In vitro aggregation assays of GAPDH and OmpA

Aggregation of denatured GAPDH from rabbit muscle (Sigma; G-2267) was measured as described previously (Saio et al., 2014 (link)). 125 µM GAPDH was denatured by 3 M guanidine-HCl in 20 mM potassium phosphate, pH 7.0, 100 mM KCl, 4 mM β-mercaptoethanol, 0.5 mM EDTA, and 0.05% NaN₃ for 12 hr at 4°C. The denatured GAPDH was diluted 50-fold into the buffer that does not contain guanidine-HCl and aggregation was monitored by 90° light scattering at 620 nm on a spectrofluorometer (FP-8500, JASCO Corporation) in the absence or presence of TF or TF^mon at the concentration of 0.5 µM or 1 µM. The experiment was carried out at 20°C. The reproducibility was confirmed by independent assays repeated three times.
In anti-aggregation assay on OmpA^1-192, 62 µM OmpA^1-192 in 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 400 mM imidazole, and 8 M urea was diluted 20-fold into 20 mM potassium phosphate, pH 7.0, 100 mM KCl, 4 mM β-mercaptoethanol, 0.5 mM EDTA, and 0.05% NaN₃. Aggregation was monitored by 90° light scattering at 620 nm on a spectrofluorometer (FP-8500, JASCO Corporation) in the absence or presence of TF or TF^mon at the concentration of 4 µM. The experiment was carried out at 25°C.