FIGURE 1.

Isolation of recombinant Tau RD fibrillar assemblies by SEC. A, recombinant Tau RD fibrils labeled with AF647 were sonicated for different periods of time (10, 50, and 100 min) to create assemblies in a range of sizes. Assemblies were resolved by SEC with a Sephacryl S500 column. B, the size of each fraction's content was estimated based on the molecular weight of gel filtration standards. C, selected chromatography fractions were resolved by SDS-PAGE probed with a Tau polyclonal antibody. The left lane shows recombinant RD fibril prior to fractionation. Fractions are indicated in the table, as are estimated Tau assembly sizes (where n represents the putative number of Tau units). D, fluorescence of fractions correlates well with protein content of each using a Micro BCA assay. E, selected fractions cross-linked with PFA were resolved by SDS-PAGE and probed with a Tau polyclonal antibody. F, mass spectrometry analysis of cross-linked monomer fraction indicates a molecular mass of 15,376 Da. Analysis of cross-linked trimer fraction indicates three detected species of 45,716 Da (trimer), 30,751 Da (dimer), and 15,376 Da (monomer). H, stability of Tau assemblies was tested by rerunning three fractions (monomer, trimer, and ∼40-mer) separately through the SEC column. Each assembly was stable through the isolation protocol. I, two fractions were recombined (trimer and ∼40-mer) and reisolated using SEC. There was no evidence of interassembly interaction.