Measuring protein concentration is a common requirement
in biotechnology, food science and drug discovery laboratories.
The Bradford assay is very easy to run and produces
a dark blue color that is proportional to the protein
The Bradford procedure
is a colorimetric assay for protein concentration: the
level of blue color developed in the test solution is
proportional to the concentration of protein.
assay is based on the formation of a complex between
the dye Coomassie Brilliant Blue (see Figure 1) and
proteins in solution. The reagent is prepared under
acidic conditions, causing the dye to assume a doubly
protonated cationic structure. In this form, the dye
is red. When it binds to proteins, it is converted to
a stable anionic form, which is blue. The dye binds
particularly to basic (arginine) and aromatic amino
acids residues (Compton and Jones 1985). The protein-dye
complex causes a shift in the absorption maximum of
the dye from 465 nm (red) to 595 nm (blue) (forms
of Coomassie molecule).
1. Coomassie brilliant blue G250 dye is used in
the Bradford assay for measuring protein concentration
in solution. The dye turns blue in the presence
of proteins. A higher concentration of protein produces
a darker blue solution.
reagent requires no dilution and is accurate over a
concentration range from 10 to 1000 micrograms per ml.
Color development is rapid, with a five minute incubation
before the sample is read The light absorbed by the
solution is proportional to the protein concentration,
as described in the Beer-Lambert law.
Tissue, BM. (2000). Beer-Lambert
. Retrieved December 23, 2009 from http://www.files.chem.vt.edu/chem-ed/spec/beerslaw.html.
Bradford MM, A rapid and sensitive
method for the quantitation of microgram quantities
of protein utilizing the principle of protein-dye
binding, Anal Biochem, 72, 248, 254 (1976)
Compton SJ and Jones CG, Mechanism
of dye response and interference in the Bradford
protein assay, Anal Biochem 151, 369-374 (1985)
Sedmak JJ and Grossberg SE, A rapid,
sensitive and versatile assay for protein using
Coomassie Brilliant Blue G-250, Anal Biochem 79,
Spector T, Refinement of the Coomassie
blue method of protein quantitation. A simple and
linear spectrophotometric assay for less than or
equal to 0.5 to 50 micrograms of protein, Anal Biochem
86, 142-146 (1978)
set up for Bradford Assay: orange LED (LD200CWY3KH-30D),
L2F sensor (TSL235).
500 ml, available from Boston Bioproducts # BPA-200,
Thermo Scientific # 1856209
Graduated, Overall Length 5 1/2", Capacity
3.0 mL, Pk 100. For example Carolina Biological
item # 736984.
Graduated, Overall Length 6", Capacity 5.0
mL, Pk 100. For example Carolina Biological
item # 736986
cylinder, 10 ml
cylinder, 500 ml
12 ounce, 12
15 ml, 36
595 nm yellow-orange, LD200CWY3KH-30D
Light to Hz
1. Photometer set-up for Bradford assay.
Bradford reagent contains phosphoric acid and
is corrosive. Wear splash-resistant goggles and gloves.
Bradford reagent: Causes eye and skin burns. Harmful
if inhaled, absorbed through skin or swallowed. Do not
ingest. Wash thoroughly after handling
method uses skim milk to make the protein standards.
This is not the usual protocol: the most commonly used
protein standard is bovine serum albumin (BSA). (BSA
is a single protein, albumin, purified from the blood
of cows). Milk has two major advantages over BSA: milk
proteins are more complex and thus represent a more
natural protein sample than purified BSA, and milk is
cheap and readily available.
from cows typically contains about 8 grams of protein
in a 240 ml serving, with surprisingly little variation.
The protein concentration measured in milk is not as
accurate as the concentration of BSA in commercial standards,
but it is fine for determining relative concentrations
(actually the BSA standard only yields accurate results
if you are measuring pure BSA in your samples!).
the protein standards.
standards will be made by performing a series of 2-fold
and record the protein content of the skim milk.
plastic cups as follows, for micrograms/ml:
10 ml of milk into the 500 ml graduated cylinder.
the 10 ml graduated cylinder to transfer 10 ml.
water to the 366 ml mark on the 500 ml cylinder.
does not need to be exact, just get it as close
the bottom of the meniscus to
line up with the marks on the cylinder.
diluted milk has a protein concentration of approximately
1024 micrograms/ml if the milk has 8.0 grams in
400 ml of the milk solution into the cup labeled
200 ml of water into the cups labeled 512, 256,
128, 64, 32, 16, 0.
