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.

淀粉样β（amyloid-β, Aβ）蛋白的聚集过程在阿尔茨海默病中居于核心地位。由于蛋白质聚集属于浓度依赖过程，严谨的相关研究需依赖精准的浓度测定。鉴于Aβ蛋白具有极高的聚集倾向，其工作液通常处于低微摩尔浓度范围。因此，理想的Aβ定量方法需兼具高灵敏度与快速、准确的特性。280 nm吸光度法常被用于测定Aβ浓度，但该方法灵敏度较低——因Aβ序列中仅含1个酪氨酸残基，且无色氨酸残基。本文报道一种基于荧光胺（fluorescamine）的Aβ定量荧光法：该试剂可与伯胺发生反应并产生高强度荧光。我们以鸡蛋清溶菌酶（hen egg white lysozyme）作为标准品，证实荧光强度与伯胺浓度呈线性相关，且该线性范围覆盖62.5至1000 µM的荧光胺浓度区间。当荧光胺浓度达到250 µM及以上时，检测灵敏度可达最优。该荧光胺法兼容二甲基亚砜（dimethyl sulfoxide, DMSO）的存在——而二甲基亚砜是制备Aβ寡聚体的常用试剂，且280 nm吸光度法因背景信号过高而应用受限。通过聚集动力学实验，我们证实荧光胺法可在低微摩尔浓度范围内实现精准的浓度测定，并可得到高度一致的聚集数据。我们推荐将该荧光胺检测法用于Aβ寡聚化与纤维化实验中，作为常规且即时的浓度测定手段。