Concentration of total ascorbic acid was calculated from the absorbance difference and standard curves of 5.25 mM dehydro-L--ascorbic acid dimer reduced with 265 mL 1 M DTT in 400 mL 100 mM phosphate buffer (pH 5.6) and monitored at 265 nm as described for the samples.
Elution was performed at 3 ml/min with equilibration buffer and spectrophotometrically monitored at 280 nm.
The proteins were eluted using the same buffer and simultaneously monitored at 280 nm.
Kinetic experiments were carried out at 25 °C, either in water with 50 m m buffer at 0.1 m ionic strength (NaNO3) or in anhydrous methanol with 50 m m buffer and monitored using UV/Vis spectroscopy to measure the change in absorbance at 400 nm (water) or 320 nm (methanol).
At 48 h after transfection, cells were lysed in luciferase reporter lysis buffer and monitored for luciferase and renilla activity using a Dual-Luciferase Reporter Assay SysteMadisonega, MadisoNY NY USA).
Proteins were eluted at 0.5 mL/min in buffer A and monitored by absorbance at 280 nm.
The column was equilibrated in buffer C. Samples were eluted isocratically in buffer C, monitoring at 220 and 280 nm.
The decline in the buffer TMRM concentration was monitored at 575 nm with fluorescent spectrophotometer (NOVOstar, BMG Labtech).
The electrode was soaked in 8 mL PBS buffer (pH 7.4) and monitored continuously for QCM frequency change at 25°C.
The column was washed with 100 ml of Buffer A containing 100 mM NaCl at a flow rate of 8 ml/min and the protein was eluted with a 250 ml linear gradient of NaCl (100 600 mM) in Buffer A. The elution was monitored at 280 nm and fractions were collected.
