Beta-amyloid protein aggregation.
The beta-amyloid peptide aggregates via a nucleation pathway where micellar aggregates propagate to form oligomers (protofibrils), which then polymerize into insoluble fibrils. This fibrillogenic process has been linked to the pathogenesis associated with Alzheimer's disease. One purpose of this chapter is to provide a protocol for reliably producing monomeric Abeta as a starting point for physical and biological studies. Many research groups have used organic solvents to disaggregate pre-seeded Abeta in an attempt to acquire monomeric starting materials. Others have used instrumental techniques such as size exclusion chromatography to isolate monomer, structural intermediates, and fibrils and study their affects on Abeta nucleation. This chapter discusses a modified method of Abeta preparation using organic solvents followed by dissolution into aqueous phosphate buffer systems that renders monomeric Abeta starting solutions for kinetic experiments. Additionally, this chapter details a number of physical techniques such as scanning force microscopy, circular dichroism spectroscopy, transmission electron microscopy, fluorescence spectroscopy, fluorescence photobleaching recovery, and dynamic light scattering, together with physiological techniques such as cell viability assays to characterize Abeta nucleation, aggregation, and fibrillization and the potential biological activity of the various Abeta particles.
Etienne
Marcus A
MA
Department of Chemistry, Louisiana State University, Baton Rouge, USA.
Edwin
Nadia J
NJ
Aucoin
Jed P
JP
Russo
Paul S
PS
McCarley
Robin L
RL
Hammer
Robert P
RP
D000544:Alzheimer Disease / Q000378:metabolism; D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000648:ultrastructure; D000818:Animals; D002470:Cell Survival; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002942:Circular Dichroism; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods; D006801:Humans; D066298:In Vitro Techniques; D008027:Light; D018625:Microscopy, Atomic Force / Q000379:methods; D046529:Microscopy, Electron, Transmission / Q000379:methods; D008967:Molecular Biology / Q000379:methods; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000648:ultrastructure; D016716:PC12 Cells; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000648:ultrastructure; D051381:Rats; D012542:Scattering, Radiation
D016229:Amyloid beta-Peptides; D046912:Multiprotein Complexes; D010446:Peptide Fragments; C078592:amyloid beta-protein (1-40)