(A) SDS-PAGE analysis of 293F cell expressed proteins. 1 μg of gp120 (ImmunoDiagnostics), gp140, or gp140:gpD fusion proteins in sample buffer with (reduced) or without (non-reduced) 100 mM DTT were resolved on a 7.5% polyacrylamide gel. (B) Analytical size exclusion chromatography. Purified gp140 and gp140:gpD proteins were resolved on a Superdex 200 column in phosphate-buffered saline, and their retention times were compared with those of known molecular mass standard proteins of 150, 440, and 669 kDa (arrows). The main peak retention time of gp140 (18.05 min) and gp140:gpD (18.042 min) is consistent with both proteins migrating as a trimer (ferritin marker protein (440 kDa), 17.95min). (C) Blue native (BN)–PAGE analysis of gp120, and 293F cell-derived, purified gp140 and gp140:gpD proteins (5 μg).