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 ( Figure 1A, lane a). Individual proteins are poorly resolved and their extraction from individual cut-out bands is often inefficient. Several attempts were made to obtain amino acid sequence from various protein bands of louse homogenate using a variety of standard methods. This approach proved to be relatively ineffective and time-consuming.
The Mini Whole Gel Eluter was then used to fractionate crude louse homogenate. Samples of louse homogenate were separated on 12% preparative well gels and subjected to electro-elution. Two elution buffers, B and C, varying slightly in pH (7.8 v. 8.3) and in SDS content (0.005% v. 0.01%), were applied and in both experiments elution resulted in good recovery of a majority of proteins. It should be noted, however, that high molecular proteins did not elute well in buffer (B) containing low concentration of SDS (0.005%). The application of NH4HCO3/SDS buffer (A) resulted in virtually no protein elution, possibly due to the low stability of ammonium carbonate buffer.
It was obvious that the buffer (C) containing Tris and a higher SDS concentration (0.01%) was a much better choice, as it resulted in the elution of virtually all proteins present in the louse homogenate ranging from <14,000 to >100,000 daltons. Increased SDS concentration also yielded approximately 23 fold greater amount of protein eluted in each of 14 fractions (Figure 1B).
Protein fractions electro-eluted on the Mini Whole Gel Eluter with buffer C, containing 0.01% SDS, pH 8.3, were transferred on Prosorb, desalted, and subjected to automated N-terminal amino acid sequencing. Of three major proteins analyzed, sequence information was obtain ed for two. One of the proteins contained a sequence that was similar to that of a reported allergen.
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