Ninhydrin assay

The ninhydrin assay was carried out in triplicate as described previously (Starcher, 2001). As protein standard, 100 μl of 1 mg/ml BSA was hydrolyzed in 100 μl of 6 N HCl at 110°C for 24 h in a heat block. After evaporating to dryness, the hydrolysate was re-dissolved in water to yield a 1 mg/ml final solution. The ninhydrin reagent was prepared as follows: 400 mg ninhydrin (2,2-dihydroxyindane-1,3-dione) was dissolved in a mixture of 15 ml ethylene glycol and 5 ml of 4 N sodium acetate buffer (4 N sodium acetate, pH 5.5, adjusted with glacial acetic acid). Then, 500 μl of stannous chloride suspension (100 mg SnCl₂ in 1,000 μl ethylene glycol mixed well before pipetting) was added while stirring. A volume of 100 μl of ninhydrin reagent was added to 1–10 μg of protein hydrolysate (final concentration of the unknown sample should be around 1 mg/ml) in a flat-bottom microtiter plate that was sealed with aluminum sealer and floated on a boiling water bath for 10 min. The plate was removed with forceps and blotted with paper towels and the absorbance at 575 nm was measured in a Molecular Devices titer plate reader. The proteins are brought to concentrations so that their measured values were around the middle of the standard curve.