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BIO-101: Lab 4. Bradford assay

Why protein estimation is required?

Protein is, an essential bio molecule, made up of amino acid monomeric units. A variety of attributes have
been assigned to proteins like protein constitutes different components of cell (plasma membrane), and
performs different functions (enzymes). Protein makes myriad of other substances like hormones,
antibodies, transporters, muscle fibers, lens protein of the eye, feathers, spider webs, rhinoceros horn, milk
proteins, antibiotics, mushroom poisons etc. and having distinct biological activities. To characterize
protein, accurate determination of protein concentration is utterly required.
Protein estimation tells about the protein content in nutritious foods (e.g. protein powders etc).
Protein estimation indicates the consequence of disease like haemoglobin level falls in certain blood
related disease (anaemia).
Protein estimation is used as one of the tool in forensic science (crime related investigation).
Protein estimation is required prior to the analysis of Protein- protein interaction or protein ligand
interaction.
Protein estimation is the preliminary step during whole protein profiling which is being used to
compare disease status and drug treatments.
Protein estimation is one of the essential requirements in protein purification.

How can I estimate the protein in a sample?

Research on protein estimation samples has long history. Different methods have been developed and
modified time to time to obtain accuracy, minimization of time and funds. Protein estimation can be done by
Biuret reaction, Folin reaction (Lowry protein assay), Bicinchoninic acid (BCA) protein assay and Bradford
assay etc. Biuret, BCA and Lowry protein assays involve Copper-based chemistry; however Bradford assay
involves Dye-based chemistry. Another very rapid method is a direct method is used for proteins which are
rich in aromatic amino acid, in this method concentration is determined by taking the absorption at 260/280
nm. Amino acids containing aromatic side chains (i.e., tyrosine, phenylalanine and tryptophan) exhibit
strong UV-light absorption. Consequently, proteins and peptides absorb UV-light in proportion to their
aromatic amino acid content and total concentration.
Once an absorptivity coefficient has been established for a given protein (with its fixed amino acid
composition), the proteins concentration in solution can be calculated from its absorbance. Estimation of
protein concentration by UV-light absorption is not accurate for complex protein solutions (e.g., cell lysates)
because the composition of proteins with different unknown absorption coefficients is not known. In
addition, proteins are not the only molecules that absorb UV-light and complex solutions will usually
contain compounds such as nucleic acid that will interfere with determination of protein concentration.
Therefore one has to select an appropriate method to get the exact concentration.

What is Bradford assay?

Bradford assay was developed by MARION M. BRADFORD in 1976. This assay is very reproducible,
sensitive and rapid with the dye binding process virtually complete in approximately 2 min with good color
stability for 1 hr.

What is the principle behind Bradford assay?

This assay works on the principle of color change of dye during its binding to the protein. Acidic solution
(see the preparation of reagent) of dye (Coomassie Brilliant Blue G-250) shifts its absorption maximum
from 465 to 595 after binding with protein. Electrostatic and hydrophobic interaction occurs in between dye
and protein molecule. These interactions stabilize the anionic form of dye which produces blue color (one
can tell whether protein is in the sample by visualizing the color). The assay is useful since the extinction
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coefficient of a dye-albumin complex solution is constant over a 10-fold concentration range (i.e. 10
microgram to 100 microgram).

How to prepare Bradford Reagent?

Coomassie Brilliant Blue G-250 (100 mg) is dissolved in 50 ml 95% ethanol. To this solution 100 ml 85%
(w/v) phosphoric acid is added. The resulting solution was diluted to a final volume of 1 litre. The Bradford
reagent should be of light brown in color. Filtration may be repeated to rid the reagent of blue components.

What is the procedure of Bradford assay?

1. Start the spectrophotometer and let the lamp warm up for some time.
2. Select a wavelength (e.g. 595 nm).
3. Prepare standard curve by taking known concentration of BSA (Bovine serum albumin).
4. First set the appropriate blank which would be the solvent + Bradford reagent, ZERO (0.00) will be
on the screen.
5. Now start taking the absorbance of known and unknown concentrations of protein by using cuvette
in spectrophotometer.
6. Prepare the standard curve (concentration/ amount taken (it is not necessary that is should be in
Molarity) vs absorbance) and determine the concentration of unknown.

Why standard curve is needed for protein estimation?

Standard curves represent the relationship between two quantities. A standard curve is used to quantify (to
know the amount/concentration of sample) the unknown sample. In this, multiple samples with known
concentration are measured, which allows the concentration to be determined for unknown samples by
interpolating the graph.

Why to set blank?

A spectrophotometer must be calibrated at each wavelength so that the solvent of the solution absorbs no
light energy (i.e., transmittance is 100%). A blank is used to calibrate the machine. The blank contains
everything except the specific compound for which the absorption is being determined. After blanking
using the blank, the test solution is inserted, and the absorbance is noted. Here, absorbance is proportional
to solute concentration.

Why we use BSA as a standard, can we use other proteins?

