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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PROTEIN DETECTION: GEL ELECTROPHORESIS

RAPID YEAST PROTEIN PREPARATION FOR SDS-PAGE AND WESTERN ANALYSIS

Rapid Yeast Protein Preparation for SDS-PAGE and Western Analysis
Contributor: The Laboratory of Steve Hahn at the Fred Hutchinson Cancer Research Center
 
Overview
This protocol is used to prepare an extract of yeast proteins for further analysis.
 
Procedure
1. Grow a cell culture overnight until the density is about 10 million cells per ml (i.e. the absorbance at 600 nm is about 0.7).

2. Collect about 1.5 ml of the cell culture (adjust this volume according to the cell density of the culture) in a 1.5 ml microcentrifuge tube and centrifuge the cells in a microcentrifuge at 14,000 X g (full speed) for 1 min (see Hint #1).

3. Remove the supernatant and resuspend the cell pellet in 1 ml of ddH2O.

4. Centrifuge the cells again at full speed in a microcentrifuge for 1 min and remove the supernatant.

3. Resuspend the cells in 100 μl of Sample Buffer.

4. Heat the cell suspension to 95°C for 5 min.

5. Centrifuge the sample at 14,000 X g (full speed) for 5 min. Load 15 μl of this sample per lane on a SDS polyacrylamide gel

Solutions
Sample Buffer   0.0025% (w/v) Bromophenol Blue
5% (v/v) 2-Mercaptoethanol
2% (w/v) SDS
10% (v/v) Glycerol
0.06 M Tris-HCl, pH 6.8
YPD   10 g/liter Yeast Extract
Autoclave 20 min, cool to room temperature.
20 g/liter Peptone
20g/liter Glucose (Dextrose)
 
BioReagents and Chemicals
SDS
Peptone
Glucose
2-Mercaptoethanol
Glycerol
Tris-Cl
Bromophenol Blue
Yeast Extract
 
Protocol Hints
1. This method will not work well if the culture is grown to too high a density. Ten μl of a saturated overnight culture in YPD inoculated to 5 ml SD plus essential amino acids for about 16 hr gives an absorbance value at 600 nm of between 0.5 and 1.0 for wild-type cells grown at 30°C.