166

Molecular Probes NanoOrange assay performed on a BMG LABTECH microplate reader

Tracey Madgett, Faculty of Health and Life Sciences, University of the West of England, 01/2008
  • 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

Introduction

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.

Fig. 1: Excitation and emission spectra of the NanoOrange reagent (Copyright Invitrogen Corporation. Used with permission).

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

Reagents:

  • 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:



Excitation filter: 485-12
Emission filter: 570-10
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).

Fig. 2: 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 3).

Fig. 3: 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 BMG LABTECH microplate reader. 

Conclusion

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
Newsletter Sign-up