Purification of plant-derived Nef. Western-blot analysis of purified Nef extracted from: (A) transgenic Nicotiana tabacum leaves or (B) Nicotiana benthamiana agroinfiltrated leaves with Nef/AMCV-P19. Total leaf protein homogenates were purified using a two step immobilized-metal affinity chromatography (IMAC) protocol consisting of a nickel-nitrilotriacetic acid (Ni-NTA) column and a cobalt column. Aliquots (20 μl) from four elution fractions (F1-F4) were loaded on reducing SDS-12% (w/v) polyacrylamide gel (PAGE) and analysed by Western-blot using a monoclonal antibody (mAb) against Nef. F1-F4 Nef: elution fractions of Nef transgenic tobacco (A) or agroinfiltrated N. benthamiana plants (B). C+: 20 ng of E. coli-derived Nef. Bands at about 30 kDa correspond to the monomeric form of Nef, while the bands at 66 kDa indicate the presence of Nef dimers. (C) Western-bolt analysis of Nef purified from agroinfiltrated N. benthamiana leaves (10 g of fresh weight). Fractions F2, F3 and F4 (0.5 ml each) were pooled and a 20 μl aliquot was loaded on reducing SDS-12% (w/v) polyacrylamide gel (PAGE). Two distinct bands at about 30 kDa are indicated by asterisks. The upper band corresponds to monomeric Nef, while the lower band proved to be a degradation product as revealed by mass spectrometry. Plant-derived Nef yield (average of 250 ng per gram of fresh leaf weight) was estimated by densitometric analysis of the upper band obtained in Western-blot.