Preparation of samples for SDS-PAGE by precipitation with Acetone
1. Precipitate the proteins in acetone (1 vol of proteins solution : 4 vol of acetone) at -20C for 12 hr.
2. Spin the samples at 14 000 rpm for 30 min in a table centrifuge at 0C.
Every time put the tubes in the centrifuge in the same orientation!
3. Remove acetone and add 200 l of ice-cold 20 TCA.
Spin for 10 min at 14 000 rpm, at 0C.
4. Remove TCA and add 200 l of ice-cold diethylether.
Spin for 10 min at 14 000 rpm, at 0C.
5. Remove diethylether very carefully!
Add 200 l of H2O and spin for 10 min at 14 000 rpm, at 0C.
6. Remove H2O very carefully!
Add 10-15 l of Sample buffer containing 5 of -mercaptoethanol, and vortex.
7. Heat the samples for 10 min at +97C. Spin the samples for several seconds in a bench centrifuge, combine them and apply on a gel. Do not forget to add 3-5 l of molecular weight standard at each side of the gel. Run the gel at 20 mA until the front has migrated to the bottom of the gel.
2003 Ove, Linn & Julius