- NanoOrange® Assay for protein quantitation performed on a BMG LABTECH microplate reader
- High and small concentration range evaluated
- Kit can easily be adapted for high throughput measurements
Table of contents
New developments in the field of proteomics require higher sensitivity and specificity in determining protein concentration. Fluorescent dyes such as NanoOrange® fulfil these requirements since being capable of measuring down to a concentration of 10 ng/ml in a high volume reaction (2.5 ml). Performing the assay in microplates reduces the reaction volume to 200 µl.
For verification of the microplate-based quantification assay, dilutions of bovine serum albumin (BSA) were measured on a multi-mode reader by BMG LABTECH. The detected fluorescence intensity was linear in a concentration range from 200 ng/ml to 10 µg/ml.
Consequently, the microplate reader enables miniaturization and increased throughput of the NanoOrange® protein quantification assay while being more sensitive than competing absorbance assays.
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, the Bradford assay, Lowry assay, BCA method and more sensitive assays such as Fluoroproﬁle® (Sigma-Aldrich) and NanoOrange® (Molecular Probes®).
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 spectrum of the NanoOrange® reagent is given in Figure 1.
Materials & Methods
The following materials were supplied by the manufacturers as detailed:
- NanoOrange® Protein Quantitation Kit
- MJ Research PTC200 Peltier Thermal Cycler
- BMG LABTECH microplate reader
- Microplates, black 96 well (Greiner Bio-One)
- General laboratory consumables included pipette tips and microcentrifuge tubes
- NanoOrange® Protein Quantitation reagent A
- NanoOrange® Protein Quantitation diluent B
- Bovine Serum Albumin (BSA) Standard (2 mg/mL)
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:
|Gain:||plate assessed, well with the highest intensity selected|
|Number of cycles:||1|
|Number of flashes per well:||10|
The data was evaluated using the BMG LABTECH MARS data analysis software. The average value of the blank measurement was subtracted from the measurements and the standard curve was plotted.
Results & Discussion
A four parameter ﬁt was performed on the data which yielded a very high R-value of 0.9998. (Figure 2).
The small concentration range was also measured separately with optimized gain on the highest protein concentration (Figure 3).
A linear relationship between protein concentration and ﬂuorescence units is obtained indicating that small concentrations of protein in samples can be determined using the BMG LABTECH microplate reader.
The NanoOrange® Protein Quantitation Kit can be easily adapted for use with a ﬂuorescent microplate reader such as BMG LABTECH´s and used in a high throughput manner.
The instrument allows measurement of endpoint or slow and high kinetics at a user-deﬁned temperature and can be easily equipped with injectors.
Nano Orange is a registered trademark of ThermoFisher Scientific