Premium
Investigation of Fasciola hepatica sample preparation for two‐dimensional electrophoresis
Author(s) -
Jefferies James R.,
Brophy Peter M.,
Barrett John
Publication year - 2000
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200011)21:17<3724::aid-elps3724>3.0.co;2-k
Subject(s) - chromatography , chemistry , fasciola hepatica , sample preparation , protein precipitation , trichloroacetic acid , sodium dodecyl sulfate , precipitation , gel electrophoresis , acetone , extraction (chemistry) , biochemistry , biology , zoology , physics , meteorology , helminths
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.