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J.Health Sci., 51(6), 702-707, 2005
Specific Binding of beta2-Microglobulin with Trypan Blue
Mara Teresa Tan Panlilio,a Christina Pascual Espiritu,b Noel Samson Quiming,c Rex Bugante Vergel,a Maria Fritzie Garcia Reyes,a and James Amador Villanueva*, a, b
aRoom 2200, Institute of Chemistry, bNatural Sciences Research Institute, University of the Philippines Diliman, Quezon City 1101, Philippines, and cDepartment of Physical Sciences and Mathematics, University of the Philippines Manila, Padre Faura Street, Ermita Manila 1000, Philippines
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 beta2-microglobulin (beta2m) 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 beta2m 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 lambdamax 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 A beta in Alzheimer's disease and the prion protein in transmissible spongiform encephalopathy.
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