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Standard protein quantitation is an involved process, often including serial dilution of standards and plotting of sample values on a curve of values obtained from the dilution series. Through integration of fluorescent reagents and a low cost fluorescent instrument programmed with a curve fitting algorithm, we have developed a fast and accurate method of protein quantitation that eliminates both calibration standard preparation and manual calculation. In the Quant-iT Protein Assay for the Qubit fluorometer, a fluorescent reagent is mixed with a dilution buffer, and the mixture is added to only three premixed calibration standards and to all samples. Instrument readings of the three standards are automatically fit to a sigmoidal curve, allowing concentration of the sample in μg/mL to be the only output. The assay is accurate for initial sample concentrations from 12.5 μg/mL to 5 mg/mL and exhibits low protein-to-protein variation compared to other protein quantitation methods. The assay is performed at room temperature, and the signal is stable for three hours. Common contaminants, such as reducing agents, salts, free nucleotides, amino acids, solvents, buffers, and DNA or RNA, but not detergents, are well tolerated in the assay. Samples in standard protein 1D, but not 2D, gel loading buffer may be diluted and then quantitated in the assay. The entire assay takes about 30 min to perform.