Extraction of cell surface-associated proteins from living yeast cells.
To extract cell surface-associated proteins from living fungal cells, reducing agents such as beta-mercaptoethanol and dithiothreitol are often used. We show here that both compounds are moderately lipophilic and may perturb the plasma membrane, thus causing the release of cytosolic proteins, especially at high extraction temperatures. To avoid artifacts, we recommend using (a) a low concentration of the reducing agent for only a short period of time, and (b) an extraction temperature of 4 degrees C to protect the integrity of the plasma membrane. Similarly, biotinylation of cell surface proteins should be carried out at low temperatures in the absence of dimethylsulphoxide.
Klis
Frans M
FM
Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands. F.M.Klis@uva.nl
de Jong
Marian
M
Brul
Stanley
S
de Groot
Piet W J
PW
D019921:Biotinylation; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008623:Mercaptoethanol; D009172:Mycology / Q000379:methods; D019163:Reducing Agents; D012441:Saccharomyces cerevisiae / Q000254:growth & development* / Q000378:metabolism; D013696:Temperature
D008565:Membrane Proteins; D019163:Reducing Agents; D008623:Mercaptoethanol; D004229:Dithiothreitol