Barry Richard C, Alsaker Britt L, Robison-Cox James F, Dratz Edward A
Thermal Biology Institute, Montana State University, Bozeman, MT 59717, USA.
Electrophoresis. 2003 Oct;24(19-20):3390-404. doi: 10.1002/elps.200305591.
The use of cup-loading for sample application has become widely used in two-dimensional electrophoresis (2-DE) for resolution of basic proteins, but no side-by-side quantitative study has been published which compares cup-loading with the alternative passive and active rehydration methods to fully promote one type of loading method over another. Replicate 2-D gels from each loading method were quantitatively evaluated for gel-to-gel reproducibility using IPG 6-11 strips and semipreparative protein loads (300 microg). Gels were stained with SYPRO Ruby and analyzed with PDQuest. An inexpensive home-made assembly for cup-loading was used with the Protean IEF Cell for separation of whole cell extracts from the archaeon, Sulfolobus solfataricus. Cup-loading was determined to be far superior for IPG 6-11 separations than active or passive rehydration methods. Cup-loading consistently produced the greatest number of detectable spots, the best spot matching efficiency (56%), lowest spot quantity variations (28% coefficient of variation, CV), and the best-looking gels qualitatively. The least satisfactory results were obtained with active rehydration, followed closely by passive rehydration in off-line tubes. Passive rehydration experiments, performed using an on-line isoelectric focusing (IEF) tray, produced comparable spot numbers to cup-loading (84%), with 55% of the spots having higher intensity but 10% more spot quantity variance than cup-loading.
在二维电泳（2-DE）中，杯上样法用于上样已被广泛应用于碱性蛋白质的分离，但尚未发表过并排定量研究，比较杯上样法与替代的被动和主动水化方法，以充分推广一种上样方法优于另一种。使用IPG 6-11胶条和半制备蛋白上样量（300微克），对每种上样方法的重复二维凝胶进行凝胶间重复性的定量评估。凝胶用SYPRO Ruby染色，并用PDQuest分析。一种用于杯上样的廉价自制装置与Protean IEF Cell一起用于分离古菌嗜热栖热菌的全细胞提取物。结果表明，对于IPG 6-11分离，杯上样法远比主动或被动水化方法优越。杯上样法始终产生最多的可检测斑点、最佳的斑点匹配效率（56%）、最低的斑点数量变化（变异系数28%，CV），并且定性地凝胶外观最佳。主动水化得到的结果最不理想，离线管中的被动水化紧随其后。使用在线等电聚焦（IEF）托盘进行的被动水化实验产生的斑点数量与杯上样法相当（84%），55%的斑点强度更高，但斑点数量变异比杯上样法多10%。
