Protein Extraction - Membrane-bound


SAFETY WARNING: ß-mercaptoethanol is hazardous.  Gloves, safety glasses, and other protection should be warn throughout this procedure.
1.Grow cells to mid-log phase.
2.Harvest 10 OD600 of cells (1x108 cells).  Add cells to yellow-capped glass tubes.
3.Centrifuge cells.  Discard supernatant, resuspend pellet in 2 ml of ddH2O, and centrifuge cells again.  Discard supernatant, and resuspend pellet in 2 ml of ddH2O.
4.Transfer cells to 13 x 100 mm disposable glass tubes.
5.Add 0.2 g of 0.5 mm glass beads
6.Add 50 µl Thorner buffer (40 mM Tris-HCl pH 6.8, 5% SDS, 8 M urea, 0.1 mM EDTA)
7.Vortex 90 seconds full speed immediately.  Place tubes in ice bath for 90 seconds.  Repeat vortexing and incubation in ice 3-5 times.
8.Place in 70°C heating block or water bath for 5 minutes.
9.Add 0.25 ml Laemmli sample buffer containing 6 M urea (1.1 ml H2O, 400 µl 5x LSB, 720 mg urea, and 50 µl ß-mercaptoethanol)
10.Return to 70°C for one minute.
11.Transfer liquid to 1.5 ml microfuge tube.
12.Centrifuge at full speed for ten minutes.
13.Transfer supernatant to new tube.  This can be used immediately or frozen at -20°C.
14.Heat sample at 70°C for five minutes prior to loading on gel.