Publications (2)5.48 Total impact
Article: Reversibility of beta-amyloid self-assembly: effects of pH and added salts assessed by fluorescence photobleaching recovery.[show abstract] [hide abstract]
ABSTRACT: The 40-residue peptide isoform beta-amyloid (Abeta(1-40)) is associated with Alzheimer's disease. Although found in the tangles and fibrous mats that characterize the brain in advanced stages of the disease, the toxic form of Abeta is believed to be oligomers or "protofibrils". Characterization of these fairly small structures in solution, especially in the presence of the much larger assemblies they also form, is a daunting task. Additionally, little is known about the rate of Abeta assembly or whether it can be triggered easily. Perhaps most importantly, the conditions for reversing assembly are not fully understood. Fluorescence photobleaching with modulation detection of the recovery profile is a sensitive and materials-efficient way to measure diffusers over a wide range of hydrodynamic sizes. The method does require attachment of a fluorescent label. Experiments to validate the use of 5-carboxyfluorescein-labeled Abeta(1-40) as a representative of the unlabeled, naturally occurring material included variation of photobleaching time and mixture of labeled and unlabeled materials. A dialysis cell facilitated rapid in situ changes in pH and salt conditions. Multiple steps and complex protocols can be explored with relative ease. Oligomeric aggregates were found by fluorescence photobleaching recovery to respond readily to pH and salt conditions. Changing these external cues leads to formation or disassembly of aggregates smaller than 100 nm within minutes.Biomacromolecules 02/2010; 11(2):341-7. · 5.48 Impact Factor
Article: Beta-amyloid protein aggregation.[show abstract] [hide abstract]
ABSTRACT: 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.Methods in molecular biology (Clifton, N.J.) 02/2007; 386:203-25.