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