Figure 2.
Time-Dependent Changes in the Association of α2M with αS Oligomers
αSA90C-AF488 (70 μM) and α2M-AF647 (0.7 μM) were co-incubated in PBS (pH 7.4) at 37°C, with shaking at 200 rpm. The formation of αS-α2M complexes was quantified by single-molecule TCCD.
(A and B) Contour plots of the apparent number of αS monomers constituting a given oligomer as a function of the Zα2M/αS value for samples taken from the aggregation reaction after 6 hr (A) and 48 hr (B). Zα2M/αS represents the logarithm of the apparent ratio of α2M to αS in each oligomer. The numbers in the inserts indicate the number of complexes represented in the plot.
(C and D) Frequency histograms of the number of oligomers at different Zα2M/αS values (for the data shown in A and B, respectively). The dotted lines each indicate a specific α2M:αSA90C stoichiometry (as shown on the upper x axis). Data are representative of three independent experiments.
(E) αSA90C-AF488 (70 μM) and α2M-AF647 (0.7 μM) were co-incubated in PBS at 37°C, with shaking at 200 rpm, for the indicated time. For each time point, the average numbers of apparent monomers of α2M and αS per oligomer were calculated. The data were fitted to linear regressions.
(F) The gradients of each linear regression for each time point shown in (A). A time-dependent linear increase in the amount of α2M found bound to αS can be observed. Data shown are means ± SEM (n = 3).