Stoichiometry of the individual polypeptides in each purified holotoxin variant was confirmed experimentally by densitometric scanning of the gel in the lower panel of Fig. 2 . To adjust for differences in loading of the purified holotoxins (ranging from 0.6 g in lane 6 to 2.9 g in lane 2), the observed density for each band was expressed as a fraction of the total density for all bands in the same lane. The observed fractional densities were then compared with the expected values based on the predicted molecular mass of each polypeptide (CTA, 27.2 kDa; wt CTB, 11.6 kDa; and tagged CTB, 15.3 kDa) and the assumption that binding of Coomassie blue is proportional to the mass of each peptide. The results (see Table S1 in the supplemental material) were generally within 15% of expected values, but all observed values for tagged B subunits were higher than expected, suggesting that the tagged subunits bound proportionately more Coomassie blue stain than native CTA or CTB. Corrected for loading differences, the expected stoichiometric ratios for the tagged B subunit bands in lanes 3 versus 4, 6 versus 7, and 8 versus 9 were 1:2, and the corresponding observed ratios were 1:2.1, 1:1.7, and 1:1.9, respectively.