Bradford Assay Performed on BMG LABTECH´s

FLUOstar Omega with new Evaluation Software

Franka Ganske, BMG LABTECH, Offenburg, Germany
E.J. Dell, BMG LABTECH, Durham, USA

Application Note 158 Rev. 08/2007

Materials and Methods

Fast and homogeneous assay to determine the protein
concentration of samples 96 well transparent microplates from Greiner, Frickenhausen, Germany
Dye shift can be followed with the spectrometer tool in the FLUOstar Omega, BMG LABTECH, Offenburg, Germany
new FLUOstar Omega Bovine Serum Albumin (BSA, Cat. No. A-9647) from Sigma-Aldrich,
Taufkirchen, Germany
New easy to use Omega evaluation software is introduced
Bradford Reagent (Cat. No. B6919) from Sigma-Aldrich, Taufkirchen,
Germany
The Bradford Reagent was bought ready to use. A stock solution of
bovine serum albumin in distilled water (10 mg/ml) was prepared
Introduction as a protein standard. For the measurements, a dilution of bovine
serum albumin was done starting with 1 mg/ml. Bradford reagent,
Determining the protein concentration of samples is a necessary and 290 µl, was pipetted into a transparent 96 well microplate. 10 µl of
often used method in biochemistry. Different colorimetric protein the protein dilution was added followed by mixing in the wells. After
assays have been developed. The most commonly used methods are the 5 min of incubation at room temperature, the plate was read at 595
Bradford assay, the Lowry assay and the BCA assay. In this application nm or in spectrum mode in the FLUOstar Omega.
note we demonstrate how to determine the protein concentration of
Instrument settings
samples by using the Bradford assay and the new FLUOstar Omega.
Number of flashes: 20
The Bradford assay is based on the binding of protein to a dye, leading to
Wave-length range: 380-800 nm (or discrete wavelength at 595 nm)
a shift in the absorbance maximum of the dye1. After creating a standard
Wave-length step width: 1 nm
curve of protein solutions with known concentrations, the protein
concentration of unknown samples can be calculated. The dye used for The progress of the measurement can be followed using the Current
the Bradford assay is Coomassie® Brilliant Blue G-250 (Figure 1). State Window (Figure 3).
Current State - Microplate View
O O
S BRADFORD IDs Bradford spectrum
O
1 2 3 4 5 6 7 8 9 10 11 12
+ A
H 3C N
B

H 3C CH3 C
H 3C O
O O D
S
N N ONa
H E
H 3C
F
Fig. 1: Chemical structure of Coomassie® Brilliant Blue G-250
G

The acidic solution of this dye has an absorbance maximum at 465 H

nm. After the addition of protein, hydrophobic amino acid residues and Well: H2
Absorbance values [mOD]

arginine residues bind to the dye. As a result, the absorbance maximum Wavelengths: 380 800 Options Timing Save Print Close Help

of the dye shifts from 465 nm to 595 nm (Figure 2).

Fig. 3: Current State Window of Bradford measurements monitoring spectra
Spectrum from 380 to 800 nm. Standards are indicated as red lines, blanks are
465 nm indicated as blue lines. Samples were run in triplicates.
0.900
0.800 595 nm Furthermore, during the measurement, it is possible to magnify a
0.700 selected well and get information about the measured values over
0.600 the spectral range (Figure 4).
0.500
OD

Current State - Well B4: S1

0.400
BRADFORD IDs Bradford spectrum
0.300
900
Values at cursor position:
0.200
Wavelength Meas. value
800
0.100 380 nm 344 mOD
700
0.000
600
400 450 500 550 600 650 700 750 800
Wavelength in nm 500
mOD

400

300
Fig. 2: The spectrum from unbound (red line) and protein bound (green line) 200
Coomassie® Brilliant Blue. After binding the absorbance maximum of 100
the dye shifts from 465 nm to 595 nm. 0
λ[nm] 400 500 600 700 800

The new FLUOstar Omega features high speed full spectrum absorbance. + - Save Print Close Help
The spectrometer tool allows measuring the whole spectrum of a sample
from 220-850 nm with selectable resolution in about 1 sec per well. In Fig. 4: Magnified current state picture of one selected well. The spectrum is
case the optimal wavelengths are already known, it is also possible to taken from 380 to 800 nm. The cursor can be set to any wavelength for
measure up to 8 pre-selected wavelengths at once. checking OD values during the measurement.
 Results and Discussion Conclusion

After measurements are taken, the data is transferred to the evaluation
software. Pre-defined templates can be used to do the calculations
needed instantaneously, i.e. average of raw data, blank correction,
performing curve fits and much more.
For the Bradford assay the blank corrected values are used for the
standard curve (Figure 5).
The Bradford assay was successfully performed on the FLUOstar
Omega (Fig. 6). According to the manufacturers protocol2 this
protein assay is linear in the range of 0.1 – 1.4 mg/ml. Because of
its homogeneous and fast nature, the assay is a preferred method to
determine the protein concentration of samples.

0.700
0.650
0.600
0.550
0.500
0.450
0.400
OD

0.350
0.300
0.250 Fig. 6: BMG LABTECH’s FLUOstar Omega
0.200
0.150 The FLUOstar Omega offers entirely new possibilities with its
0.100 spectrometer tool. A whole absorbance spectrum can be read in about
0.050
1 sec per well. Furthermore, the new Omega Evaluation Software
0.000
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 allows for the absorbance maximum or minimum to be recognized
BSA [mg/mL] at once after clicking on the spectral curve. Any wavelength can be
selected to give the values for the optical density in any well. The
Fig. 5: BSA standard curve (linear regression fit performed with the new Omega
speed of the spectrometer and the easy work-up in the software
Evaluation Software)
provide users with unmatched flexibility that can be used to optimize
absorbance settings for all experiments.
With the help of the standard curve the software calculates the protein
concentration for unknown samples automatically. If the option “path
length correction” is used, the measured data is multiplied by a factor References
that depends on the type of microplate and volume used. With the
help of this calculation, the data is normalized to a path length of 1 Bradford, MM. (1976) A rapid and sensitive method for the
1 cm, thereby allowing a comparison to be made between absolute quantification of microgram quantities of protein utilizing the
data obtained from a microplate reader with data obtained from a principle of protein-dye-binding. Anal Biochem. 72, 248-254.
cuvette-based spectrometer. 2 www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/B6916

Germany: BMG LABTECH GmbH Tel: +49 781 96968-0

Australia: BMG LABTECH Pty. Ltd. Tel: +61 3 59734744

France: BMG LABTECH SARL Tel: +33 1 48 86 20 20
Japan: BMG LABTECH JAPAN Ltd. Tel: +81 48 647 7217
UK: BMG LABTECH Ltd. Tel: +44 1296 336650
USA: BMG LABTECH Inc. Tel: +1 919 806 1735

Internet: www.bmglabtech.com info@bmglabtech.com