Figure 3.

Analysis of the purification and preparation steps of 2C for crystallization. (A) Ni²⁺-agarose-purified 2C was purified by gel filtration (Sephacryl-S-100) to remove aggregates and monomers. (B) Gel filtration-purified 2C was treated with carboxypeptidases A and B and purified by HIC. The progress of the reaction is monitored by HIC. Partially (Upper Left) and fully digested (Lower Left) 2C clearly separate by HIC and 2D nonequilibrium, pH-gradient electrophoresis (Upper Right). The fully digested 2C is purified by HIC and migrates on reducing and nonreducing SDS/PAGE at a position approximately 5 kDa smaller than the digested band (Lower Right).