Fulgentini Lorenzo, Marangoni Roberto, Colombetti Giuliano
Istituto di Biofisica del CNR, Pisa, Italy.
Electrophoresis. 2008 Jun;29(11):2411-2. doi: 10.1002/elps.200700838.
An efficient protein extraction methodology is quite important for sample preparation and subsequent 2-D PAGE and MS analysis. Cell lysis is the first step in protein extraction and purification. Many techniques are available for cell disruption, including physical and detergent-based methods. Here, we report on a very fast and efficient detergent-free Tris-based method to extract the soluble fraction proteins of extremophile ciliates, comparing it with a detergent-based protocol. This comparison has been carried out by means of 2-D PAGE and subsequent MALDI-compatible silver staining of protein samples obtained from the intensely pigmented hypersaline ciliate Fabrea salina and the Antarctic hypotrich ciliate Euplotes focardii. Our results indicate that this fast and easy extraction method allows to obtain more clear crude extracts and more spot-abundant polyacrylamide gels.
一种高效的蛋白质提取方法对于样品制备以及后续的二维聚丙烯酰胺凝胶电泳（2-D PAGE）和质谱（MS）分析非常重要。细胞裂解是蛋白质提取和纯化的第一步。有许多技术可用于细胞破碎，包括物理方法和基于去污剂的方法。在此，我们报告一种非常快速且高效的无去污剂的基于Tris的方法，用于提取嗜极端微生物纤毛虫的可溶性组分蛋白质，并将其与基于去污剂的方案进行比较。这种比较是通过二维聚丙烯酰胺凝胶电泳以及对从色素沉着强烈的高盐纤毛虫法氏嗜盐菌（Fabrea salina）和南极腹毛类纤毛虫福氏真核草（Euplotes focardii）获得的蛋白质样品进行与基质辅助激光解吸电离（MALDI）兼容的银染来进行的。我们的结果表明，这种快速简便的提取方法能够获得更清亮的粗提物和斑点更丰富的聚丙烯酰胺凝胶。
