Separation Analysis of Amyloid Fibrils Utilizing a PTFE Tube as a Separation Field

We developed an easy-to-use analytical method for amyloid fibrils by high-performance liquid chromatography (HPLC) with post column fluorescent labeling. Thioflavin T (Th T), which was used widly as a labeling agent for amyloid fibrils, is a traditional probe for the confirmation of amyloid fibril formation. Although the traditional fluorescence spectroscopy measurements are used for monitoring the aggregation reaction, they have no applicability to separation analysis of the amyloid fibrils. In order to understand the fibril formation process, the development of an efficient method that enables to separate and detect the amyloid fibrils is required. In this work, Th T labeling of amyloid fibrils in 1 mM hen egg white lysozyme (HEWL) solution was performed following separation of different-sized amyloid fibrils in a PTFE tube (5000 X 0.50 mm i.d.) as a separation field. The fluorescence was monitored at 485 nm, with the maximum excitation wavelength at 435 nm. The novel method's sensitivity for the detection of amyloid fibrils was high and compared well with the traditional fluorescence spectroscopy measurements. In addition, the developed method detected more than 5 peaks within 20 mM under the optimum separation conditions. These results suggest that this analytical method is used to understand the amyloid fibril formation process.