Klis Frans M, de Jong Marian, Brul Stanley, de Groot Piet W J
Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands.
Yeast. 2007 Apr;24(4):253-8. doi: 10.1002/yea.1476.
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.
为了从活的真菌细胞中提取细胞表面相关蛋白，常使用β-巯基乙醇和二硫苏糖醇等还原剂。我们在此表明，这两种化合物都具有中等亲脂性，可能会扰乱质膜，从而导致胞质蛋白释放，尤其是在高提取温度下。为避免假象，我们建议：(a) 仅在短时间内使用低浓度的还原剂；(b) 在4℃的提取温度下以保护质膜的完整性。同样，细胞表面蛋白的生物素化应在低温且不存在二甲基亚砜的条件下进行。
