protein quantitation
tech note 2634
Quantitation of Proteins by the Bradford, Lowry, and BCA Protein
Assays Using the SmartSpec 3000 Spectrophotometer
™
Introduction
UV/visible spectrophotometry is among the most mature
analytical techniques used in the laboratory. Since the
commercialization of the spectrophotometer in the 1940s,
methods have been developed for its use in many areas of
scientific research. Today, spectrophotometers may be found
in laboratories focusing on all major areas of research, such
as analytical chemistry, functional genomics, and proteomics.
Up until 1999, most laboratory spectophotometers were large
instruments that relied on light emission and signal collection
technology that has not changed dramatically in 50 years.
Method
Instruments
Two different spectrophotometers were compared to the
SmartSpec 3000. One instrument, referred to as Spec A,
is a benchtop spectrophotometer. Spec A has a user
interface, operational features, and optics technologies that
are similar to those of the SmartSpec 3000.
The other instrument compared to the SmartSpec 3000 is a
high-end analytical spectrophotometer. This instrument,
Spec B, has a more complex optics system and a traditional
design. Spec B has a tungsten/deuterium lamp, a
photomultiplier detection system, and dual-beam optics.
Instruments like Spec B typically have a greater degree of
accuracy and precision over a wider wavelength and
absorbance range. In general, such instruments are used for a
wider range of applications because they have more complex
operating software and accessories packages.
New advances in optics technologies have allowed the
development of more affordable, compact spectrophotometers
for routine laboratory use. Bio-Rad’s SmartSpec 3000
spectrophotometer is one such benchtop instrument.
In this paper, results of colorimetric protein assays were
compared for 2 different benchtop spectrophotometers and
a more traditional high-end spectrophotometer. Protein
concentration standards were assayed using the following
protocols: Bradford,1 Lowry,2 and bicinchoninic acid (BCA).3
Standard curves were generated using each spectrophotometer,
and performance of the spectrophotometers was compared.
Assay Types
Three popular colorimetric protein assay methods were used
to compare spectrophotometer performance. They are:
•
•
•
1. Bradford M, Anal Biochem 72, 248–254 (1976)
2. Lowry O et al., J Biol Chem 193, 265–275 (1951)
3. Smith PK et al., Anal Biochem 150, 76–85 (1985)
Bio-Rad protein assay kit I (Bradford)
Bio-Rad DC protein assay kit I (modified Lowry)
Pierce Chemical Company’s BCA protein assay
The Bio-Rad protein assay kit is based on the Bradford
dye-binding procedure.1 The DC (detergent compatible)
protein assay kit is an assay for protein solubilized in
detergent. The reaction is similar to the well-documented
Lowry assay,2 modified to save time. The BCA protein assay
is a third colorimetric assay method.3
Assay Protocols
Standards of bovine gamma globulin (provided in the Bio-Rad
protein assay kits) were prepared at 7 concentrations, ranging
from 0.2 mg/ml to 2.0 mg/ml. For the Bio-Rad assay kits, all
7 solutions were assayed using each instrument. For the BCA
assay, 4 of the protein standard dilutions were assayed using
each instrument.
Standards were prepared in triplicate for each assay method.
The solutions were made and the assays performed
according to the manufacturer’s recommended protocol for
preparation of a standard curve.
The SmartSpec 3000 spectrophotometer.
After color development, the samples were transferred to 1.5 ml
disposable polycarbonate cuvettes. All 3 replicates of each
standard were read using the 3 spectrophotometers included
in the study. Each of the 3 protein assay methods used in this
performance evaluation was strictly timed to ensure that
changes in absorbance would not affect measurements.
A
1.0
SmartSpec 3000
Spec A
Spec B
A595
0.8
Results
Bio-Rad Protein Assay Kit I
The standard curves generated using each instrument have
very similar slopes (Figure 1A). The standard curve generated
by samples read in the SmartSpec 3000 falls between the
curves generated by the other spectrophotometers. The
correlation coefficient of the standard curve obtained using
each instrument is >0.98. The standard deviation for each
data point was <0.01 absorbance unit (AU).
Bio-Rad DC Protein Assay Kit I
For Bio-Rad’s DC protein assay, the results using the
SmartSpec 3000 and the high-end spectrophotometer,
Spec B, are very similar (Figure 1B). The correlation coefficient
calculated for each standard curve is >0.99. As with the
Bio-Rad protein assay, the standard deviation for all data points
from each instrument was <0.01 AU.
0.6
0.4
0.2
BCA Assay (Pierce Chemicals)
0.0
0.0
B
0.5
1.0
1.5
2.0
2.5
0.6
0.5
A750
0.4
0.3
0.2
0.1
0.0
0.0
C
0.5
1.0
1.5
2.0
2.5
2.0
Discussion
For assays with a desired absorbance range between 0.1 and
1.5 AU, the benchtop spectrophotometers compare favorably
to more traditional, high-end instruments. Although greater
precision and accuracy can be achieved using high-end
instruments for other applications, protein quantitation is
limited more by the sensitivity of the assays than by the
sensitivity of the spectrophotometer.
These results indicate that each of the spectrophotometers is
acceptable for accurate protein quantitation by any of the
3 popular methods. The SmartSpec 3000 spectrophotometer
performed similarly to the high-end instrument for all 3 assays.
1.5
A562
All 3 instruments showed values that were very close for the
BCA assay. Figure 1C shows that the differences in the
readings from each instrument were minor. The correlation
coefficient calculated from the standard curve >0.99 for each
instrument. The standard deviation calculated from data
obtained in the BCA assay was <0.02 AU for all 3 instruments.
1.0
Ordering Information
0.5
Catalog #
0.0
Spectrophotometer and Accessories
170-2501
SmartSpec 3000 Spectrophotometer
223-9950
Disposable Polystyrene Cuvettes, 3.5 ml, 100
223-9955
Disposable Polystyrene Cuvettes, 1.5 ml, 100
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Protein concentration, mg/ml
Fig. 1. A, Bio-Rad protein assay. B, Bio-Rad DC protein assay. C, BCA assay.
Standard curves are from triplicate standards at each concentration (error bars
showing standard deviation are smaller than the size of the symbols).
Description
Bio-Rad Protein Assays
500-0001
Bio-Rad Protein Assay Kit I, 450 standard 5 ml assays, bovine
gamma globulin standard, based on the method of Bradford
500-0111
Bio-Rad DC Protein Assay Kit I, 500 standard 4 ml assays,
bovine gamma globulin standard
500-0121
RC DC Protein Assay Kit I, reducing agent/detergent compatible
protein assay, includes DC protein assay kit I components and
RC reagents
Bio-Rad
Laboratories, Inc.
Life Science
Group
Bulletin 2634
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US/EG
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Sig 0101
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