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Rapid Protein Prep

Horvath and Riezman, Yeast, 1994
Stuff you need:

Sample Buffer: 0.06M Tris-HCl, pH 6.8
10% (v/v) glycerol
2% (w/v) SDS
5% (v/v) 2-mercaptoethanol
0.0025% (w/v) bromophenol blue

1. Grow cells overnight (~1E7 cells/ml) and collect 1.5 ml cells in 1.5 ml microfuge tube (1 minute, 14000xg).

2. Wash cells 1X with water and collect again by centrifugation.

3. Resuspend cells in 100 Ál sample buffer.

4. Heat at 95 deg C for 5 minutes.

5. Centrifuge 14000xg for 5 minutes.

6. Load ~25 Ál per lane on an SDS polyacrylamide gel.


Glass Bead Prep

Stuff you need:

Sample Buffer: 0.06M Tris-HCl, pH 6.8
10% (v/v) glycerol
2% (w/v) SDS
5% (v/v) 2-mercaptoethanol
0.0025% (w/v) bromophenol blue

0.1 M PMSF

0.5 M Benzamidine

1. Grow 25 ml of cells to mid-log.

2. Spin down (2500 rpm for 5 minutes). Wash 1X with water and spin again.

3. Resuspend in 1 ml of water and transfer to 1.5 ml microfuge tube. Spin down for 5 seconds and pour off water.

4. Resuspend in 0.5 ml of ice cold Sample Buffer with freshly added PMSF (0.5 mM) and benzamidine (0.5 mM).

5. Add glass beads (~0.5 ml).

6. Vortex on high 4X for 45 seconds with 30 seconds on ice in between each mixing.

7. Spin for 5 minutes in microfuge at 4 deg C.

8. Transfer supernatant to a new tube and boil for 5 minutes.

9. Load 10-15 Ál on a protein gel.

 
Prepared By:
Michael McMurray
(206) 667-6660
Last Modified: Aug 29, 2001


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