FIG. 7.

Cross-linking of VP22.159-301. VP22.159-301 was expressed and purified exactly as previously described (31). Protein samples (12 μg) were incubated with the homobifunctional chemical cross-linking agents disulfosuccinimidyl tartrate (sulfo-DST, lanes 2 to 4), bis(sulfosuccinimidyl)suberate (BS³, lanes 5 to 7), or ethylene glycolbis(sulfosuccinimidylsuccinate) (sulfo-EGS, lanes 8 to 9), in increasing concentrations (60, 150, and 300 μM) of the cross-linkers in citrate buffer on ice for 2 h. The reactions were quenched by the addition of 50 mM Tris for 30 min on ice, and samples were then denatured in SDS lysis buffer and separated by SDS-PAGE under reducing conditions. The positions of the VP22.159-301 in the absence of cross-linker (lane 1) and the major cross-linked species are indicated by arrows.