Quantification of amyloid fibrils using size exclusion chromatography coupled with online fluorescence and ultraviolet detection

An amyloid fibrils investigation within biofilm samples requires distinguishing the amyloid beta-sheet structure of these proteins and quantifying them. In this study, the property of amyloids to incorporate the fluorescent dye Thioflavin T has been exploited to propose a method of quantification. The experimental protocol includes the preparation of amyloids from commercial K-casein (kappa CN) and their fractionation through size exclusion chromatography (SEC) to provide calibration curves from fluorescence and absorbance signals. Finally, a bacterial biofilm extract was injected into SEC, and the amyloid fibrils could be expressed as equivalent kappa CN, representing approximately 21% of the total proteins. (C) 2015 Elsevier Inc. All rights reserved.