Investigation of Fasciola hepatica sample preparation for two-dimensional electrophoresis

This paper investigates the preparation of Fasciola hepatica samples for tw o-dimensional electrophoresis (2-DE). Whole samples were prepared by both h ot sodium dodecyl sulfate (SDS) solubilisation and precipitation using tric hloroacetic acid (TCA) to remove nonprotein contaminants and to inactivate endogenous proteases. Sample preparation had a marked influence on the 2-DE gel profile. TCA precipitation resulted in no measurable improvement in th e profile observed, compared to the untreated control. Solubilisation of sa mple with hot SDS increased the number of protein spots, as did TCA precipi tation with the addition of phosphotungstic acid. The preparation of excret ory-secretory (ES) products poses problems due to both high salt concentrat ions and low protein concentration. All precipitation methods used to overc ome this gave similar profiles, except acetone alone, which caused depletio n of the larger proteins. TCA in acetone gave the best result, similar to t hat obtained by centrifugal filtration of the sample. Overcrowding of spots in some regions of the 2-DE gel occurred in the whole Fasciola hepatica sa mple. This problem was alleviated by differential solubilisation, which als o resulted in the enrichment of some proteins.