Spectramax m2e multi mode plate reader

Purified protein stored at −80 °C (from multiple batches of purification) was allowed to thaw on ice, following which it was pooled. The pooled protein was concentrated using Amicon® ultra centrifugal filters (Merck) and then subjected to SEC as described before (section 4.2). Only the monomeric protein fraction obtained using SEC was quantified using bicinchoninic acid (BCA) assay and utilized for setting up the amyloid aggregation assay. Monomeric m-OLF at a concentration of 0.5 mg mL⁻¹ (15 μM) in reaction buffer was taken in 1.5 mL micro-centrifuge vials with/without the presence of ECG (Cayman Chemical) and incubated at 37 °C with a steady agitation rate of 200 rpm. The experiment was performed in triplicates, all reaction vials were sampled at regular intervals by aliquoting 10 μL of the reaction mix and diluting it with 90 μL of Thioflavin T (ThT) prepared in the same buffer. ThT was employed at a final dilution of 10 μM; fluorescence emission values were documented at 485 nm of wavelength following excitation at a wavelength of 450 nm in SpectraMax® M2e multimode plate reader (Molecular Devices), using a 1 cm path length quartz cuvette. OriginPro 8.5 was used for plotting and fitting the data, sigmoidal fit for the scatter points was obtained by utilizing the curve fitting Boltzmann function.