Diaz-Griffero Lab

Diagram of the binding assay using stabilized HIV-1 capsid tubes. (A) Assembly of stabilized HIV-1 capsid tubes. Recombinant capsid protein bearing the mutations A14C and E45C was purified from bacterial extracts, as previously described (Pornillos et al., 2010). The purified monomeric capsid protein is assembled into hexamers, which are subsequently stabilized by oxidation of adjacent cysteines 14 and 45 to a disulfide bond or disulfide bridge. Cysteines are depicted in red. Oxidized hexamers assemble into large stable tubes(stabilized HIV-1 capsid tubes). These tubes are readily stable in solution, and washing does not affect the integrity of the complexes. (B) Binding assay using stabilized HIV-1 capsid tubes. The tubes are incubated for 1 h with specific and nonspecific protein binders. Subsequently the tubes are washed in a buffer of choice to eliminate nonspecific binding preserving specific binding. The proteins that remain associated to the stabilized tubes are analyzed by Western blotting or mass spectrometry.