TY - JOUR
T1 - Investigation of Fasciola hepatica sample preparation for two-dimensional electrophoresis
AU - Jefferies, James R.
AU - Brophy, Peter
AU - Barrett, John
PY - 2000/11/1
Y1 - 2000/11/1
N2 - 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
AB - 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
KW - Fasciola hepatica
KW - trichloroacetic acid precipitation
KW - two-dimensional electrophoresis
KW - excretory-secretory product
KW - protease
UR - http://hdl.handle.net/2160/45272
U2 - 10.1002/1522-2683(200011)21:17<3724::AID-ELPS3724>3.0.CO;2-K
DO - 10.1002/1522-2683(200011)21:17<3724::AID-ELPS3724>3.0.CO;2-K
M3 - Article
SN - 0173-0835
VL - 21
SP - 3724
EP - 3729
JO - Electrophoresis
JF - Electrophoresis
IS - 17
ER -