will be performed from the same chromatin preparation, place the entire volume for the number of desired immunoprecipitations in one large tube that will be able to accommodate a volume of 1.1 mL for each IP.
# Add 1 μg primary antibody. Mix gently by tapping--'''Do not vortex beads'''.
• Each 100 μL will contain ~1 x 106 cell equivalents of chromatin.
# Gently mix on a rotator platform for at least '''2 hours''' at 4°C.
3. Add 900 μL of Dilution Buffer containing Protease Inhibitor Cocktail II into each tube containing 100 μL of chromatin.
# Wash beads 3 times (steps b-c), resuspending the beads by inverting the tubes during each wash.
• Alternatively, if multiple immunoprecipitations will be performed from the same chromatin preparation, use the appropriate volume of Dilution Buffer containing Protease Inhibitor Cocktail II for the correct number of immunoprecipitations.
# Resuspend in 100 μl  PBS/BSA (for each sample you plan to use this antibody with), and proceed to Step 2.
4. Add 60 μL of Protein G Agarose for each IP.
 
• The Protein G Agarose is a 50% slurry. Gently mix by inversion before pipetting.
==== Incubate bead-antibody complex with fragmented, cross-linked chromatin====
• This step serves to “preclear” the chromatin, i.e., to remove proteins or DNA that may bind nonspecifically to the Protein G agarose.
# Add 100 μl of antibody-coupled beads from above to each 25ug chromatin of preparation (from Sonication protocol) and incubate on a rotator for '''1 hour at room temperature''', followed by '''1 hour at  4°C'''.
• Alternatively, if multiple immunoprecipitations will be performed from the same chromatin preparation, use the appropriate volume of Protein G Agarose for the correct number of immunoprecipitations.
# Collect beads containing immuno-bound chromatin by placing the microfuge tube on a magnet rack.
5. Incubate for 1 hour at 4°C with rotation.
# Remove and discard supernatant. ''Turn water bath to 65°C before next step''.
6. Pellet agarose by brief centrifugation (3000-5000 x g for 1 minute).
# Wash beads 5 times with 1 mL LiCl Wash Buffer, mixing 3 minutes for each wash on a rotator.
• Do not spin Protein G Agarose beads at high speeds. Applying excessive g-force may crush or deform the beads and cause them to pellet inconsistently.
# Add 1 ml TE Buffer to beads. Mix 1 minute on rotator and then place tubes on magnet rack to collect beads and discard supernatant.
7. Remove 10 μL (1%) of the supernatant as Input and save at 4°C until Section D, step 1.
# Resuspend the bead pellet in 200 μl IP Elution Buffer (at room temperature). Vortex to mix.
• If different chromatin preparations are being carried together through this protocol, remove
 
1% of the chromatin as Input from each.
==== Reverse cross-linking and recover ChIP DNA ====
8. Collect the remaining supernatant and dispense 1 mL aliquots into fresh microfuge tubes. Discard agarose pellet.
# Incubate beads from above in a 65°C water bath for '''1 hour''', shake or vortex every 15 minutes to elute the immuno-bound chromatin from the beads.
9. Add the immunoprecipitating antibody to the supernatant fraction:
# Spin at 14,000 rpm in a microfuge at room temperature for 3 minutes.
• For the positive control, anti-RNA Polymerase, add 1.0 μg of antibody per tube.
# Collect the supernatant, which contains the ChIP’d DNA. The tubes can be placed on the magnet to facilitate supernatant recovery.
• For the negative control, Normal Mouse IgG, add 1.0 μg of antibody per tube.
# Incubate the supernatant containing the ChIP’d DNA in a 65°C water bath overnight to complete the reversal of the formaldehyde cross-links.
• For user-provided antibody and controls, add between 1-10 μg of antibody per tube. The appropriate amount of antibody needs to be determined empirically.
10. Incubate overnight at 4°C with rotation.
• It may be possible to reduce the incubation time of the IP. This depends on many factors
(antibody, gene target, cell type, etc.) and will have to be tested empirically.
11. Add 60 μL of Protein G Agarose to each IP and incubate for 1 hour at 4°C with rotation.
• This serves to collect the antibody/antigen/DNA complex.
12. Pellet Protein G Agarose by brief centrifugation (3000-5000 x g for 1 minute) and remove the
supernatant fraction.
13. Wash the Protein G Agarose-antibody/chromatin complex by resuspending the beads in 1 mL each of the cold buffers in the order listed below and incubating for 3-5 minutes on a rotating platform followed by brief centrifugation (3000-5000 x g for 1 minute) and careful removal of the supernatant fraction:
a. Low Salt Immune Complex Wash Buffer (Catalog # 20-154), one wash
b. High Salt Immune Complex Wash Buffer (Catalog # 20-155), one wash
c. LiCl Immune Complex Wash Buffer (Catalog # 20-156), one wash
d. TE Buffer (Catalog # 20-157), two washes
D. Elution of Protein/DNA Complexes
Prior to starting this section:
• Bring 1 M NaHCO3 to room temperature. A precipitate may be observed but will go into solution once room temperature is achieved. The 1 M NaHCO3 can be vortexed.
• Set water bath to 65°C for use in Section E.
1. Make Elution Buffer for all IP tubes as well as all Input tubes (see Section C, step 7).
• For each tube, prepare 200 μL of elution buffer as follows: 10 μL 20% SDS, 20 μL 1 M NaHCO3 and 170 μL sterile, distilled water.
2. Alternatively, make a large volume to accommodate all tubes. For example, if there are 10 tubes mix together 105 μL 20% SDS, 210 μL 1M NaHCO3 and 1.785 mL sterile, distilled water.
3. For Input tubes (see Section C, step 7), add 200 μL of Elution Buffer and set aside at room temperature until Section E.
4. Add 100 μL of Elution Buffer to each tube containing the antibody/agarose complex. Mix by flicking tube gently.
5. Incubate at room temperature for 15 minutes.
6. Pellet agarose by brief centrifugation (3000-5000 x g for 1 minute) and collect supernatant into new microfuge tubes.
7. Repeat steps 4-6 and combine eluates (total volume = 200 μL).
E. Reverse Crosslinks of Protein/DNA Complexes to Free DNA
1. To all tubes (IPs and Inputs) add 8 μL 5 M NaCl and incubate at 65°C for 4-5 hours or overnight to reverse the DNA – Protein crosslinks. After this step the sample can be stored at -20°C and the protocol continued the next day.
2. To all tubes, add 1 μL of RNase A and incubate for 30 minutes at 37°C.
3. Add 4 μL 0.5M EDTA, 8 μL 1M Tris-HCl and 1 μL Proteinase K to each tube and incubate at 45°C for
1-2 hours.


==== Purification of ChIP DNA ====
==== Purification of ChIP DNA ====
Retrieved from "http://bridgeslab.sph.umich.edu/protocols/index.php/Chromatin_Immunoprecipitation"