SDS-PAGE and Western Blot Analysis

Protein samples were denatured in NuPAGE sample buffer (Invitrogen) supplemented with 5% (v/v) 2-mercaptoethanol and by subsequent heating to 95°C for 5 min. Denatured proteins were resolved on 12% (w/v) NuPAGE polyacrylamide gels run with MOPS buffer (Invitrogen). See Blue Plus, protein molecular weight markers (Invitrogen) were run on all NuPAGE gels. Instant Blue stain (Expedeon Ltd.) was used to visualize the protein bands. Western blotting to detect either TMV coat protein with an anti-TMV antibody, (AS-72203-2ML, Austral Biologicals, San Ramon, CA, United States) or the six C-terminal histidine residues with a monoclonal antibody prepared to six histidine residues, (Cat 34660, Qiagen) was performed as described by Saunders and Lomonossoff (2015) (link).