(565c) Optimization of Sample Preparation for Two-Dimensional Protein Electrophoresis

Effective sample preparation is essential to capture the sample proteome and maximize utility of subsequent electrophoresis.  The large number of interacting variables impacting protein solubility and the relatively low-throughput nature of most electrophoretic methods constrains the systematic study of sample preparation.  We employed a design of experiments approach to optimize primary protein recovery by altering solution compositions during sample preparation.  The impact of four components (urea, DTT, CHAPS, and SDS) was examined, with protein recovery quantified by protein concentration assays, SDS-PAGE, and two-dimensional electrophoresis.  Recovery results were obtained for a mixture of model proteins, as well as CHO-K1 and E. coli cell lysates, and favorable solution conditions for each sample type are discussed.