Nuclear extracts protocol

Nuclear extracts protocol (adapted from Dignam et al, 1983, NAR)Harvest cells by spinning at 1000 rpm for 5 min at 4C if possible.Wash cells with PBS (preferably cold)Resuspend cells in ice cold buffer A, wash again with buffer A (spin at 1000 rpm, 5 min4C).Lyse cells with buffer A + 0.1% NP-40, incubate 5 min on ice (this tie depends on thecell type, you can test if cells are lysed by trypan blue staining). Volume of bufferA+NP=40 should be 2-3X volume of cell pellet. You can also use a dounce homogeniser.Spin at max speed in microcentrifuge for 10 min at 4C. Supernatant is your cytoplasminextract.Resuspend pellet in Buffer C (1-2 volumes of pellet). Pipette up and down to achieve agood homogenous mix, avoid bubbles. Rock for 15 min at 4C. Spin down at max speedfor 15 min. Supernatant is your nuclear extract. You dilute with buffer D for reducing thesalt concentration.Freeze on dry ice.Buffer A10 mM Hepes pH 7.91.5 mM MgCl210 mM KCL1 mM DTT0.5 mM PMSF, plus protease and phosphatase inhibitorsBuffer C20mM Hepes pH 7.9420 mM KCl1.5 mM MgCl21 mM DTT25% Glycerol0.5 mM PMSF, plus protease and phosphatase inhibitorsBuffer D20mM Hepes pH 7.90.5 mM DTT 1

20% Glycerol0.5 mM PMSF, plus protease and phosphatase inhibitors 2