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A mix-and-click method to measure amyloid-? concentration with sub-micromolar sensitivity.


ABSTRACT: Aggregation of amyloid-? (A?) protein plays a central role in Alzheimer's disease. Because protein aggregation is a concentration-dependent process, rigorous investigations require accurate concentration measurements. Owing to the high aggregation propensity of A? protein, working solutions of A? are typically in the low micromolar range. Therefore, an ideal A? quantification method requires high sensitivity without sacrificing speed and accuracy. Absorbance at 280?nm is frequently used to measure A? concentration, but the sensitivity is low with only one tyrosine and no tryptophan residues in the A? sequence. Here we present a fluorescence method for A? quantification using fluorescamine, which gives high fluorescence upon reaction with primary amines. We show that, using hen egg white lysozyme as a standard, fluorescence correlates linearly with primary amine concentration across a wide range of fluorescamine concentrations, from 62.5 to 1000?µM. The maximal sensitivity of detection is achieved at a fluorescamine concentration of 250?µM or higher. The fluorescamine method is compatible with the presence of dimethyl sulfoxide, which is commonly used in the preparation of A? oligomers, and limits the use of absorbance at 280?nm due to its high background reading. Using aggregation kinetics, we show that the fluorescamine method gives accurate concentration measurements at low micromolar range and leads to highly consistent aggregation data. We recommend the fluorescamine assay to be used for routine and on-the-fly concentration determination in A? oligomerization and fibrillization experiments.

SUBMITTER: Xue C 

PROVIDER: S-EPMC5579099 | BioStudies | 2017-01-01

REPOSITORIES: biostudies

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