PVY and VLP samples were analyzed under reducing conditions with SDS-PAGE using NuPAGE 4 to 12% bis-tris protein gel (Invitrogen). Wet electroblotting was carried out to transfer proteins from the gel to the polyvinylidene difluoride membrane. The transfer sandwich (filter paper–gel–membrane–filter paper) was placed into the transfer box (XCell SureLock, Invitrogen) at constant voltage of 30 V at 5°C for 60 min in NuPAGE transfer buffer with 10% methanol. After the transfer, the membrane was washed for 5 min in 50% methanol; then stained for 1 min in 0.1% Coomassie Blue R-250, 50% methanol, and 5% acetic acid; and then destained in 50% methanol until bands were visible. The membrane was left to air dry, and the protein bands were cut out and submitted for N-terminal Edman sequencing on Procise protein sequencing system 492A (PE Applied Biosystems at Jožef Stefan Institute, Ljubljana, Slovenia). Edman chemistry using pulsed-liquid blot was performed for preparation of phenylthiohydantoin amino acid derivatives. Analysis of the derivatives was performed on high-performance liquid chromatography (HPLC) system 140C (PE Applied Biosystems) using RP C18 column Spheri-5 (5 μm, 220 mm by 2.1 mm) (Brownlee).

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