For limited sample quantities, protein quantification is usually performed by measuring the absorption at 280 nm in a microvolume spectrophotometer which allows the quantification of a total protein using as little as 1 µL of sample. To calculate the concentration of mixtures of proteins using absorbance at 280 nm the relationship 1 O.D. unit = 1 mg/mL of protein is often used. However, for known purified proteins dividing the absorbance by the absorbance coefficient of the protein will give much better results.
Even though a majority of biological and biochemical laboratories has applied one of these methods the detection and determination of some protein concentrations might not be satisfactory or possible at all. Fluorescent labelling of proteins and subsequent detection of the label via fluorescence measurement has proven to be the method of choice for many types of assays. In this case a microplate fluorescence reader is usually the instrument of choice.
Application Notes related to protein quantification
SingleQuant protein quantification assay with the Colibri Microvolume protein quantification based on Amido Black (SingleQuant Assay) with the Colibri Microvolume Spectrophotometer
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