162
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Changes
→Couple the primary antibody for each transcription factor or chromatin protein to magnetic beads
* Dynabeads (Invitrogen cat#)
* PBS with 5 mg/mL BSA and 1x Protease inhibitor (cold)
*[[Dilution Buffer]]*[[Low Salt Immune Complex Wash Buffer]] * [[High Salt Immune Complex Wash Buffer]] * [[LiCl Immune Complex Wash Buffer]] * [[TE Buffer]]
* [[ChIP Elution Buffer]] make fresh
* RNase A (10ug/ul;-20C)
* Proteinase K (10ug/ul; -20C)
* QIAquick PCR Purification Kit
* 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.
* Each 100 μL will contain ~1 x 106 cell equivalents of chromatin.
18. Add 900 μL of Dilution Buffer containing Protease Inhibitor Cocktail II into each tube containing 100 μL of chromatin.
* 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.
19. Add 60 μL of Protein G Agarose for each IP.
* The Protein G Agarose is a 50% slurry. Gently mix by inversion before pipetting.
* This step serves to “preclear” the chromatin, i.e., to remove proteins or DNA that may bind nonspecifically to the Protein G agarose.
* 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.
20. Incubate for '''1 hour ''' at 4°C with rotation.
21. Pellet agarose by brief centrifugation (3000-5000 x g for 1 minute).
* 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.
22. Remove 10 μL (1%) of the supernatant as Input and save at 4°C until Section D, step 1.
* If different chromatin preparations are being carried together through this protocol, remove
1% of the chromatin as Input from each.
23. Collect the remaining supernatant and dispense 1 mL aliquots into fresh microfuge tubes. Discard agarose pellet.
24. Add the immunoprecipitating antibody to the supernatant fraction:
* For the positive control, anti-RNA Polymerase, add 1.0 μg of antibody per tube.
* For the negative control, Normal Mouse IgG, add 1.0 μg of antibody per tube.
* 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.
25. 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.
26. 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.
supernatant fraction.
28. 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:** [[Low Salt Immune Complex Wash Buffer]] (Catalog # 20-154), '''one wash'''** [[High Salt Immune Complex Wash Buffer]] (Catalog # 20-155), '''one wash'''** [[LiCl Immune Complex Wash Buffer]] (Catalog # 20-156), '''3-5 washes'''** [[TE Buffer]] (Catalog # 20-157), '''two washes''' ''Note: for TE washes use a pipette to carefully aspirate, the beads seem to come off of the magnet easily with this wash''
=== 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 Elater.29. 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.
* OR can make this way [[ChIP Elution Buffer]]
* 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.
30. For Input tubes (see Section C, step 7), add 200 μL of Elution Buffer and set aside at room temperature.
31. Add 100 μL of Elution Buffer to each tube containing the antibody/agarose complex. Mix by flicking tube gently.
32. Incubate at room temperature for '''15 minutes'''.
33. Pellet agarose by brief centrifugation (3000-5000 x g for 1 minute) and collect supernatant into new microfuge tubes.
=== Reverse Crosslinks of Protein/DNA Complexes to Free DNA===
36. 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.
37. To all tubes, add 1 μL of RNase A and incubate for '''30 minutes ''' at 37°C.
38. 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 ====
