3.3. TTR Stability Assay

Recombinant wtTTR was incubated alone or in the presence of different compounds: IDIF as a reference positive control, the veterinary drug sulfaquinoxaline as a negative control, and the BBM drug at a molar ratio of 1:10 (TTR:drug) for 1 h at 37 °C. Then, urea was added at 6 M and samples were further incubated at 37 °C, overnight. The cross-linking reaction was performed by adding 2.5% glutaraldehyde for 4 min and then the reaction was quenched by adding 0.1% sodium borohydride. Samples were then run in a 13.5% acrylamide gel prepared with SDS, and transferred onto a nitrocellulose membrane (Amersham NC Protran^TM 0.2 μm Amersham GE Healthcare, Buckinghamshire, UK) using a wet system (Bio-Rad Criterion Blotter). The membranes were blocked for 1 h at RT with 5% nonfat dry milk (DM) in PBS containing 0.05% Tween-20 (PBS-T) and then incubated with primary antibody antihuman TTR (Dako; 1:1000 in 3% DM/PBS-T, Dako, Glostrup, Denmark). Then, washed membranes were incubated for 1 h at RT with sheep antirabbit immunoglobulins conjugated with horseradish peroxidase (Binding Site; 1:5000 in 3% DM/PBS-T). The blots were developed using Clarity^TM Western ECL substrate (Bio-Rad), and levels of folded (tetramer + trimer + dimer) and monomeric TTR were detected and visualized using a chemiluminescence detection system (ChemiDoc, Bio-Rad).