Rapid and reliable preparation of total cell lysates from fission yeast
Related Journal & Article Information
Journal: Nature Biotechnology
Article Title: ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe
Introduction
In the biochemical and molecular biological studies of the fission yeast Schizosaccharomyces pombe, the current methods that are mainly used for preparation of the total cell lysates are mostly laborious and sometimes even ineffective. We propose a method for successful and reproducible extraction of proteins from S. pombe cells by slightly modifying the V. V. Kushnirov’s protocol that previously demonstrated effective extraction of proteins from the two yeast species Saccharomyces cerevisiae and Hansenula polymorpha.
Materials
Reagents
0.7 N NaOH solution
SDS-PAGE sample buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 4% 100 mM DTT, 10% glycerol, 0.1% bromophenol blue) #Note: Glycerol and bromophenol blue are not necessarily required.
Equipment
Microcentrifuge
Heating block (or alternatives for heating samples at nearly 100˚C)
Procedure
1. Culture S. pombe cells in an appropriate medium.
2. Harvest cells in a collection tube.
3. Suspend the cells with medium that was used in the culture of the cells (do not exceed the half volume of the tube).
4. Add an equal amount of 0.7 N NaOH solution and mix well.
5. Incubate at room temperature for 3 minutes.
6. Centrifuge the tube (5,000 rpm, 1 min) and discard the supernatant.
7. Add SDS-PAGE sample buffer and mix well.
8. Heat at 95-100˚C for 5 min.
9. Centrifuge at at least 3,500 rpm, and use the supernatant as the total cell lysate.
Troubleshooting
Longer incubation at 95-100˚C will reduce a protein yield.
Critical Steps
Anticipated Results
Almost all proteins are expected to be extracted.
Since proteins are extracted with SDS-PAGE sample buffer, you can directly use the samples for SDS-PAGE or other experiments.
References
Kushnirov, V.V. Rapid and reliable protein extraction from yeast. Yeast 16, 857-860 (2000).
Acknowledgements
Keywords
Total cell lysate, SDS-PAGE, Protein microarray, Protein macroarray, Reverse array, Schizosaccharomyces pombe
Figure 1
S. pombe cells were grown on minimum solid medium, harvested in MM liquid medium, and pretreated with indicated concentration of NaOH. Proteins were subsequently extracted with SDS-PAGE sample buffer, resolved by SDS-PAGE and stained with Coomassie blu
