Rapid Yeast Protein Prep for SDS PAGE and Western

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.