Figure 3. Size Exclusion Chromatograms (a) Gel filtration chromatogram using a S75 column, of the wild type tDGC protein (red) that elutes as a c-di-GMP-bound dimer and of the R158A mutant (green) that purifies as a monomer and is devoid of nucleotides.

(b) Following incubation of the wild type tDGC bound to c-di-GMP with RocR (in the gel filtration buffer containing 25 mM Tris-HCl, 300 mM NaCl, 5% (v/v) glycerol, 10 mM MgCl₂ for 2 hrs at room temperature), c-diGMP is converted to pGpG and tDGC elutes predominantly as a monomer. SDS PAGE analysis of the dimeric and monomeric fractions demonstrates that both peaks contain tDGC.