Investigation of Fasciola hepatica sample preparation for two-dimensional electrophoresis

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Investigation of Fasciola hepatica sample preparation for two-dimensional electrophoresis. / Jefferies, James R.; Brophy, Peter; Barrett, John.

In: Electrophoresis, Vol. 21, No. 17, 01.11.2000, p. 3724-3729.

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Jefferies, James R. ; Brophy, Peter ; Barrett, John. / Investigation of Fasciola hepatica sample preparation for two-dimensional electrophoresis. In: Electrophoresis. 2000 ; Vol. 21, No. 17. pp. 3724-3729.

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@article{c842afdd63724cf1ba3872d4cc7cf66b,
title = "Investigation of Fasciola hepatica sample preparation for two-dimensional electrophoresis",
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",
keywords = "Fasciola hepatica, trichloroacetic acid precipitation, two-dimensional electrophoresis, excretory-secretory product, protease",
author = "Jefferies, {James R.} and Peter Brophy and John Barrett",
year = "2000",
month = nov,
day = "1",
doi = "10.1002/1522-2683(200011)21:17<3724::AID-ELPS3724>3.0.CO;2-K",
language = "English",
volume = "21",
pages = "3724--3729",
journal = "Electrophoresis",
issn = "0173-0835",
publisher = "Wiley",
number = "17",

}

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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

VL - 21

SP - 3724

EP - 3729

JO - Electrophoresis

JF - Electrophoresis

SN - 0173-0835

IS - 17

ER -

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