Yan J X, Tonella L, Sanchez J C, Wilkins M R, Packer N H, Gooley A A, Hochstrasser D F, Williams K L
Australian Proteome Analysis Facility, Macquarie University, Sydney, NSW, Australia.
Electrophoresis. 1997 Mar-Apr;18(3-4):491-7. doi: 10.1002/elps.1150180325.
The cellular slime mold Dictyostelium discoideum is a eukaryotic microorganism which has developmental life stages attractive to the cell and molecular biologist. By displaying the two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) protein map of different developmental stages, the key molecules can be identified and characterised, allowing a detailed understanding of the D. discoideum proteome. Here we describe the preparation of reference gel of the D. discoideum multicellular aggregate, the slug. Proteins were separated by 2-D PAGE with immobilised pH gradients (pH 3.5-10) in the first dimension and sodium dodecyl sulfate (SDS)-PAGE in the second dimension. Micropreparative gels were electroblotted onto polyvinylidene difluoride (PVDF) membranes and 150 spots were visualised by amido black staining. Protein spots were excised and 31 were putatively identified by matching their amino acid composition, estimated isoelectric point (pI) and molecular weight (M(r)) against the SWISS-PROT database with the ExPASy AAcompID tool (http:// expasy.hcuge.ch/ch2d/aacompi.html). A total of 25 proteins were identified by matching against database entries for D. discoideum, and another six by cross-species matching against database entries for Saccharomyces cerevisiae proteins. This map will be available in the SWISS-2DPAGE database.
细胞黏菌盘基网柄菌是一种真核微生物，其发育生命阶段对细胞生物学家和分子生物学家具有吸引力。通过展示不同发育阶段的二维聚丙烯酰胺凝胶电泳（2-D PAGE）蛋白质图谱，可以鉴定和表征关键分子，从而详细了解盘基网柄菌的蛋白质组。在此，我们描述了盘基网柄菌多细胞聚集体蛞蝓体的参考凝胶制备方法。蛋白质在第一维通过固定化pH梯度（pH 3.5 - 10）进行2-D PAGE分离，在第二维通过十二烷基硫酸钠（SDS）-PAGE分离。微量制备凝胶被电印迹到聚偏二氟乙烯（PVDF）膜上，并用酰胺黑染色可视化150个斑点。切除蛋白质斑点，通过使用ExPASy AAcompID工具（http://expasy.hcuge.ch/ch2d/aacompi.html）将其氨基酸组成、估计的等电点（pI）和分子量（M(r)）与SWISS-PROT数据库进行匹配，推定鉴定出31个斑点。通过与盘基网柄菌的数据库条目匹配，共鉴定出25种蛋白质，另外6种通过与酿酒酵母蛋白质的数据库条目进行跨物种匹配鉴定出来。该图谱将在SWISS-2DPAGE数据库中提供。
