This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Rapid and reliable preparation of total cell lysates from fission yeast
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Figure 1
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.
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.
Microcentrifuge
Heating block (or alternatives for heating samples at nearly 100˚C)
- Culture S. pombe cells in an appropriate medium.
- Harvest cells in a collection tube.
- Suspend the cells with medium that was used in the culture of the cells (do not exceed the half volume of the tube).
- Add an equal amount of 0.7 N NaOH solution and mix well.
- Incubate at room temperature for 3 minutes.
- Centrifuge the tube (5,000 rpm, 1 min) and discard the supernatant.
- Add SDS-PAGE sample buffer and mix well.
- Heat at 95-100˚C for 5 min.
- Centrifuge at at least 3,500 rpm, and use the supernatant as the total cell lysate.
Within 20 minutes
Longer incubation at 95-100˚C will reduce a protein yield.
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.
Kushnirov, V.V. Rapid and reliable protein extraction from yeast. Yeast 16, 857-860 (2000).
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Version History
CURRENT STATUS: Posted
Version 1
Posted 21 Jul, 2006
Metrics
Comments: 2
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Subject Areas
Biological techniques
Associated Publications
ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe
by Matsuyama, A. et al.
Nature Biotechnology (06 June, 2006)
Method Article
Rapid and reliable preparation of total cell lysates from fission yeast
Version History
CURRENT STATUS: Posted
Version 1
Posted 21 Jul, 2006
Metrics
Comments: 2
HTML Views: ...
Subject Areas
Biological techniques
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Figure 1
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.
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.
Microcentrifuge
Heating block (or alternatives for heating samples at nearly 100˚C)
- Culture S. pombe cells in an appropriate medium.
- Harvest cells in a collection tube.
- Suspend the cells with medium that was used in the culture of the cells (do not exceed the half volume of the tube).
- Add an equal amount of 0.7 N NaOH solution and mix well.
- Incubate at room temperature for 3 minutes.
- Centrifuge the tube (5,000 rpm, 1 min) and discard the supernatant.
- Add SDS-PAGE sample buffer and mix well.
- Heat at 95-100˚C for 5 min.
- Centrifuge at at least 3,500 rpm, and use the supernatant as the total cell lysate.
Within 20 minutes
Longer incubation at 95-100˚C will reduce a protein yield.
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.
Kushnirov, V.V. Rapid and reliable protein extraction from yeast. Yeast 16, 857-860 (2000).
2 comments flagged for moderation
If you posted a comment and think it was mistakenly removed, please contact customer support at [email protected].
Associated Publications
ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe
by Matsuyama, A. et al.
Nature Biotechnology (06 June, 2006)