Application of proteomics for determining protein markers for wool qualitytraits

The technique of two-dimensional electrophoresis (2-DE) has been under inve stigation for its usefulness in identifying protein markers for wool qualit y traits in sheep. However, before this could be achieved, unique problems relating to the detection and quantitation of wool proteins needed to be ov ercome so that 2-DE protein maps could be examined using computational prog rams like Melanie II. Four protein staining regimes were examined. Colloida l Coomassie Blue G-250 was found to be superior to Coomassie Blue R-250 and gave satisfactory staining of all protein classes. Silver staining detects minor strings of keratinous proteins, but unfortunately it negatively stai ns intermediate filament proteins, the major high sulphur proteins (HSPs) a nd the high glycine tyrosine proteins and the latter two classes can only b e seen by overstaining the background of the gel. in contrast, labeling red uced keratins with [C-14]iodoacetamide, followed by autoradiography detecti on, results in a protein map with tow background and all protein spots stai ned positively. 2-DE has been used to obtain wool protein maps of Lincoln/M erino chimeric sheep to examine wool originating from two genotypes grown w ith different crimp frequencies within the same fleece. Between fleece, var iations have also been examined. Work to date suggests that several major H SPs may be associated with the fibre curvature trait known as crimp frequen cy. From matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) mass spectral mapping, one of these proteins has been identified as be ing from the B2A family from the HSP class.