Beschreibung
ProteOrange Kit with a fluorescent dye allows protein quantification with higher sensitivity than that of spectrophotometry and other conventional methods used for protein quantification (Lowry, Bradford, BCA). ProteOrange reagent, the key kit ingredient, exhibits almost no fluorescence in the free form. In a solution it forms complexes with protein molecules and exhibits increased fluorescence upon binding to proteins. The excitation maximum of ProteOrange in the complex with protein is at ~485 nm, whilst the emission maximum is at ~590 nm. The kit can be used with any fluorometer that has a suitable excitation source and detection channel. In general, the working range of measured protein concentration is limited by the equipment sensitivity. The recommended range of concentrations that can be measured is 10 ng/mL to 10 µg/mL for cuvette fluorometers and 100 ng/mL to 10 µg/mL for plate fluorometers. The kit contains ProteOrange fluorescent dye, buffer for preparation of dye working solution, and protein concentration standard (BSA, 2 mg/mL). To quantify the test protein, prepare the dye working solution and use it to dilute test samples and samples with known protein concentration to generate a calibration curve. Protein concentration standards are prepared using bovine serum albumin from the kit or (if available) experimental protein identical to that in the test samples. Incubate prepared samples for 10 min at 95 °C to denature proteins, allow them to cool down to room temperature and measure fluorescence intensity. Depending on sample volume, the number of measurements varies from 200 (sample volume 2.5 mL and assay volume 2 mL for a standard fluorometric cuvette) to 2000 (sample volume 250 µL and assay volume 200 µL for a 96-well plate).
