Rapid and reliable preparation of total cell lysates from fission yeast

doi:10.1038/nprot.2006.191

Method Article

Rapid and reliable preparation of total cell lysates from fission yeast

https://doi.org/10.1038/nprot.2006.191

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Version 1

posted 21 Jul, 2006

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Biological techniques

Total cell lysate

SDS-PAGE

Protein microarray

Protein macroarray

Reverse array

_Schizosaccharomyces pombe_

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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posted 21 Jul, 2006

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Rapid and reliable preparation of total cell lysates from fission yeast

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