- For each of the following steps,
Use a clean beaker.
Mix gently. Use
a clean plastic spoon.
200 ml from the cup labeled 1024 into the cup labeled
200 ml from the cup labeled 512 into the cup labeled
200 ml from the cup labeled 256 into the cup labeled
200 ml from the cup labeled 128 into the cup labeled
200 ml from the cup labeled 64 into the cup labeled
200 ml from the cup labeled 32 into the cup labeled
not add any milk to the blank.
the sample so that its protein concentration
falls within the middle of the standards. For example,
the sample could be diluted 10-fold several times to
bring it into the correct range.
four test tubes as follows:
10 ml of the test sample into tube labeled 1.
9.0 ml of water into the tubes labeled 1/10, ;1/100
1.0 ml of the sample from the "1" tube
into the tube labeled 1/10.
the top of the tube, or plastic wrap to cover
a fresh pipet, pipet 1.0 ml from the sample labeled
1/10 into the tube labeled 1/100.
a clean pipet, pipet 1.0 ml from the sample labeled
1/100 into the tube labeled 1/1000.
the standard protein solutions:
8 test tubes (15 ml) as follows:
16, 32, 64, 128, 256, 512 and 1024.
- These are the concentrations
of the protein standards in micrograms per ml.
- These will be used to
make a standard curve.
the test sample (the unknown)
4 test tubes (15 ml) as follows:
1/10, 1/100 and 1/1000.
- These are suggested
dilutions of the test sample.
- The test sample is diluted
to bring the protein concentration within the
linear range of the assay.
- Use your judgment to
determine which dilutions to use – it
may involve some trial and error.
4 ml Bradford reagent to the 15 ml test tubes labeled
the protein standards to the Bradford reagent:
50 microliters from the standards (labeled 0,
16, 32, 64, 128, 256, 512 and 1024) into the test
tubes with Bradford reagent.
plastic wrap to cover the top, if needed.
the diluted test sample to the Bradford reagent:
50 microliters from the diluted samples (labeled
1X, 10X, 100X and 1000x) into the test tubes with
the solutions into cuvettes.
the Bradford reagent/protein complexes into cuvettes,
3 ml per cuvette.
a pipet to transfer the solutions.
you can carefully pour the solution into the appropriate
cuvette should be about ¾ full.
readings for the blank.
- Turn on the photometer.
- Insert the blank into the photometer.,
blank is the sample labeled 0 in the standards.
It has Bradford reagent without any protein.
Adjust the brightness of the LED to get a reading
between 150 and 200 uW/cm2
the sensor reading for the blank solution.
value is called Io, since it
is the reading with no protein in the sample.
readings for the standards and samples.
each cuvette in the photometer and record the
irradiance reading. This is called "I".
reading each cuvette once, repeat all of the readings
(blank, standards, and samples) two more times.
Io/I for each reading.
= the average sample reading
= the average reading for the blank solution.
value is the absorbance and
is proportional to the concentration
the concentration of the standards on the x-axis
and the absorbance (log Io/I) on the y-axis.
the graph to estimate the concentration of protein
in the unknown sample.
a horizontal line on the graph showing the value
of log Io/I for the sample.
the concentration on the x-axis that corresponds
to this absorbance.
for the dilution factor.
example, if the unknown sample has a concentration
of 400 micrograms per ml after it was diluted
100 X, the original solution must have been
100 times more concentrated. See Equation 5.
the value obtained for the protein concentration
of the unknown sample or samples.
Objective: Determine protein concentration in dairy products.
Terms and definitions
colorimetric assay: a procedure (assay) that depends on a change in color in the solution.
anion: negatively charged atom or molecule
cation: positively charged atom or molecule
protonated: having an extra proton (positive charge)
amino acids: building blocks of proteins