(a) Analytical ultracentrifugation sedimentation velocity analysis of the CLL183 Fab. Two primary species are present in solution with sedimentation coefficients of 3.7 and 5.0 S, respectively (1 S=10−13 s). The appearance of the larger species is concentration dependent, consistent with the presence of a monomer–dimer equilibrium. (b) Fitting of SE-AUC data with a self-association model. The points represent the absorbance at a specific position in the cell, and the curve show the fitting of the chosen model. In this experiment, the protein concentration was 0.26 mg ml−1, and the cell was subjected to speeds of 8,000 (blue points and line), 12,000 (cyan), 16,000 (green) and 22,000 r.p.m. (violet). The residuals are small and show no systematic trends (see also Supplementary Fig. 3 and Supplementary Table 3). (c,d) For both the CLL183 Fab epitope (Glu16HAla) and paratope (Arg106HAla/Tyr107HAla) mutants a single, monomeric species is present in solution even at protein concentrations of 1 mg ml−1 (corresponding to 20 μM).