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Methodologies & Further Information on the FishProm Project

Rainbow trout were maintained in freshwater as described elsewhere (Martin et al. 2003) and were fed diets containing varying levels of corn gluten meal, wheat gluten, extruded peas, rapeseed meal, extruded whole wheat, and supplemental amino acids.

Protein extraction was performed in urea-based lysis buffer as described by Martin et al. (2001) followed by centrifugation at 50,000×g to remove particulates. Samples were stored at -70°C until gel electrophoresis was performed.

Electrophoresis was performed at the Aberdeen University Proteome Facility using standard protocols (Cash et al. 1999). Briefly, after reswelling, samples added onto pH 4-7 immobilised pH Gradient strips and isoelectrofocussed in three stages with a ramped voltage change between each step. The strips were then laid onto a 10-15% gradient polyacrylamide slab gel and the proteins electrophoresed in the second dimension. The resolved proteins were detected using Collodial Coomassie Blue G250 staining (Anderson et al. 1991).

Gels were scanned at a resolution of 200dpi using a Hewlett Packard Scanjet 3p flat bed scanner and the gel images analysed using the software package Phoretix 2-D version 5.01 (NonLinear Dynamics, Gateshead, UK). The liver proteome reference map was created from a representative 2DE gel image of a liver protein extract from a rainbow trout fed a standard diet to satiation, as described in Martin et al. (2001). Each spot was automatically assigned a reference number by the Phoretix program. As new spots are discovered on other gels, a virtual "spot" is added in the corresponding place on the reference map. Molecular masses and isoelectric points are read off the reference map after calibration as per the software manufacturer's instructions. Individual protein spot abundance were determined by spot volume after background subtraction and normalisation against total gel volume, as per the software manufacturer's instructions. Five replicate gels were used for each treatment group. Significance of abundance variation was determined by a Student's t test.

Vilhelmsson, O.T., Martin, S.A.M ., Médale, F., Kaushik, S.J. & Houlihan, D.F. (2004). Dietary plant protein substitution affects hepatic metabolism, but does not invoke a stress response, in rainbow trout. British Journal of Nutrition 92 : 71-81

Martin, S. A., Vilhelmsson, O., Medale, F., Watt, P., Kaushik, S. & Houlihan, D. F. (2003). Proteomic sensitivity to dietary manipulations in rainbow trout. Biochim Biophys Acta 1651, 17-29.

Martin, S. A. M., Cash, P., Blaney, S. & Houlihan, D. F. (2001). Proteome analysis of rainbow trout (Oncorhynchus mykiss) liver proteins during short term starvation. Fish Physiol Biochem 24, 259-270.

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