Pridmore A M, Devine D A, Bonass W A, Silley P
Division of Oral Biology, Leeds Dental Institute, University of Leeds, Shipley, UK.
Lett Appl Microbiol. 1999 Apr;28(4):245-9. doi: 10.1046/j.1365-2672.1999.00544.x.
Sample preparation methods were compared for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of cellular proteins from the proteolytic bacterium Porphyromonas gingivalis. Standard solubilization buffer yielded poorly resolved protein spots, but pre-treatment of cells with trichloroacetic acid or inclusion of the protease inhibitor TLCK during solubilization improved definition and separation. The latter approach allowed reliable detection of a 55 kDa immunodominant surface antigen by Western immunoblotting. Further improvements in resolution occurred when SDS was included in the sample preparation. Thus, controlling proteolysis and optimizing protein solubilization were essential for reproducible separations and maximal protein recovery during 2D-PAGE of P. gingivalis.
针对牙龈卟啉单胞菌这种蛋白水解细菌的细胞蛋白二维聚丙烯酰胺凝胶电泳（2D-PAGE），对样品制备方法进行了比较。标准溶解缓冲液产生的蛋白质斑点分辨率较差，但用三氯乙酸对细胞进行预处理或在溶解过程中加入蛋白酶抑制剂TLCK可改善清晰度和分离效果。后一种方法通过蛋白质免疫印迹能够可靠地检测到一种55 kDa的免疫显性表面抗原。当样品制备中加入SDS时，分辨率进一步提高。因此，在牙龈卟啉单胞菌的2D-PAGE过程中，控制蛋白水解和优化蛋白质溶解对于可重复的分离和最大程度的蛋白质回收至关重要。
