Chromatin Immunoprecipitation: Difference between revisions
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* 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: | 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 | ||