Post-alkaline Extraction for Western Time-course Experiments
Can be kept at room temperature but after a month check that ß-mercaptoethanol
is okay by smelling buffer.
|– 0.06 M Tris-HCl pH 7.0
– 5% glycerol
– 2% SDS
– 4% ß-mercaptoethanol
– Few grains of bromophenol blue
- Collect 2.5 OD and spin down.
- Resuspend in 200 µl dH2O.
- Incubate 3 min at room temperature.
- Spin 15 s at 12000 rpm and discard supernatant.
- Resuspend in 70 µl sample buffer.
- Boil 3 min.
- Spin 15 s at 12000 rpm and retain supernatent in new tube (can
be frozen and reboiled) or loaded immediately (cool for a bit,
- Load about 5 µl per lane.