Anti-aggregation assays

Hi, thank you for your interest on our work. I added notes to the method desription in the paper.

I hope this helps.

Aggregation of denatured GAPDH from rabbit muscle (Sigma; G-2267) was measured as described previously (Saio et al., 2014).

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 h at 4°C. 

Note: The denatured GAPDH solution is stable at least for 1 week 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.

Note: Step-by-step instruction of the assay. 

1.     During the assay, the 125 µM GAPDH solution is placed on ice, and the buffer solution as well as TF solutions are placed in the sand bath with the temperature set to 20°C. 

2.     The buffer (20 mM KPi (pH 7.0), 100 mM KCl, 4 mM bME, 0.5 mM EDTA, and 0.05% NaN3) or TF solution is placed in the cuvette and incuvated in the the spectrometer for 10 minutes. Note that the tenperature regulation is critical in this experiment. The spectromer needs to be equipped with peltier thermostatted cell holder. 

3.     The GAPDH solution was added into the buffer or TF solution, then mixed gently by pipette for ~20 sec, followed by the measurement of light scattering.

Note: Between each run, the cuvette needs to be carefully washed using detergent. Even slight precipitation of protein or detergent left on the cuvette wall can destroy the assay. We usually soak the cuvette in the detergent for ~10 min and wash with detergent using transfer pipette. Then the cuvette is rinsed with water followed by ethanol (or methanol).

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