The field of proteomics has expanded dramatically in recent years with research on a whole host of organisms. Techniques such as two dimensional difference gel electrophoresis (2D DIGE) require the accurate quantitation of protein, as up to three labelled samples can be loaded on a single gel for comparison. Assay methods for determining protein quantitation include absorbance at 280 nm(1), the Bradford assay(2), Lowry assay(3), BCA method(4) and more sensitive assays such as Fluoroprofile® (Sigma-Aldrich) and NanoOrange® (Molecular Probes®)5(). The measurement of solution absorbance at 280 nm (A280) has problems with variability between samples and interference from nucleic acids and other contaminants. Detergents and reducing agents can cause problems with the other assays mentioned above – these agents are present in samples for 2D gel electrophoresis. Fluorescent methods are more sensitive for quantitating proteins than absorbance methods. The NanoOrange® protein quantitation assay from Molecular Probes® is a highly sensitive assay with the useful range being between 100 ng/mL and 10 µg/mL for fluorescence-based microplate readers. The spectra of the NanoOrange® reagent is given in Figure 1.
Fig. 1: Excitation and emission spectra of the NanoOrange® reagent. Copyright Invitrogen Corporation. Used with permission.
The sensitivity persists over 6 hours and there is low protein-to-protein variability. Tolerance levels are as high as 100 mM for reducing agents such as DTT and β-mercaptoethanol and 0.01% for detergents such as SDS. This application note investigates a method for using the NanoOrange® assay on the BMG LABTECH FLUOstar OPTIMA microplate reader.
Materials and Methods
The following materials were supplied by the manufacturers as detailed:
A description of the preparation for microplate assays has been noted earlier
The working NanoOrange® reagent was made by diluting reagent A 500-fold in a 1:10 dilution of diluent B. A stock BSA solution at 10 µg/mL was made by diluting 1:200 in working NanoOrange® reagent. A serial dilution of the stock BSA solution was performed, yielding further concentrations ranging from 0.2 µg/mL to 10 µg/mL. The blank solution was working NanoOrange® reagent alone. Dilutions of samples were made in working NanoOrange® reagent (for example, 1:100, 1:500, 1:1000). Following denaturation of the standards and samples, 100 µL of each was placed into a microplate for measurement. The prepared 96-well plate was inserted into the instrument and read in fluorescence mode with the following parameters:
|Excitation filter:||485 BP12|
|Gain:||plate assessed, well with the highest intensity selected|
|Optics:||2mm combination optic|
|Number of cycles:||1|
|Number of flashes per well:||10|
The data was evaluated using the BMG LABTECH FLUOstar evaluation package. The average value of the blank measurement was subtracted from the measurements and the standard curve was plotted.
Results and Discussion
A four parameter fit was performed on the data which yielded a very high R-value of 0.9998. (Figure 3).
Fig.3: BSA standard curve obtained with the NanoOrange® Protein Quantitation Kit. A BSA concentration range of 0.2-10 µg/mL is used. A gain of 1967 was optimized on the highest protein concentration.
The small concentration range was also measured separately with optimized gain on the highest protein concentration (Figure 4).
Fig.4: Small concentration range BSA standard curve obtained with the NanoOrange® Protein Quantitation Kit. A gain of 2721 was optimized on the highest protein concentration (12 replicates).
A linear relationship between protein concentration and fluorescence units is obtained indicating that small concentrations of protein in samples can be determined using the FLUOstar OPTIMA. Protein concentrations below 200 ng/mL can be determined with the help of the 3 mm fluorescence optic.
The NanoOrange® Protein Quantitation Kit can be easily adapted for use with a fluorescent microplate reader such as the FLUOstar OPTIMA and used in a high throughput manner. NanoOrange® can also be performed on other BMG LABTECH microplate readers, including the FLUOstar and POLARstar Omegas, as well as the NOVOstar and PHERAstar.
The FLUOstar OPTIMA is a flexible multidetection microplate reader that has four different measurement modes: fluorescence intensity, time-resolved fluorescence, luminescence and absorbance. The instrument allows measurement of endpoint or slow and high kinetics at a user-defined temperature and can be easily equipped with injectors.