Amyloid, The Amyloid B -Peptide and Alzheimer’s Disease: Structural Considerations - Molecules: Nucleation, Aggregation and Crystallization - page 294

solution can be incubated for days. In order to remove potential seeds that
have survived the initial treatment, centrifugation or filtration is frequently
used. Each step in the preparation procedure leads to sample loss, and it is
necessary to measure the concentration of A β in the final working solution.

is a very “sticky peptide,” it is important to select tubes, well
plates and pipette tips with low affinity for the peptide. Several factors, such
as temperature, buffer ions, pH, detergents, surface tension and shaking,
affect the polymerization rate. Depending on the goal of the polymerization
studies, different techniques are used for studying A

Since A β

-polymerization.
Here we will discuss the merits and drawbacks of the most frequently used.

β

Thioflavin T binding assay

Thioflavin T binding (ThT) is a fluorescent molecule that has a high affin-
ity for amyloid fibrils. Upon binding, the fluorescence spectrum is shifted,
and thus, the polymerization can be followed by monitoring the fluores-
cence of ThT (LeVine, 1993). The method is rapid, easy to use and suit-
able for well plate readings. However, it does not give information on the
size or morphology of the aggregates, and ThT can bind to other aggre-
gates than fibrils (Tjernberg et al ., 1999). Therefore, EM is usually used
as a complement to ThT for showing the morphology of the aggregates.
The ThT assay is frequently used for finding and studying polymerization
inhibitors, but gives only limited information on at what stage the poly-
merization is inhibited, although it can be inferred that an increase in the
lag phase would indicate that the inhibitor interferes with seed formation.
In certain cases the binding sites for ThT can be blocked, for instance
when fibril bundles are formed or when the studied molecule competes for
the ThT binding site, resulting in a low signal (Söderberg et al ., 2005).

Gel Electrophoresis

Polyacrylamide gel electrophoresis (PAGE) has been used to visualize
oligomers of different size. Using the strong ionic detergent SDS in the