Investigation of Fasciola hepatica sample preparation for two-dimensional electrophoresis

James R. Jefferies, Peter Brophy, John Barrett

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34 Citations (SciVal)

Abstract

This paper investigates the preparation of Fasciola hepatica samples for two-dimensional electrophoresis (2-DE). Whole samples were prepared by both hot sodium dodecyl sulfate (SDS) solubilisation and precipitation using trichloroacetic 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 the profile observed, compared to the untreated control. Solubilisation of sample with hot SDS increased the number of protein spots, as did TCA precipitation with the addition of phosphotungstic acid. The preparation of excretory-secretory (ES) products poses problems due to both high salt concentrations and low protein concentration. All precipitation methods used to overcome this gave similar profiles, except acetone alone, which caused depletion of the larger proteins. TCA in acetone gave the best result, similar to that obtained by centrifugal filtration of the sample. Overcrowding of spots in some regions of the 2-DE gel occurred in the whole Fasciola hepatica sample. This problem was alleviated by differential solubilisation, which also resulted in the enrichment of some proteins
Original languageEnglish
Pages (from-to)3724-3729
Number of pages6
JournalElectrophoresis
Volume21
Issue number17
DOIs
Publication statusPublished - 01 Nov 2000

Keywords

  • Fasciola hepatica
  • trichloroacetic acid precipitation
  • two-dimensional electrophoresis
  • excretory-secretory product
  • protease
  • Protease
  • Excretory-secretory product
  • Two-dimensional electrophoresis
  • Trichloroacetic acid precipitation

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