Sucrose gradient centrifugation of polymerization reactions. Polymerization reactions containing ARA, ThS and either mock-phosphorylated (minus GSK-3β), or phosphorylated tau were centrifuged through a discontinuous sucrose gradient containing 1.0, 1.5, and 2.0 M sucrose. Filament clusters sedimented at the interface of the 1.5 and 2.0 M sucrose concentrations. Panels A, and C are TEM micrographs of gradient fractions from reactions induced with a low inducer:tau ratio (25 μM ARA:2 μM tau, panels B and D with an optimal ratio (75 μM ARA:2 μM tau). Grids were prepared without glutaraldehyde fixation of polymerization samples. Samples were concentrated five fold on the TEM grids.