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 ''Note: for TE washes use a pipette to carefully aspirate, the beads seem to come off of the magnet easily with this wash'' | ** [[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 === | ||
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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. | ||