Linear quantitation of Abeta aggregation using Thioflavin T: reduction in fibril formation by colostrinin.
Thioflavin T (ThT) fluorescence is a commonly used method to monitor Abeta protein fibril formation. This method is particularly attractive since ThT fluoresces only when bound to fibrils, the reaction is completed within 1min and ThT does not interfere with aggregation of Abeta fibrils. One of the drawbacks of this method is the lack of a strict quantitative relationship between ThT fluorescence and fibril content. It was observed that, when the same gram molecular weight of Abeta (1-40) is dissolved into varying amounts of base then placed into a constant volume of aqueous buffer, a non-linear fluorescent response is obtained. By maintaining a strict relationship between Abeta content and the volume of base, this anomalous result can be alleviated and a linear dose response curve is obtained at much lower Abeta concentrations than is typically observed. In addition, differences in Abeta batch to batch preparations are alleviated. It was previously reported that colostrinin (CLN), a proline-rich peptide derived from colostrum, reduces fibril content and protects neuroblastoma cells against Abeta peptide-induced toxicity. The newly developed ThT fluorescence protocol was used to quantify Abeta fibril content after treatment with CLN. We also demonstrate that CLN, can solubilize Abeta fibrils in a dose and time-dependent fashion.
Bourhim
Mustapha
M
Department of Cancer Chemoprevention, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Kruzel
Marian
M
Srikrishnan
Thamarapu
T
Nicotera
Thomas
T
D000544:Alzheimer Disease / Q000378:metabolism; D016229:Amyloid beta-Peptides / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000737:chemistry; D052160:Benzothiazoles; D001681:Biological Assay / Q000379:methods*; D015415:Biomarkers / Q000032:analysis / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D016014:Linear Models; D008856:Microscopy, Fluorescence / Q000379:methods; D008970:Molecular Weight; D009448:Neurochemistry / Q000379:methods*; D018696:Neuroprotective Agents / Q000494:pharmacology; D010446:Peptide Fragments / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000737:chemistry; D010455:Peptides / Q000494:pharmacology*; D058225:Plaque, Amyloid / Q000737:chemistry* / Q000187:drug effects; D012995:Solubility / Q000187:drug effects; D013844:Thiazoles / Q000737:chemistry*
D016229:Amyloid beta-Peptides; D052160:Benzothiazoles; D015415:Biomarkers; D005456:Fluorescent Dyes; D036341:Intercellular Signaling Peptides and Proteins; D018696:Neuroprotective Agents; D010446:Peptide Fragments; D010455:Peptides; D013844:Thiazoles; C078592:amyloid beta-protein (1-40); C105874:colostrinine; C009462:thioflavin T