Quantification of Viral Proteins in Yeast Lysates

250 YU aliquots from N or N-PbCS cultures were lysed and clarified as described above and stored at −80 °C for western blot (WB) analysis of antigen concentration after each immunization. Stored samples contained 28 μl of N-PbCS lysate or of a 1/125 dilution of N lysate, 10 μl of XT Sample Buffer 4× (161-0791, Bio-Rad) and 2 μl of XT Reducing Agent 20× (161-0792, Bio-Rad). WB was performed as previously described [6 (link)] in denaturing conditions on 4–12% Bis–Tris polyacrylamide gels with XT MOPS buffer (Criterion 345-0123, Bio-Rad) using the Color Plus Prestained Protein Ladder (7-200 kDa; P7711 BioLabs). Anti-N monoclonal antibody (3E1, Abnova) was used at 1/2000 dilution to detect N-PbCS and N proteins in yeast lysate samples.