Nuclear cytoplasmic fractionation

Treat 500,000 BMDM’s with DMSO or 50nanomolar (nM) MLi-2 for 6h 0m 0s.

After the treatment, wash the cells 3X in PBS.

Harvest the cells in 500µL of ice-cold hypotonic buffer.

Homogenize the cells with 20 strokes of a Dounce homogenizer.

To 100µL of this homogenate, add SDS (1% final) and 25U of Benzonase (Novagen). Use this as total lysate.

Spin the rest of the homogenate at 14000rpm,4°C.

Collect the supernatant (cytoplasmic fraction) into a new tube.

Resuspend the pellet (nuclear fraction) in 200µL of high-salt buffer and solubilize with SDS (1% final) in the presence of 25U of Benzonase.

Measure protein concentration with the BCA reagent (Thermo Scientific), and subsequently analyze the samples by immunoblotting.