Supplementary Figure 3: Bivalent anti-FLAG antibodies are ineffective at disassembling FLAG-tag cages.

A. Light scattering vs. time in disassembly reactions with +0AA FLAG cages run as in fig. 2J but with the indicated concentrations of anti-FLAG IgG rather than Fab. Traces are averages from 7-9 repetitions. B: The same data as in A, but plotted with error bars giving +/- s.e. range for each experiment (error bars removed in A for clarity due to data overlap). C. Plot of the 1st 15 seconds of the reactions to better resolve the initial antibody binding step. Note that the amplitude of the scattering increase (~40%) is similar to that seen with Fab (fig. 2J-K), and is therefore consistent with bivalent binding of the antibody since monovalent binding would result in binding of similar numbers of Ab and Fab molecules and with a larger scattering increase for the former due to its larger MW. D: Gel analysis confirms that anti-FLAG IgG binds cages bivalently. Plot of IgG/CHC ratios vs. [IgG] (at 1, 3 and 9 μM; errors are +/- s.e. for n=3) shows that 0.45-0.65 IgG molecules per CHC bind to cages, while 0.8-1.2 Fab molecules per CHC are seen to bind in identical experiments shown in figure 5.