| 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.
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Guanidine Hydrochloride 