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

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Abstract
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 μg). 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.

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