Horvath and Riezman, Yeast, 1994; Gottschling Lab
|Sample Buffer:||10 ml|
|0.06M Tris-HCl, pH 6.8||0.6 ml 1M Tris 6.8|
|10% (v/v) glycerol||2 ml 50% glycerol|
|2% (w/v) SDS||2 ml 10% SDS|
|5% (v/v) 2-mercaptoethanol||0.5 ml 2-mercaptoethanol|
|0.0025% (w/v) bromophenol blue||0.1 ml Sat. Bromphenol blue|
|4.9 ml H2O|
Make sample Buffer fresh before use. Can store buffer frozen at 20 degrees for ~ 6 months.
1. Grow cells overnight (~1x107 cells/ml; A600 = 0.7) and collect 1.5 ml cells (adjust volumes according to cell density of cultures) in 1.5 ml microfuge tube (1 minute, 14000xg). It is important not to grow the cells to a high density as this method will not work well.
10 microliters of a saturated overnight culture in YPD innoculated to 5 ml SD + essential amino acids for ~16 hrs gives A600 of 0.5 to 1.0 for wild-type cells grown at 30 degrees
150 microliters of YPD saturated culture diluted to 5 ml YPD and grown for ~5 hrs at 30 degrees gives an A600 of ~0.8 for wild-type cells.
2. Wash cells 1X with water and collect again by centrifugation.
3. Resuspend cells in 100 microliters sample buffer.
4. Heat at 95 deg C for 5 minutes.
5. Centrifuge 14000xg for 5 minutes. Load 15 microliters per lane on an SDS polyacrylamide gel.