Measurement of protein concentration in solution has since long been of essential importance in biological laboratories. The most commonly used ways to determine protein concentrations are the Bradford, Lowry and BCA methods. These methods, however, have definite limitations speaking of sensitivity, dynamic range and - in the case of the Bradford and Lowry assay - compatibility with reducing agents. Even though a majority of biological and biochemical laboratories has at least at some point applied one of these methods, the detection and determination of some protein concentrations might not be satisfactory, if possible at all.

Fluorescent labelling of proteins and subsequent detection of the label via fluorescence measurement has proven to be the method of choice in many types of assays.

The NanoOrange^® protein quantitation kit from Invitrogen is based on the binding of the reagent to detergent covered and hydrophobic regions of proteins. The dye is fluorescent only when bound. This assay provides a number of advantages over the traditional methods mentioned above. The bound or unbound reagent is stable for hours and there is little variability between protein types. It shows a high selectivity towards proteins with little interference by sample contaminants.

All reagents and buffers to be used come with the kit. For the standard curve BSA (bovine serum albumin) standard is diluted to concentrations ranging from 0,01 µg/ml to 10 µg/ml. The NanoOrange^® solution is added as described in the kit-protocol. The samples are excited at 485nm and the resulting emission is collected at 580nm. Black 96 well plates were used to get optimum signal-to-noise ratio.

Using the Twinkle microplate fluorometer it is possible to detect concentrations from 12 ng/ml to 10 µg/ml BSA in solution. The lowest concentration detected corresponds to the detection limit stated by the kit manufacturer. The Twinkle is ideally suited to measure protein concentrations with the NanoOrange^® protein quantitation kit.