ANS and ThT Binding Fluorescence Analysis

Fluorescence analysis of ANS and ThT binding to F/Tinduced
aggregated sample was performed at room temperature
on a FluoroMax-3 spectrofluorometer (Horiba Jobin
Yvon, Edison, NJ). A quartz cell with 5 mm path length (Part
number 4ES5X5 Precision Cells Inc., Farmingdale, NY) was
used. For ANS binding analysis, 360 μL LDH samples
(0.1 mg/ml) were mixed with 40 μL of 100 μM ANS stock
solution. Mixed samples were excited at 390 nm, and the
emission spectrum was recorded over a wavelength range of
410–600 nm. For ThT binding analysis, 360 μL 0.1 mg/ml
LDH samples were mixed with 40 μL of 100 μM ThT stock
solution. The excitation wavelength was 446 nm, and the
emission spectrum was recorded over a wavelength range of
460–560 nm. All the measurements were performed within
5 min of sample mixing for both ANS and ThT binding, and
three scans were performed to obtain an average spectrum for
each sample.