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Cell Fixation Protocol for ChIP Assays
- Fix cells for each cell population to be tested. To fix, add 1/10 volume of freshly prepared Formaldehyde Solution* (see Reagents below) to the existing media in each container of cells (culture flask, plate, or tube). Do NOT remove existing media.
For example, to a flask containing 10 ml of media, add 1 ml of 11% Formaldehyde Solution. Cap and agitate for exactly 15 min. at room temperature.
- Stop fixation by adding 1/20 volume Glycine Solution* to the existing media in each container.
For example, if the flask in Step 1 now contains 11 ml, add 0.55 ml 2.5 M glycine. Let set at room temperature for 5 min. After the glycine incubation, if cells are adherent, scrape them thoroughly from culture surface.
- Wash cells by transferring contents of each container to a conical tube (15 ml or 50 ml tube, depending on the volume). Keep samples on ice for the remainder of the procedure. Centrifuge tubes at 800 x g in a refrigerated centrifuge for 10 min. to pellet the cells. Re-suspend cells in 10 ml chilled PBS-Igepal* per tube by pipeting up and down. If cells from any one population are contained in multiple centrifuge tubes, combine together at this step.
- Centrifuge tubes again to pellet the cells, and add 10 ml chilled PBS-Igepal* to each tube. Add 100 μl PMSF (100 mM in ethanol*; final concentration will be 1 mM) to each tube and pipet up and down to resuspend the cells.
- Centrifuge tubes a third time to pellet the cells, and decant supernatant thoroughly from cell pellets.
- Snap-freeze cell pellets on dry ice and store at –80° C.
- Ship on dry ice.
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Chromatin and Methylated DNA Immunoprecipitation
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