Karadzic Ivanka M, Maupin-Furlow Julie A
Department of Microbiology and Cell Science, University of Florida, Gainsville, FL 32611, USA.
Proteomics. 2005 Feb;5(2):354-9. doi: 10.1002/pmic.200400950.
Proteins of haloarchaea are remarkably unstable in low-ionic-strength solvents and tend to aggregate under standard two-dimensional (2-D) gel electrophoresis conditions, causing strong horizontal streaking. We have developed a new approach to generate 2-D maps of halophilic proteins which included washing cells with 1.5 M Tris-HCl buffer. In addition, proteins were precipitated with acetone, solubilized with urea and thiourea in the presence of the sulfobetaine detergent 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS), reduced with tributylphosphine (TBP), and separated with microrange strips of immobilized pH gradients (pH 3.9-5.1). This combination enabled the construction of highly reproducible 2-D maps of Haloferax volcanii proteins.
嗜盐古菌的蛋白质在低离子强度溶剂中非常不稳定，并且在标准二维（2-D）凝胶电泳条件下容易聚集，导致强烈的水平条纹。我们开发了一种新方法来生成嗜盐蛋白质的二维图谱，该方法包括用1.5 M Tris-HCl缓冲液洗涤细胞。此外，蛋白质用丙酮沉淀，在磺基甜菜碱去污剂3-[(3-胆酰胺丙基)二甲基氨基]-1-丙烷磺酸盐（CHAPS）存在下用尿素和硫脲溶解，用三丁基膦（TBP）还原，并用固定化pH梯度的微范围条带（pH 3.9-5.1）分离。这种组合能够构建高度可重复的沃氏嗜盐富饶菌蛋白质二维图谱。
