Sedimentation velocity analysis of amyloid oligomers and fibrils.
The different aggregation states of amyloid oligomers and fibrils have been associated with distinct biological properties and disease pathologies. These various amyloid species are distinguished by their different molecular weights and sedimentation coefficients and can be consistently resolved, separated, and analyzed using sedimentation velocity techniques. We first describe the theoretical background and use of the preparative ultracentrifuge to separate amyloid fibrils and their oligomeric intermediates from monomeric subunits as well as the factors and limits involved in such methods. The approach can be used to monitor the kinetics of fibril formation as well as providing purified fractions for functional analysis. Secondly, we describe the use of analytical ultracentrifugation as a precise and robust system for monitoring the rate of sedimentation of amyloid fibrils under different solution conditions. Sedimentation velocity procedures to characterize the size, interactions, and tangling of amyloid fibrils as well as the binding of nonfibrillar components to form heterologous complexes are detailed.
Mok
Yee-Foong
YF
University of Melbourne, Biochemistry and Molecular Biology, Melbourne, Victoria, Australia.
Howlett
Geoffrey J
GJ
D000682:Amyloid / Q000737:chemistry* / Q000302:isolation & purification; D016229:Amyloid beta-Peptides / Q000302:isolation & purification; D053304:Apolipoprotein C-II; D001056:Apolipoproteins C / Q000302:isolation & purification; D052160:Benzothiazoles; D002498:Centrifugation / Q000379:methods; D006801:Humans; D010446:Peptide Fragments / Q000302:isolation & purification; D020836:Protein Structure, Quaternary; D013844:Thiazoles / Q000032:analysis; D014461:Ultracentrifugation / Q000379:methods*
D000682:Amyloid; D016229:Amyloid beta-Peptides; D053304:Apolipoprotein C-II; D001056:Apolipoproteins C; D052160:Benzothiazoles; D010446:Peptide Fragments; D013844:Thiazoles; C078592:amyloid beta-protein (1-40); C009462:thioflavin T