Additional file 2: Figure S2. Effect of peptide concentrations on the SrtA coupling reaction. (A) Comparison of the C-terminal sequence of PapMV WT, PapMV-SrtA and PapMV-SrtA(short). In PapMV-SrtA, a linker of 5 amino acids (TSTTR) was added before the SrtA recognition motif LPETGG, while in PapMV-SrtA(short) the recongnition motif was included directly in the native PapMV sequence by deleting two proline residues. (B) SDS-PAGE and western blot of SrtA reactions on PapMV-SrtA and PapMV-SrtA(short) nanoparticles. PapMV nanoparticles (25 µM) were incubated with SrtA (50 µM) and GGG-M2e peptide (50 µM) for 2.5 hours at room temperature. Reactions were stopped with EGTA (10 µM) and passed through a 100 kDa centrifugal filter unit to eliminate excess peptide and contaminating SrtA. PapMV nanoparticles retained by the 100 kDa filter unit were diluted to 0.1 µg/µL in migration buffer supplemented with 30% of SDS loading buffer and 4 µL was loaded onto 15% Tris-Glycine SDS-PAGE. PapMV-SrtA, PapMV-SrtA(short) and SrtA controls correspond to lanes 1, 10 and 11, respectively. Lanes 2-5 and lanes 6-9 represent four experimental replicates of SrtA conjugation on PapMV-SrtA or PapMV-SrtA(short), respectively. Efficient SrtA labelling of GGG-M2e peptide onto PapMV nanoparticles was assessed by SDS-PAGE (top panel), and immunoblotting with a specific antibody against the M2 (bottom panel).