Specific Binding of [beta]2-Microglobulin with Trypan Blue

Several staining methods have been developed to monitor protein fibril formation. Two widely used dyes that are now utilized in standard staining assays are Congo red and Thioflavin T (ThT). However, non-specificity, false negative results and a requirement for expensive instrumentation have precluded the use of these dyes in the characterization of amyloidogenic proteins. In this study, we developed a simple method to follow specific binding of β2-microglobulin (β2m) fibrils using UV-visible (Vis) spectroscopy with the Trypan blue (TB) dye. The use of UV-Vis spectroscopy as a technique for amyloid fibril demonstration serves as an advantage due to the availability of the instrument in most laboratories. Binding of β2m fibrils was achieved by combining a solution of TB with a concentrated fibril solution followed by UV-Vis spectroscopy. Here we observed a significant shift of the λmax towards a longer wavelength when TB specifically binds with the fibrils. Also, the observed increase in absorbance upon binding of TB was dependent on the amount of fibrils. This new and simple assay adds to the variety of staining methods which may potentially be used to analyze other protein fibrils like the Aβ in Alzheimer's disease and the prion protein in transmissible spongiform encephalopathy.