Preparing synthetic Aβ in different aggregation states.
This chapter outlines protocols that produce homogenous preparations of oligomeric and fibrillar amyloid-β peptide (Aβ). While there are several isoforms of this peptide, the 42 amino acid form is the focus because of its genetic and pathological link to Alzheimer's disease (AD). Past decades of AD research highlight the dependence of Aβ42 function on its structural assembly state. Biochemical, cellular and in vivo studies of Aβ42 usually begin with purified peptide obtained by chemical synthesis or recombinant expression. The initial steps to solubilize and prepare these purified dry peptide stocks are critical to controlling the structural assembly of Aβ. To develop homogenous Aβ42 assemblies, we initially monomerize the peptide, erasing any "structural history" that could seed aggregation, by using a strong solvent. It is this starting material that has allowed us to define and optimize conditions that consistently produce homogenous solutions of soluble oligomeric and fibrillar Aβ42 assemblies. These preparations have been developed and characterized by using atomic force microscopy (AFM) to identify the structurally discrete species formed by Aβ42 under specific solution conditions. These preparations have been used extensively to demonstrate a variety of functional differences between oligomeric and fibrillar Aβ42. We also present a protocol for fluorescently labeling oligomeric Aβ42 that does not affect structure, as measured by AFM, or function, as measured by a cellular uptake assay. These reagents are critical experimental tools that allow for defining specific structure/function connections.
Stine
W Blaine
WB
Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, IL, USA.
Jungbauer
Lisa
L
Yu
Chunjiang
C
LaDu
Mary Jo
MJ
D016229:Amyloid beta-Peptides / Q000138:chemical synthesis* / Q000737:chemistry* / Q000633:toxicity; D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D018625:Microscopy, Atomic Force
D016229:Amyloid beta-Peptides