Biotek epoch 2 microplate reader

All C. albicans isolates are listed in Table 1. C. albicans cells were cultured in liquid YPD medium [2% bacto-peptone, 1% yeast extract, and 2% dextrose (filter-sterilized)] overnight at 30°C with continuous shaking (200 rpm), unless otherwise specified. The number of cells in each culture was determined by measuring the optical densities of culture dilutions (OD₆₀₀ nm) in sterile water using a Biotek Epoch 2 microplate reader (Agilent Technologies). The cultures were then diluted to the desired concentrations in sterile water.

The activity of the purified proteins (DHY-SC-VUT5 and DHY-G-VUT7) were carried out according to the protocol by Li et al. [14 (link)] for assessing ALDH activity. The standard reaction mixture for the oxidation reaction (200 μl) contained hexanal (Merck, USA) as the substrate, 50 mM Nicotinamide adenine dinucleotide (NAD) in 50 mM Tris-HCl (pH 8.0) and an appropriate amount of enzyme. Varying concentrations of hexanal substrate (25 mM, 50 mM, 100 mM, 150 mM and 200 mM) and enzyme concentrations (0.001 mg/ml, 0.003 mg/ml, 0.007 mg/ml and 0.014 mg/ml) were tested respectively in order to determine the optimal concentrations for each. ALDH activity was assayed spectrophotometrically at 340 nm in a 96 well plate by determining the Nicotinamide adenine dinucleotide phosphate (NADH) produced or oxidized, using the BioTek® Epoch 2 microplate reader (Agilent Technologies, USA). The enzymes were added to the reaction mixture to start the reaction. This was mixed thoroughly and immediately placed in the microplate reader. The absorbance values were recorded at twenty second intervals for 3 min at 30 °C. The enzymatic activity of the purified proteins was measured by the increase in A₃₄₀ due to the formation of NADH. The relative activity for each enzyme was calculated as a percentage by dividing the absorbance range by the highest absorbance value.