Quantitative evaluation of sample application methods for semipreparative separations of basic proteins by two-dimensional gel electrophoresis

he 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 mug). 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.