The Bradford Method for Protein Quantitation
Nicholas J. Kruger
1. Introduction
A rapid and accurate method for the estimation of protein concentration is essential
in many fields of protein study. An assay originally described by Bradford (1) has
become the preferred method for quantifying protein in many laboratories. This technique
is simpler, faster, and more sensitive than the Lowry method. Moreover, when
compared with the Lowry method, it is subject to less interference by common reagents
and nonprotein components of biological samples (see Note 1).
The Bradford assay relies on the binding of the dye Coomassie Blue G250 to protein.
Detailed studies indicate that the free dye can exist in four different ionic forms
for which the pKa values are 1.15, 1.82, and 12.4 (2). Of the three charged forms of the
dye that predominate in the acidic assay reagent solution, the more cationic red and
green forms have absorbance maxima at 470 nm and 650 nm, respectively. In contrast,
the more anionic blue form of the dye, which binds to protein, has an absorbance maximum
at 590 nm. Thus, the quantity of protein can be estimated by determining the
amount of dye in the blue ionic form. This is usually achieved by measuring the absorbance
of the solution at 595 nm (see Note 2).
The dye appears to bind most readily to arginyl and lysyl residues of proteins (3,4).
This specificity can lead to variation in the response of the assay to different proteins,
which is the main drawback of the method (see Note 3). The original Bradford assay
shows large variation in response between different proteins (5–7). Several modifications
to the method have been developed to overcome this problem (see Note 4). However,
these changes generally result in a less robust assay that is often more susceptible
to interference by other chemicals. Consequently, the original method devised by
Bradford remains the most convenient and widely used formulation. Two types of assay
are described here: the standard assay, which is suitable for measuring between 10 and
100 μg of protein, and the microassay, which detects between 1 and 10 μg of protein.
The latter, although more sensitive, is also more prone to interference from other compounds
because of the greater amount of sample relative to dye reagent in this form of
the assay.
2. Materials
1. Reagent: The assay reagent is made by dissolving 100 mg of Coomassie Blue G250 in
50 mL of 95% ethanol. The solution is then mixed with 100 mL of 85% phosphoric acid
and made up to 1 L with distilled water (see Note 5).
The reagent should be filtered through Whatman no. 1 filter paper and then stored in an
amber bottle at room temperature. It is stable for several weeks. However, during this time
dye may precipitate from solution and so the stored reagent should be filtered before use.
2. Protein standard (see Note 6). Bovine γ-globulin at a concentration of 1 mg/mL
(100 μg/mL for the microassay) in distilled water is used as a stock solution. This should
be stored frozen at –20oC. Since the moisture content of solid protein may vary during
storage, the precise concentration of protein in the standard solution should be determined
from its absorbance at 280 nm. The absorbance of a 1 mg/mL solution of γ-globulin, in a
1-cm light path, is 1.35. The corresponding values for two alternative protein standards,
bovine serum albumin and ovalbumin, are 0.66 and 0.75, respectively.
3. Plastic and glassware used in the assay should be absolutely clean and detergent free.
Quartz (silica) spectrophotometer cuvettes should not be used, as the dye binds to this
material. Traces of dye bound to glassware or plastic can be removed by rinsing with
methanol or detergent solution.
3. Methods
3.1. Standard Assay Method
1. Pipet between 10 and 100 μg of protein in 100 μL total volume into a test tube. If the
approximate sample concentration is unknown, assay a range of dilutions (1, 1:10, 1:100,
1:1000). Prepare duplicates of each sample.
2. For the calibration curve, pipet duplicate volumes of 10, 20, 40, 60, 80, and 100 μL of
1 mg/mL γ-globulin standard solution into test tubes, and make each up to 100 μL with
distilled water. Pipet 100 μL of distilled water into a further tube to provide the reagent
blank.
3. Add 5 mL of protein reagent to each tube and mix well by inversion or gentle vortexmixing.
Avoid foaming, which will lead to poor reproducibility.
4. Measure the A595 of the samples and standards against the reagent blank between 2 min
and 1 h after mixing (see Note 7). The 100 μg standard should give an A595 value of about
0.4. The standard curve is not linear, and the precise absorbance varies depending on the
age of the assay reagent. Consequently, it is essential to construct a calibration curve for
each set of assays (see Note 8).
3.2. Microassay Method
This form of the assay is more sensitive to protein. Consequently, it is useful when
the amount of the unknown protein is limited (see also Note 9).
1. Pipet duplicate samples containing between 1 and 10 μg in a total volume of 100 μL into
1.5-mL polyethylene microfuge tubes. If the approximate sample concentration is
unknown, assay a range of dilutions (1, 1:10, 1:100, 1:1000).
2. For the calibration curve, pipet duplicate volumes of 10, 20, 40, 60, 80, and 100 μL of
100 μg/mL γ-globulin standard solution into microfuge tubes, and adjust the volume to
100 μL with water. Pipet 100 μL of distilled water into a tube for the reagent blank.
3. Add 1 mL of protein reagent to each tube and mix gently, but thoroughly.
4. Measure the absorbance of each sample between 2 and 60 min after addition of the protein
reagent. The A595 value of a sample containing 10 μg γ-globulin is 0.45. Figure 1 shows
the response of three common protein standards using the microassay method.
4. Notes
1. The Bradford assay is relatively free from interference by most commonly used biochemical
reagents. However, a few chemicals may significantly alter the absorbance of the reagent
blank or modify the response of proteins to the dye (Table 1). The materials that are
most likely to cause problems in biological extracts are detergents and ampholytes (3,8).
