title: Quantification of amyloid fibrils using size exclusion chromatography coupled with online fluorescence and ultraviolet detection.
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.
authors.affiliation: Université de Toulouse, UPS, Laboratoire de Biotechnologies Agroalimentaire et Environnementale (LBAE), EA 4565, Institut Universitaire de Technologie, F-32000 Auch, France.
authors.forename: Irina
authors.initials: I
authors.lastname: Randrianjatovo-Gbalou
authors.affiliation: Université de Toulouse, UPS, Laboratoire de Biotechnologies Agroalimentaire et Environnementale (LBAE), EA 4565, Institut Universitaire de Technologie, F-32000 Auch, France.
authors.forename: Claire-Emmanuelle
authors.initials: CE
authors.lastname: Marcato-Romain
authors.affiliation: Université de Toulouse, UPS, Laboratoire de Biotechnologies Agroalimentaire et Environnementale (LBAE), EA 4565, Institut Universitaire de Technologie, F-32000 Auch, France. Electronic address: elisabeth.neuhauser@iut-tlse3.fr.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Girbal-Neuhauser
mesh_terms: D000465:Algorithms; D000682:Amyloid / Q000032:analysis* / Q000737:chemistry; D000818:Animals; D001407:Bacillus / Q000737:chemistry / Q000502:physiology; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D052160:Benzothiazoles; D018441:Biofilms; D002138:Calibration; D002364:Caseins / Q000032:analysis / Q000737:chemistry; D002417:Cattle; D002850:Chromatography, Gel; D005456:Fluorescent Dyes / Q000737:chemistry; D005602:France; D008958:Models, Molecular; D008970:Molecular Weight; D066329:Protein Aggregates; D066263:Protein Aggregation, Pathological / Q000175:diagnosis*; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013844:Thiazoles / Q000737:chemistry
chemical_list: D000682:Amyloid; D001426:Bacterial Proteins; D052160:Benzothiazoles; D002364:Caseins; D005456:Fluorescent Dyes; D066329:Protein Aggregates; D013844:Thiazoles; C009462:thioflavin T
keywords: Amyloids; Quantification; Size exclusion chromatography; Thioflavin T