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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PURIFICATION: EXTRACT FROM CELLS

RAPID YEAST PROTEIN PREPARATION

Rapid Yeast Protein Preparation
Contributor: The Laboratory of R. Daniel Gietz at the University of Manitoba.
URL: Lab Web Site
 
Procedure
1. Grow cells overnight to approximately 1 X 107 cells/ml (See protocol on Growth of Yeast Cells).

2. Pellet cells by spinning down 1.5 ml of the cell culture in a 1.5 ml microcentrifuge tube for 1 minute at maximum speed in a microcentrifuge.

3. Aspirate the supernatant.

4. Resuspend the cell pellet in 1 ml of ddH2O.

5. Spin down the cells for 1 minute at maximum speed in a microcentrifuge.

6. Resuspend cell pellet in 100 μl of Sample Buffer.

7. Heat the samples for 5 minutes at 95°C.

8. Centrifuge sample for 5 minutes at maximum speed in the microcentrifuge.

9. Analyze 25 μl by SDS-PAGE (see Protocol on SDS-PAGE).

Solutions
SDS-PAGE Sample Buffer   0.0025% (w/v) Bromophenol Blue
60 mM Tris, pH 6.8
5% (v/v) 2-Mercaptoethanol
2% (w/v) SDS
10% (v/v) Glycerol
 
BioReagents and Chemicals
2-Mercaptoethanol
Bromophenol Blue
Glycerol
Tris
SDS
 
Protocol Hints
No hints are associated with this bioProtocol
 
Citation and/or Web Resources
1. Horvath A, Riezman H. Rapid Protein Extraction from Saccharomyces cerevisiae. Yeast 1994, 10:1305-1310