Preparation of nuclear extract for EMSA

(after Wadman et al., EMBO J. 16:3145, 1997)

Buffer A :

- HEPES pH 7.9 10 mM

- MgCl2 1.5 mM

- KCl 10 mM

- DTT 0.5 mM

Including Protease inhibitors : - Aprotinin 100 μg/ml

- Leupeptin 5 μg/ml

- Pepstatin 1 μg/ml

- PMSF 0.5 mM

Buffer C :

- Hepes pH 7.9 20 mM

- MgCl2 1.5 mM

- NaCl 420 mM

- EDTA 0.2 mM

- Glycerol 25% v/v

Including Protease inhibitors : - Aprotinin 100 μg/ml

- Leupeptin 5 μg/ml

- Pepstatin 1 μg/ml

- PMSF 0.5 mM

50.106 cells are washed twice in 10 ml cold PBS and resuspended in 500 μl of buffer A

and incubated on ice for 15 min. Add NP-40 to a final concentration of 0.5% and vortex

the cells for 10 seconds. Pellet the nuclei at 6500 rpm (tabletop Eppendorf microfuge) for

20 seconds and add 150 μl of buffer C to resuspend the nuclear pellet. Incubate on ice

with a vigourous agitation: use a small, magnetic stirr bar for 30 min. Nuclear extracts

(supernatants) are recovered after centrifugation for 10 min at 12000 rpm at 4 degrees.

Protein concentration is determined by Bradford assay.