Resuspend nuclei pellet in 5 ml digestion buffer and check absorbance ratios at 260 nm and 280 nm for a diluted sample of the nuclei suspension calculate the approximate DNA concentration from the A260 reading (the ratio of A260/A280 should be about 1.1). Discard supernatant and wash nuclei pellet in 5 ml 25% sucrose/TBS centrifuge samples (10,000 x g, 15 min, 4☌). 
Resuspend nuclei pellet in 25% sucrose/TBS at 4 x 10 6 nuclei/ml and underlay with 0.5 vol of 50% sucrose/TBS centrifuge the samples (10,000 g, 15 min, 4☌). Keep cells on ice between the rounds of homogenization. You may have to increase or decrease this homogenization step to maximize the yield of nuclei depending on cell line. Check that nuclei have been released by phase-contrast microscopy intact cells should have the central dark region of the nucleus surrounded by a halo, which is the less dense cytoplasm. Transfer cell lysate to an all-glass homogenizer and homogenize 7 ml aliquots with seven strokes using an ‘A’ or ‘tight’ pestle. Leave stirring gently on ice for 1 hr (Transfer the suspension into a 50 ml tube with a small magnetic bar or flea place the tube in ice on top of a magnetic stirrer). Add PMSF to a final concentration of 0.5 mM. Resuspend cell pellet in TBS (Tris buffered saline) at 2 x 10 7 cells/ml and add an equal volume of 1.0% v/v Tween 40 in TBS. 
It is essential that 5 mM Na butyrate is present in all solutions throughout chromatin isolation when using antibodies to acetylated histones to prevent deacetylation.
Harvest cells: centrifuge samples (1,000 g, 10 min, 4☌) and wash the cell pellet 3 x with ice cold PBS (Phosphate buffered saline). HL-60 or lymphoblastoids) are grown to a density of approximately 1 x 10 6 cells/ml, until they are in log phase. 
easy-to-use ChIP Kit ab500, or another kit from the ChIP kit product range.
flexible Chromatin Extraction Kit ab117152 which is used to extract native, cross-linked, sheared or unsheared chromatin carefully validated ChIP antibodies, including recombinant rabbit monoclonals designed to give exactly the same performance year-after-year Sucrose containing solutions must be made up fresh on the day.Īdd protease inhibitors to all lysis solutions before use (0.1mM PMSAF and complete mini protease inhibitors commercially available).Īfter reading this ChIP protocol, review more ChIP assay /chromatin immunoprecipitation resources and products, or go straight to getting great ChIP data with: The preparation of native chromatin from cultured human cells
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