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

Abstract

Aggregation of amyloid-β (Aβ) 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 Aβ protein, working solutions of Aβ are typically in the low micromolar range. Therefore, an ideal Aβ quantification method requires high sensitivity without sacrificing speed and accuracy. Absorbance at 280 nm is frequently used to measure Aβ concentration, but the sensitivity is low with only one tyrosine and no tryptophan residues in the Aβ sequence. Here we present a fluorescence method for Aβ 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 Aβ 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 Aβ oligomerization and fibrillization experiments.

Early Effects of Aβ Oligomers on Dendritic Spine Dynamics and Arborization in Hippocampal Neurons

“…Hexafluoroisopropanol was totally removed under vacuum in a speed vac system and the peptide film was stored dried at −80 • C. For the aggregation protocol, the peptide was first resuspended in anhydrous DMSO (Sigma-Aldrich, Saint Louis, MO, USA) to a concentration of 5 mM, to finally bring the peptide to a final concentration of 100 µM in Hams F-12 (PromoCell, Heidelberg, Germany) and to incubate it at 4 • C for 24 h. The preparation was then centrifuged at 14,000 g for 10 min at 4 • C to remove insoluble aggregates and the supernatants containing soluble Aβ 1-42 were transferred to clean tubes and kept at 4 • C. The concentration of Aβ peptides in the soluble phase of the preparation was quantified by absorbance at 280 nm. The measured Aβ 42 concentration at 100 µM showed a perfect agreement with the calculated concentration based on dilution, as was previously reported (Xue et al, 2017). The contribution of monomers, low molecular and high molecular weight (HMW) oligomers of the Aβ preparation was 40.5 ± 5%, 16 ± 2%, and 43.5 ± 7%, respectively of total Aβ (Figures 1A,B).…”

Aβ42 fibril formation from predominantly oligomeric samples suggests a link between oligomer heterogeneity and fibril polymorphism

“…We took aliquots of the Aβ42 sample immediately after the buffer exchange step and filtered through ultrafiltration filters with seven molecular weight cutoffs: 10, 30, 50, 100, 300, 1000 kDa and 0.2 µm. Then we measured the concentration of the different filtrate samples using the fluorescamine method [40]. If all Aβ42 proteins are monomers, which are 4.5 kDa in size, then the filtrate concentration from various filters would be very similar.…”

“…The concentration of Ab42 in CG buffer was 50 mM for all nine tubes. After buffer exchange to PBS buffer, two aliquots of 400 ml from each sample were filtered through 0.2 mm or 100 kDa filters, and the concentration of the filtrate was measured using the fluorescamine method [40]. The results of measured concentrations are shown in figure 2.…”

Alzheimer’s Aβ42 and Aβ40 form mixed oligomers with direct molecular interactions

“…HFIP treated samples were solubilized in dimethyl sulfoxide (DMSO) at 5 mM and bath-sonicated for 5 min. The Aβ concentration was determined using a fluorescamine method [32]. We prepared four samples of Aβ globulomers.…”