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Protein aggregation monitoring on a BMG LABTECH microplate reader

Stephen Poon, Robert C. Rivers Department of Chemistry, University of Cambridge 12/2005

Protein aggregation is the cause of many debilitating and often incurable human diseases. Moreover, aggregation during drug formulation invariably leads to reduced efficacy, low yield, poor storage capacity, and increased production costs. Two assays were applied on a BMG LABTECH microplate reader to assess protein aggregation. One is based on the increase in fluorescence intensity of Thioflavin T (ThT) when binding β-sheet-rich fibrils; the other is based on turbidity changes induced by precipitated protein.

 

Using a BMG LABTECH multi-mode reader, differences in aggregation of wildtype and mutated protein were determined by heating the samples up to 60 °C and monitoring absorbance at 340 nm. These differences were confirmed using a ThT end point assay. High fluorescence was observed for the wild-type self-aggregating protein whereas some mutated forms gave low fluorescence signals.

 

We demonstrated that the BMG LABTECH microplate reader is well-suited to screen for aggregation properties in medium to high throughput as well as to follow the kinetics of protein aggregation.

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