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

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.

β淀粉样蛋白（amyloid-β, Aβ）的聚集过程在阿尔茨海默病中占据核心地位。由于蛋白质聚集属于浓度依赖的生理过程，相关严谨研究均需精准的浓度测定手段。鉴于Aβ蛋白具有极强的聚集倾向，其工作液通常处于低微摩尔浓度区间，因此理想的Aβ定量方法需在不牺牲检测速度与准确性的前提下具备高灵敏度。280 nm吸光度法常被用于Aβ浓度测定，但由于Aβ序列中仅含1个酪氨酸残基且无色氨酸残基，该方法的灵敏度较低。本文报道一种基于荧光胺（fluorescamine）的Aβ定量荧光检测法：荧光胺可与伯胺发生反应并产生高强度荧光信号。以鸡卵清溶菌酶为标准品，实验结果表明，当荧光胺浓度介于62.5至1000 µM时，荧光强度与伯胺浓度呈良好线性相关；当荧光胺浓度为250 µM及以上时，检测灵敏度可达最优水平。该荧光胺法兼容二甲基亚砜（dimethyl sulfoxide, DMSO）——这是制备Aβ寡聚体时的常用溶剂——而280 nm吸光度法因背景信号较高，应用范围受限。通过聚集动力学实验验证，该荧光胺法可在低微摩尔浓度范围内实现精准的浓度测定，并获得高度一致的聚集数据。我们推荐将该荧光胺分析法用于Aβ寡聚化与纤维化实验中的常规及实时浓度测定。