Figure 4
From: Development of a hydroxyflavone-labelled 4554W peptide probe for monitoring αS aggregation
“Aged”-αS experiment design and results. (A) Schematic describing αS aging method. Identical vials of αS monomer (500 μM) were incubated under aggregating conditions (700 rpm double orbital shaking, 37 °C) for a set period of time. It is assumed that during these varying lengths of aggregation that a range of oligomeric species will form. Aged αS samples were added to a 96-well microplate along with the labelled-peptides, and the resulting fluorescence spectra were obtained on a CLARIOstar plate reader. Figure made in BioRender.com. (B) On a single occasion (n = 1), the N peak was observed when K1 (25 μM) was added to a sample of αS (50 μM) aged for the specified length of time (0, 1, 2, 4, 6, 8, 12, or 24 h) and the spectra (λex 360 nm, λem 390–650 nm) was recoded at 0 h, after 2 h incubation, and after 24 h incubation. (C) To the aged αS sample (50 μM), either the fluorophore (25 μM) or K1 (25 μM) was added and the resulting spectra (λex = 360 nm, λem 390–650 nm) was recorded. Plotting the intensity of the T peak (540 nm) for each of the samples showed that the intensity of the fluorophore alone did not vary depending on the age of the αS sample, whereas there was a strong decreasing trend in intensity correlating to αS sample age with K1. The results are the average of three repeats, with the error bars representing standard error.
