Nuclear enrichment

Method

1. Requires large 12ml petri dishes. Prepare 1ml of Buffer A with added cocktail of protease inhibitors from a frozen stock and store on ice.
2. Add 500µl of Buffer A per large petri dish on ice and scrape thoroughly, leave on ice for 10 min (NP40 primarily lyses plasma membrane and leaves other membranes more intact than Triton X-100 does).
3. Centrifuge at 4^oC at 3000 rpm for 10 min.
4. Remove supernatant and keep (this will contain everything except large plasma membrane pieces, DNA, nucleoli), extract out 10µl for Bradford assay.
5. On ice, resuspend pellet in 374µl of Buffer C and add 26µl of 4.6M NaCl to give 300mM NaCl (high salt helps lyse membranes and forces DNA into solution).
6. Homogenise with 20 full strokes in Dounce or glass homogeniser on ice.
7. Leave on ice for 30 min
8. Centrifuge at 24,000g for 20 min at 4^oC
9. Aliquot supernatant, remove 10µl for Bradford assay and store at –70^oC.

Reagents

Buffer A

10mM HEPES

1.5mM MgCl₂

10mM KCl

0.5mM DTT

0.05% NP40 (or 0.05% Igepal or Tergitol)

pH 7.9

To prepare 250ml stock of Buffer A:

HEPES: 1M = 238.3g/litre, therefore 10mM = 0.59g/250ml

MgCl₂: 1M = 203.3g/litre, therefore 1.5mM = 0.076g/250ml

KCl: 1M = 74.5g/litre, therefore 10mM = 0.187g/250ml

DTT: 1M = 154.2g/litre, therefore 0.5mM = 0.019g/250ml

NP40 = 0.05% (v/v)

Buffer C

5mM HEPES

1.5mM MgCl₂

0.2mM EDTA

0.5mM DTT

26% glycerol (v/v)

pH 7.9

To prepare 250ml stock of Buffer C:

HEPES: 1M = 238.3g/litre, therefore 5mM = 0.295g/250ml

MgCl₂: 1M = 203.3g/litre, therefore 1.5mM = 0.076g/250ml

EDTA: 1M = 372.2g/litre, therefore 0.2mM = 0.0186g/250ml

DTT: 1M = 154.2g/litre, therefore 0.5mM = 0.019g/250ml

26% Glycerol (v/v) = 65ml

4.6M NaCl

87.66g/326ml H₂0

Example: Nuclear Enrichment

Glutamate mediated c-jun phosphorylation is abolished in the absence of extracellular calcium. Nuclear homogenates (3 mg of each) or resultant crude lysates prepared from cultured striatal neurons exposed for 15 min to: vehicle (Basal), 100 mM glutamate (Glu) or 100 mM glutamate in the absence of extracellular calcium (Glu –Ca2+) were immunoblotted with an antibody that recognizes c-Jun when phosphorylated on Ser 73 (phospho c-Jun). (courtesy of Dr R.J. Williams, King`s College London)

kindly contributed by Dr Sophie Pezet, CARD, King's College London.