Characterization of consensus Envgp140s. A) 1 μg of each purified consensus Envgp140 was loaded onto a native PAGE and then sliver stained. The top of the gel is labeled with the consensus Envgp140 present in the lane and the protein ladder values are present on the y-axis. B) Using surface plasmon resonance, the interaction of the consensus Envgp160s and b12 was investigated in solution. The x-axis gives the rates of association between b12 and the Envgp140 and the y-axis gives the dissociation between the Envgp160s and b12. The consensus trimers are indicated by the darker symbols on the graph and the primary envelope trimers are indicated by the lighter symbols. C) The consensus Envgp140s and negative control BSA was pre-incubated with Histag soluble human CD4 at 37°C then mixed in with magnetic beads that were pre-incubated with the anti-his antibody. Using immunoprecipitation (IP), sCD4 and anything bound to it was pulled down in the pellet fraction. Supernatants and pellet fraction was analyzed by SDS-PAGE and western blot using anti-his and anti- Envgp160 antibodies. Left panel: supernatants fraction. Right panel: pellet fraction (sCD4 and proteins bound). The protein ladder values on y-axis. The upper blots were probed with rabbit polyclonal anti- Envgp160 antibody and the bottom blots were probed with mouse anti-human CD4 antibody (Clone RFT-4g mouse IgG, SouthernBiotech). Lane 1: BSA, Lane 2: Con M Envgp140, Lane 3: Consensus E Envgp140, Lane 4: Consensus C Envgp140, Lane 5: Consensus B Envgp140, Lane 6: Consensus A Envgp140.
Eugene et al. Virology Journal 2013 10:102 doi:10.1186/1743-422X-10-102