Quantification of amyloid fibrils using size exclusion chromatography coupled with online fluorescence and
Irina Randrianjatovo-Gbalou1, Claire-Emmanuelle Marcato-Romain1, Elisabeth Girbal-Neuhauser1
1Université de Toulouse, UPS, Laboratoire de Biotechnologies Agroalimentaire et Environnementale (LBAE), EA 4565, Institut Universitaire de Technologie, F-32000 Auch, France.
Abstract:
An amyloid fibrils investigation within biofilm samples requires distinguishing the amyloid β-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 κ-casein (κCN) 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 κCN, representing approximately 21% of the total proteins.