These can be removed from the sample solution by gel filtration, dialysis, or precipitation
of protein with calcium phosphate (9,10). Alternatively, they can be included in the reagent
blank and calibration standards at the same concentration as that found in the sample.
The presence of base in the assay increases absorbance by shifting the equilibrium of the
free dye toward the anionic form. This may present problems when measuring protein
content in concentrated basic buffers (3). Guanidine hydrochloride and sodium ascorbate
compete with dye for protein, leading to underestimation of the protein content (3).
2. Binding of protein to Coomassie Blue G250 may shift the absorbance maximum of the
blue ionic form of the dye from 590 nm to 620 nm (2). It might, therefore, appear more
sensible to measure the absorbance at the higher wavelength. However, at the usual pH of
the assay, an appreciable proportion of the dye is in the green form (λmax = 650 nm) which
interferes with absorbance measurement of the dye–protein complex at 620 nm. Measurement
at 595 nm represents the best compromise between maximizing the absorbence due
to the dye–protein complex while minimizing that due to the green form of the free dye (2–4;
but see also Note 9).
3. The dye does not bind to free arginine or lysine, or to peptides smaller than about 3000 Da
(4,11). Many peptide hormones and other important bioactive peptides fall into the latter category,
and the Bradford assay is not suitable for quantifying the amounts of such compounds.
4. The assay technique described here is subject to variation in sensitivity between individual
proteins (see Table 2). Several modifications have been suggested that reduce this
variability (5–7,12). Generally, these rely on increasing either the dye content or the pH of
the solution. In one variation, adjusting the pH by adding NaOH to the reagent improves
the sensitivity of the assay and greatly reduces the variation observed with different proteins
(7). (This is presumably caused by an increase the proportion of free dye in the blue
form, the ionic species that reacts with protein.) However, the optimum pH is critically
dependent on the source and concentration of the dye (see Note 5). Moreover, the modified
assay is far more sensitive to interference from detergents in the sample.
Particular care should be taken when measuring the protein content of membrane fractions.
The conventional assay consistently underestimates the amount of protein in membrane-
rich samples. Pretreatment of the samples with membrane-disrupting agents such
as NaOH or detergents may reduce this problem, but the results should be treated with
caution (13). A useful alternative is to precipitate protein from the sample using calcium
phosphate and remove contaminating lipids (and other interfering substances, see Note 1)
by washing with 80% ethanol (9,10).
5. The amount of soluble dye in Coomassie Blue G250 varies considerably between sources,
and suppliers’ figures for dye purity are not a reliable estimate of the Coomassie Blue
G250 content (14). Generally, Serva Blue G is regarded to have the greatest dye content
and should be used in the modified assays discussed in Note 4. However, the quality of the
dye is not critical for routine protein determination using the method described in this
chapter. The data presented in Fig. 1 were obtained using Coomassie Brilliant Blue G
(C.I. 42655; product code B-0770, Sigma-Aldrich).
6. Whenever possible the protein used to construct the calibration curve should be the same
as that being determined. Often this is impractical and the dye response of a sample is
quantified relative to that of a “generic” protein. Bovine serum albumin (BSA) is commonly
used as the protein standard because it is inexpensive and readily available in a
pure form. The major argument for using this protein is that it allows the results to be
compared directly with those of the many previous studies that have used bovine serum
albumin as a standard. However, it suffers from the disadvantage of exhibiting an unusually
large dye response in the Bradford assay, and thus, may underestimate the protein
content of a sample. Increasingly, bovine γ-globulin is being promoted as a more suitable
general standard, as the dye binding capacity of this protein is closer to the mean of those
proteins that have been compared (Table 2). Because of the variation in response between
different proteins, it is essential to specify the protein standard used when reporting measurements
of protein amounts using the Bradford assay.
7. Generally, it is preferable to use a single new disposable polystyrene semimicrocuvette
that is discarded after a series of absorbance measurements. Rinse the cuvette with reagent
before use, zero the spectrophotometer on the reagent blank and then do not remove the
cuvette from the machine. replace the sample in the cuvette gently using a disposable
polyethylene pipet.
8. The standard curve is nonlinear because of problems introduced by depletion of the amount
of free dye. These problems can be avoided, and the linearity of the assay improved, by
plotting the ratio of absorbances at 595 and 450 nm (15). If this approach is adopted, the
absolute optical density of the free dye and dye–protein complex must be determined by
measuring the absorbance of the mixture at each wavelength relative to that of a cuvette
containing only water (and no dye reagent). As well as improving the linearity of the
calibration curve, taking the ratio of the absorbances at the two wavelengths increases the
accuracy and improves the sensitivity of the assay by up to 10-fold (15).
9. For routine measurement of the protein content of many samples the microassay may be
adapted for use with a microplate reader (7,16). The total volume of the modified assay is
limited to 210 μL by reducing the volume of each component. Ensure effective mixing of the
assay components by pipetting up to 10 μL of the protein sample into each well before adding
200 μL of the dye reagent. If a wavelength of 595 nm cannot be selected on the microplate
reader, absorbance may be measured at any wavelength between 570 nm and 610 nm. However,
absorbance measurements at wavelengths other than 595 nm will decrease the sensitivity
of response and may increase the minimum detection limit of the protocol.
10. For studies on the use of the Bradford assay in analyzing glycoproteins, see Note 9 in
Chapter 3.
References
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