The Role of Buffers in Wild-Type HEWL Amyloid Fibril Formation Mechanism: A Methodological Approach

The amyloidophilic dyes thioflavin T and Congo red are small, yet powerful, molecules that allow the in vitro and in vivo detection of amyloid fibrils in protein solutions. Even though Congo red and thioflavin T binding assays are widespread techniques for unveiling amyloid fibers and are gradually replacing the more demanding X-ray diffraction method, handling samples containing amyloid fibrils is still challenging and can lead to false-positive/negative results. Here we describe a relatively straightforward procedure of preparing hen egg-white lysozyme amyloid fibrils in different buffer solutions and their detection with thioflavin T and Congo red, supported by an indispensable method for determining the secondary structure of proteins - circular dichroism.