A label-free high-throughput protein solubility assay and its application to Aβ40

A major hallmark of Alzheimer's disease is the accumulation of aggregated amyloid peptide (Aβ) in the brain. Here we develop a solubility assay for proteins and measure the solubility of Aβ40. In brief, the method utilizes 96-well filter plates to separate monomeric Aβ from aggregated Aβ, and the small species are quantified with the amine reactive dye o-phthalaldehyde (OPA). This procedure ensures that solubility is measured for unlabeled species, and makes the assay high-throughput and inexpensive. We demonstrate that the filter plates successfully separate fibrils from monomer, with negligible monomer adsorption, and that OPA can quantify Aβ peptides in a concentration range from 44 nM to 20 μM. We also show that adding a methionine residue to the N-terminus of Aβ1–40 decreases the solubility by <3-fold. The method will facilitate further solubility studies, and contribute to the understanding of the thermodynamics of amyloid fibril formation.