Bridges Lab Protocols

Chromatin Immunoprecipitation: Difference between revisions

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* Dynabeads (Invitrogen cat#)
* Dynabeads (Invitrogen cat#)
* PBS with 5 mg/mL BSA and 1x Protease inhibitor (cold)
* PBS with 5 mg/mL BSA and 1x Protease inhibitor (cold)
* [LiCl Wash Buffer]] make fresh (cold)
* [[Dilution Buffer]]
* TE: 10 mM Tris 7.5, 0.1 mM EDTA (cold)
*[[Low Salt Immune Complex Wash Buffer]]
* [[High Salt Immune Complex Wash Buffer]]
* [[LiCl Immune Complex Wash Buffer]]  
* [[TE Buffer]]
* [[ChIP Elution Buffer]] make fresh
* [[ChIP Elution Buffer]] make fresh
* RNase A (10ug/ul;-20C)
* Proteinase K (10ug/ul; -20C)
* QIAquick PCR Purification Kit
* QIAquick PCR Purification Kit


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* 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.
* 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.
* 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.
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.
* 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.
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.
* 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.
* 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.
* 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.
 
20. Incubate for '''1 hour''' at 4°C with rotation.
 
21. Pellet agarose by brief centrifugation (3000-5000 x g for 1 minute).
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.
* 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.
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
* If different chromatin preparations are being carried together through this protocol, remove
1% of the chromatin as Input from each.
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.
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:
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 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 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.
* 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.
 
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
* 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.
(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.
 
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.
* This serves to collect the antibody/antigen/DNA complex.
27. Pellet Protein G Agarose by brief centrifugation (3000-5000 x g for 1 minute) and remove the
27. Pellet Protein G Agarose by brief centrifugation (3000-5000 x g for 1 minute) and remove the
supernatant fraction.
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


==== Elution of Protein/DNA Complexes ====
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: =====
===== 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.
* 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.
* Set water bath to 65°C for use later.
1. Make Elution Buffer for all IP tubes as well as all Input tubes (see Section C, step 7).
29. Make Elution Buffer for all IP tubes as well as all Input tubes.
• 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.
* 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.
* OR can make this way [[ChIP Elution Buffer]]
3. For Input tubes (see Section C, step 7), add 200 μL of Elution Buffer and set aside at room temperature until Section E.
* 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.
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.
30. For Input tubes, add 200 μL of Elution Buffer and set aside at room temperature.
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).
31. Add 100 μL of Elution Buffer to each tube containing the antibody/agarose complex. Mix by flicking tube gently.
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.
32. Incubate at room temperature for '''15 minutes'''.
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
33. Pellet agarose by brief centrifugation (3000-5000 x g for 1 minute) and collect supernatant into new microfuge tubes.
1-2 hours.
 
34. Repeat steps 4-6 and combine eluates (total volume = 200 μL).
 
=== 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 ====
==== Purification of ChIP DNA ====
# Add 5 volumes Qiagen Buffer PB (QIAquick PCR Purification Kit) to one volume of ChIP’d DNA. Add pH detector (at a 1:250 dilution) to samples. Upon addition of Buffer PB, the sample should be yellow, indicating the correct pH. If the sample is not yellow, the pH should be adjusted with 3M sodium acetate as recommended by the manufacturer (Qiagen). One microliter at a time, mixing between each works fine.
39. Add 5 volumes Qiagen Buffer PB (QIAquick PCR Purification Kit) to one volume of ChIP’d DNA. Add pH detector (at a 1:250 dilution) to samples. Upon addition of Buffer PB, the sample should be yellow, indicating the correct pH. If the sample is not yellow, the pH should be adjusted with 3M sodium acetate as recommended by the manufacturer (Qiagen). One microliter at a time, mixing between each works fine.
# Add half (~600 µl) of the solution to a QIAquick PCR Purification column, centrifuge for 30-60 sec @ 13,000 RPM , and then repeat with other half to bind the ~1.2 ml sample on a Qiagen column.
 
# Wash the column with 750 µl Qiagen Buffer PE, centrifuge for 30-60sec @ 13,000 RPM.
40. Add half (~600 µl) of the solution to a QIAquick PCR Purification column, centrifuge for 30-60 sec @ 13,000 RPM , and then repeat with other half to bind the ~1.2 ml sample on a Qiagen column.
# Empty the collection tube and centrifuge the column containing the bound DNA a second time to allow it to dry.
 
# Elute the DNA from the column with two 35 µl aliquots (note: this is how much you will need to run duplicates with 5 primers and may need to be adjusted based on your experiment) of warmed (~55°C) Qiagen Buffer EB, allow to sit on column for 1 minute, spin for 1 min @ 13,000 RPM, and repeat).
41. Wash the column with 750 µl Qiagen Buffer PE, centrifuge for 30-60sec @ 13,000 RPM.
 
42. Empty the collection tube and centrifuge the column containing the bound DNA a second time to allow it to dry.
 
43. Elute the DNA from the column with two 35 µl aliquots (note: this is how much you will need to run duplicates with 5 primers and may need to be adjusted based on your experiment) of warmed (~55°C) Qiagen Buffer EB, allow to sit on column for 1 minute, spin for 1 min @ 13,000 RPM, and repeat).


===Analysis of Immunoprecipitated DNA===
===Analysis of Immunoprecipitated DNA===
* See [[RT-PCR primer design for ChIP]] to design primers if analysing by qPCR
* See [[RT-PCR primer design for ChIP]] to design primers if analysing by qPCR
Retrieved from "http://bridgeslab.sph.umich.edu/protocols/index.php/Chromatin_Immunoprecipitation"