Brian J. Shiell and Wojtek P. Michalski, CSIRO Division of Animal Health, Australian Animal Health Laboratory, Geelong, Victoria, Australia
Amino acid sequence and composition analyses of proteins can be performed on samples derived from polyacrylamide gels. Protein electro-elution, the most commonly used method of recovering proteins from gels, can be performed on cut-out gel slices or using the Whole Gel Eluter recently introduced by Bio-Rad.14 The Mini Whole Gel Eluter was used here to fractionate crude homogenate of the common sheep louse with the aim of characterizing immunogenic components.
Common sheep lice, Bovicola ovis, were harvested from infested animals, homogenized by ultrasonication, and the homogenate was clarified by centrifugation on a benchtop microfuge. Homogenate samples (1 mg protein) were separated by SDS-PAGE in 12% preparative well Tris-HCl Ready Gels (Bio-Rad). Protein fractions were immediately electro-eluted at 100 mA for 25 min using the Mini-Whole Gel Eluter. Three elution buffers: (A) 100 mM NH4HCO3, 0.01% SDS, pH 8.5, (B) 10 mM phosphate buffer, 0.005% SDS, pH 7.8, and (C) 25 mM Tris, 19.2 mM glycine, 0.01% SDS, pH 8.3, were used to determine the elution efficacy. The fractions were analyzed in 12%, 15 well Tris-HCl Ready Gels (Bio-Rad) and proteins were visualized by Coomassie blue staining. Bio-Rads low molecular weight standards for SDS-PAGE are shown in Figure 1B, lane b.
Results and Discussion
Louse homogenate preparations are often rich in lipids and do not separate well on SDS-PAGE gels (