Specific Binding of .BETA.2-Microglobulin with Trypan Blue

Abstract

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, nonspecificity, 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 ␤ 2 -microglobulin (␤ 2 m) 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 ␤ 2 m 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 max 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␤ in Alzheimer's disease and the prion protein in transmissible spongiform encephalopathy.

Persistent Staining of Lattice Lines After Intraoperative Trypan Blue Use in Patients With Lattice Corneal Dystrophy

“…Trypan blue is a symmetric diazo dye that is derived from toluidine and has a structure similar to that of Congo red. 3 In the laboratory setting, trypan blue is commonly used to determine cellular viability, because it is unable to penetrate cells with an intact membrane. In ophthalmology, it is primarily used for improved visualization of the anterior lens capsule while performing capsulorhexis.…”

“…This seems to be consistent with previous observations that trypan blue stains amyloid, both in vivo and in vitro, and can be used to verify its presence. [3][4][5][6] In all our patients, there was either violation of the endothelium (combined PKP and cataract surgery or recent PKP) or direct exposure of the dye to the corneal stroma (DALK surgery) at the time of surgery. This is consistent with the fact that trypan blue is unable to penetrate cells with intact membranes.…”