Because proteins differ in their amino acid compositions, each one responds somewhat differently in each
type of protein assay. Therefore, the best choice for a reference standard is a purified, known concentration
of the most abundant protein in the samples. This is usually not possible to achieve, and it is seldom
convenient or necessary. In many cases, the goal is merely to estimate the total protein concentration, and
slight protein-to-protein variability is acceptable.
If a highly purified version of the protein of interest is not available or it is too expensive to use as the
standard, the alternative is to choose a protein that will produce a very similar color response curve in the
selected protein assay method and is readily available to any laboratory at any time. Generally, bovine serum
albumin (BSA) works well for a protein standard because it is widely available in high purity and relatively
inexpensive. Alternatively, bovine gamma globulin (BGG) is a good standard when determining the
concentration of antibodies because BGG produces a color response curve that is very similar to that of
immunoglobulin G (IgG).
For greatest accuracy in estimating total protein concentration in unknown samples, it is essential to include
a standard curve each time the assay is performed. This is particularly true for the protein assay methods that
produce non-linear standard curves. Deciding on the number of standards and replicates used to define the
standard curve depends upon the degree of non-linearity in the standard curve and the degree of accuracy
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required. In general, fewer points are needed to construct a standard curve if the color response is linear.
Typically, standard curves are constructed using at least two replicates for each point on the curve.
(http://www.piercenet.com/method/overview-protein-assays).

How to Interpolate on a Complete Standard Curve?

Consider the following example involving a set of six standard points (0, 250, 500, 1000, 1500 and 2000
g/ml). The same point-to-point relationship for this set of standards is plotted in Figures 1 and 2, but
different methods were used to determine a mathematical equation that describes this absorbance-
concentration relationship. If a test sample results in an absorbance of 0.6, then one must interpolate
between values obtained for the 500 and 1000 g/ml standards to determine the test sample concentration.
(This part is adopted from Thermo Scientific, TECH TIP # 57)
On a merely graphical basis, one can see that the test sample must be ~ 650 g/ml.
The line segment AB in the point-to-point graph is described by the equation y = 0.0006(x + 333.33).
Solving this equation for x gives x = 1666.7y - 333.33.
If y = 0.6, then x = 667 g/ml
Many researchers plot a linear regression for the entire set of standards, assuming that the overall relationship between
concentration and absorbance is best described by a straight line. The thick, straight line in Figure 1 is the linear
regression that best describes the entire set of standard points (R
2
= 0.9355).
The equation for this line is y = 0.0005x + 0.1493.
Solving for x gives x = 1770.4y - 207.91.
For y = 0.6, x = 854 g/ml.
As is obvious from the graph, this linear regression does not provide a good basis for interpolating test sample
concentrations relative to assay results for the standards. The best method for interpolation in this example is by
reference to a curvilinear regression, in this case a 3-parameter polynomial equation that can be calculated by most
plate reader software or standard spreadsheet programs.
When solved for x, the 3-parameter equation (R2 = 0.9997) is x = 1372.2y3 - 769.01y2 +1004.2y - 2.9373.
For y = 0.6, x = 619 g/ml.
On a merely graphical basis (Figure 2), this method can be seen as the most accurate for interpolating the test sample.
If one had included a 750 g/ml standard, it surely would have occurred on this 3-parameter trend line rather than on
the straight line segment AB or on the linear regression displayed in Figure 1.



Figure 1 Figure 2

Details of Figure: Figure 1. Example standard curve involving six points. The thin line is a point-to-point graph through the
plotted standards. The thick line is linear regression for the entire set of standard points. Dashed lines represent interpolations for a
test sample having absorbance 0.6.
Figure 2. Example standard curve involving six points. The thin line is a point-to-point graph through the plotted standards. The
thick line is a 3-parameter regression for the entire set of standard points. Dashed lines represent interpolations for a test sample
having absorbance 0.6.

Few Important Points to consider:

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Unfortunately, no protein assay method exists that is either perfectly specific to proteins (i.e., not
affected by any nonprotein components) or uniformly sensitive to all protein types (i.e., not affected
by differences in protein composition). Therefore, successful use of protein assays involves selecting
the method that is most compatible with the samples to be analyzed, choosing an appropriate assay
standard, and understanding and controlling the particular assumptions and limitations that remain.

Ideally, the absorbance readings obtained in your standard curve should be linearly proportional to
the protein concentration (which means that there are limits as to how much protein you can use; too
much protein will give absorbance readings too high to be meaningful). In an actual experiment, you
would run replicates for both the standard curve and the unknown samples, so as to be able to assess
the uncertainty in your data.

The Bradford Reagent is compatible with reducing agents. Reducing agents are often used to
stabilize proteins in solution. Other protein assay procedures (Lowry and BCA) are not compatible
with reducing agents. The Bradford Reagent should be used in place of these protein assays if
reducing agents are present. However, the Bradford Reagent is only compatible with low
concentrations of detergents. If the protein sample to be assayed has detergent(s) present in the
buffer, it is suggested to use the BCA protein determination procedure.

The Beer-Lambert Law may not be applicable to all solutions since solutions can ionize/polymerize
at higher concentrations, or precipitate to give a turbid suspension that may increase or decrease the
apparent absorbance. Further, the Beer-Lambert Law is most accurate between Abs of 0.05 to 0.80.
Above 0.80, the measured Abs tends to underestimate the real Abs. Below 0.02 Abs many
instruments are not accurate.



Figure 3: CBB G-250 Structure Figure 4: CBB reacts with protein

References:

http://www.ruf.rice.edu/~bioslabs/methods/protein/bradford.html
Anal Bioanal Chem (2008) 391:391403. DOI 10.1007/s00216-008-1996-x
Bradford MM (1976) Anal Biochem 72:248254
http://chem.winthrop.edu/faculty/grossoehme/link_to_webpages/courses/chem525/methods.pdf
Lehninger, principles of biochemistry.
http://www.piercenet.com/product/bovine-serum-albumin-bsa-standards
http://www.piercenet.com/method/overview-protein-assays
Thermo-scientific protein assay handbook
http://www.nature.com/protocolexchange/protocols/617
http://www.qcbio.com/pierce/23236.htm