Figure 4

Gel electrophoresis of recombinant antigen expressed in P. pastoris and analyzed by mass spectrometry and Western blot. A) Ten μg of purified protein was added to an equal volume of sample buffer (4% sodium dodecyl sulfate, 120 mM Tris pH 6.8, 0.02% bromophenol blue, 5% β-mercaptoethanol), heated for 5 minutes at 90°C, and electrophoresed on a 10% polyacrylamide gel. Following staining with Coomassie G-250 the gel was destained with water for approximately 3 d before the indicated band (arrow) was extracted and analyzed by mass spectrometry. Lane 1: protein molecular weight standards; Lane 2: empty; Lanes 3 and 4: 10 μg of purified vaccine antigen protein with calculated molecular weight of 33.9 kDa (sequence in Additional file 1: Table S1) expressed in P. pastoris; Lanes 5 and 6: 10 μg of bovine serum albumin protein standard (MW =66.4 kDa); B) Ten μg of purified protein was electrophoresed on a NuPAGE® 4-12% Bis-Tris gel and analyzed by Western blotting using standard protocols provided with the WesternBreeze Chromogenic Kit and Anti-myc-HRP antibody (Invitrogen). Lane 1: All Blue Precision Plus Protein Standards (Bio-Rad); Lane 2: Purified recombinant Aquaporin-derived vaccine antigen. The blot image was adjusted through contrast and brightness controls to enable the visualize the minor background products of approximately 60-65 kDa.