Chromatin Immunoprecipitation: Difference between revisions
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* 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). | ||
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* 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. | ||
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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: | 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 | ** [[Low Salt Immune Complex Wash Buffer]] (Catalog # 20-154), '''one wash''' | ||
** [[High Salt Immune Complex Wash Buffer]] (Catalog # 20-155), one wash | ** [[High Salt Immune Complex Wash Buffer]] (Catalog # 20-155), '''one wash''' | ||
** [[LiCl Immune Complex Wash Buffer]] (Catalog # 20-156), 3-5 washes | ** [[LiCl Immune Complex Wash Buffer]] (Catalog # 20-156), '''3-5 washes''' | ||
** [[TE Buffer]] (Catalog # 20-157), two 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 === | === 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 | * Set water bath to 65°C for use later. | ||
29. Make Elution Buffer for all IP tubes as well as all Input tubes | 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. | ||
* 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. | * 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 | 30. For Input tubes, 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. | 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. | 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. | 33. Pellet agarose by brief centrifugation (3000-5000 x g for 1 minute) and collect supernatant into new microfuge tubes. | ||
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=== Reverse Crosslinks of Protein/DNA Complexes to Free DNA=== | === 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. | 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. | 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 | 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. | '''1-2 hours'''. | ||
==== Purification of ChIP DNA ==== | ==== Purification of ChIP DNA ==== | ||