PROTEIN ASSAY BY BRADFORD METHOD Measurement of Protein Concentration

What is Bradford Assay?

• Colorimetric Assay Reagent
• Spectrophotometer blank
• Quantitative Assay
• To measure the TOTAL protein conc. in a soln. Serial dilutions

Principle of Bradford Assay

• depends on Coomassie Brilliant Blue molecule
o Unstable Cationic state (reddish-brown)
ACIDIC
o Stable Anionic state (blue) BASIC

y=mx + b
𝑦−𝑏
x= 𝑚

Nonpolar region Hydrophobic pockets

Bovine Serum Albumin
• aka BSA or "Fraction V"
Basic
• serum albumin protein isolated from cows
• stabilize some restriction enzymes during
digestion of DNA
• prevent adhesion of the enzyme to reaction
tubes, pipet tips, and other vessels.
Nonpolar region Hydrophobic pockets
o universal blocking reagent in many
applications—because BSA does not
affect the functions of other proteins
UNSTABLE CATIONIC STABLE ANIONIC (enzymes) that do not need it for
stabilization.
• commonly used to determine the quantity of other
proteins, by comparing an unknown quantity of
protein to known amounts of BSA in, for example,
the Bradford Protein Assay.
o used because of its stability to
increase signal in assays,
o its lack of effect in many biochemical
reactions
o low cost, since large quantities of it
can be readily purified from bovine
blood, a byproduct of the cattle
industry.
COOMASSIE BRILLIANT BLUE in any protocol. They do not bind equally to all
proteins, and do not react chemically with proteins
• a cation (red form) at a pH below 0 with an but merely form non-covalent complexes.
absorbance peak at 470 nm
• an anion (blue form) at a pH above 2 with an
absorbance peak at 595 nm
• neutral, green form at a pH around 1 with an
absorbance peak around 650 nm.
• The blue, anionic form is what binds with amino
acid residues, such as arginine or aromatics, to
form a stable complex—forming the basis of the
Bradford Method.

Coomassie Dyes 101: Understanding the Basics

Coomassie stains (also known as Coomassie Blue or
Coomassie Brilliant Blue)

• most popular anionic protein dyes used in

visualizing proteins in SDS-PAGE gels.
• may not be as sensitive as fluorescent and
silver stains, these provide good quantitative
linearity and medium sensitivity.
• most widely used staining technique for protein
identification and mass spectrometry
• commonly used as the basis of stains for
detection of proteins in gel electrophoresis
• Bradford-type assay reagents for protein
quantitation.
• has two variants – Coomassie R-250 and
Coomassie G-250.
o two chemical forms of a disulfonated Coomassie R-250 is the more commonly used variant for
triphenylmethane compound, and are protein detection since it can detect as little as 0.1 ug of
structurally very similar protein. When using classical technique, the protein gels
o G-250 has two additional methyl are incubated with Coomassie staining solution and de-
groups stained using acetic acid solution to visualize of the
protein bands. This relatively cheap technique involves a
• Coomassie dyes work by
simple protocol and is compatible with mass
o binding to proteins through ionic
spectrometry, but it has a low reproducibility and may
interactions between the sulfonic acid
make visualization of low abundance proteins difficult.
groups in the dye and the positive
amine groups in the protein
o Van der Waals attractions
Coomassie G-250 (also known as colloidal Coomassie
dye) also offers relatively high sensitivity and involves
a simple protocol. However, G-250 offers a faster
R-250 vs. G-250: Which One Should You Use?
staining protocol and eliminates the need for
Basically, both R-250 (R signifies the slightly reddish destaining the gel (you can easily visualize the protein
tint in the blue color of the dye) and G-250 (G signifies bands against the light amber background).
the greenish tint in the dye) have relatively high
In addition, G-250 can be used to quantify the amount
sensitivity and allow for easy detection (they develop
of protein in the solution (Bradford assay). Upon
intensely colored complexes upon binding with
binding with proteins, the dye (which has a brownish color
protein molecules). They also allow reversible
under acidic conditions) will produce a bluish tint. The
staining under the proper conditions.
optical absorbance of the solution is measured at a
Although they have basically the same chemical wavelength of 595 nm and the resulting data is used to
structure, R-250 and G-250 require different physical determine protein concentration and the actual amount of
and staining procedures and are not interchangeable protein in each solution.
 Spectrometer vs. Spectrophotometer
Spectrometer
• separates and measures spectral components of
a physical phenomenon
• measure a continuous variable of a phenomenon
where the spectral components are somehow
mixed such as a beam of white light

Spectrophotometer
• performs quantitative measurements of
reflectance or transmission properties of a
material as a function of wavelength
• a device that is used to measure the intensity of
electromagnetic radiation at various wavelengths
• Used to measure the absorbency of a certain
wavelength of the solution, reflectance of
solutions, transmittance or the transparency of
solids

Albumin
• Globular protein made by the liver.
• makes up about 60% of the total protein in the
blood
• keeps fluid from leaking out of blood vessels,
nourishes tissues, and transports hormones,
vitamins, drugs, and substances like calcium
throughout the body.

Extinction Coefficient
• a measure of the rate of transmitted light via
scattering and absorption for a medium.”
• In analytical chemistry, the quantity ϵ (epsilon) is
called the molar absorptivity (ϵmolar) or
extinction coefficient.
• ϵ has the units M-1 cm-1. Molar absorptivity
refers to the characteristics of a substance that
tells how much light is absorbed at a wavelength.
• “specific absorption coefficient (a)” refers to the
absorbance of light per unit path length, usually
expressed in cm, and per unit of mass
concentration.

The “molar absorption coefficient (ϵmolar)”

refers to the absorbance of light per unit path
length and per unit of concentration expressed in
“moles per liter.”

For proteins, an absorbance maximum near 280 nm

(A280) in the UV spectra of a protein solution is mostly
due to the presence of aromatic tryptophan and tyrosine
residues, and to a minor portion phenylalanine. For a
given protein, the A280 is proportional to its concentration
of amino acids. However, corrections may be needed to
calculate the accurate absorbance value, the type, and
the environment the amino acids are in. Using the known
amino acid sequence of a protein allows estimation of a
sufficiently accurate extinction coefficient.
In general, a 1 mg/ml solution of most proteins has an
A280 of ~ 1 ± 0.6.
The Bradford Assay: Colorimetric Protein
Determination with Coomassie Blue
The Bradford assay is based on the use of the dye
Coomassie Brilliant Blue G-250, which is frequently
abbreviated as Coomassie G-250 or Coomassie Blue.
This is one of two Coomassie dyes that are often
confused. Coomassie R-250 is used to stain protein gels
but is not used in protein assays. In addition to being used
in the Bradford assay, Coomassie G-250 can also be
used to stain protein gels, although it is less sensitive than
Coomassie R-250.
Under acidic conditions, Coomassie G-250 is cationic,
mainly doubly protonated, and is red, whereas in neutral
conditions the dye is green, and the anionic form is blue.
The Bradford reagent is an acidified solution of
Coomassie G-250; the dye is thus primarily protonated
and red.
Bradford Assay Standards
The Bradford assay uses standards to both quantify the
amount of protein in samples and to subtract any
background due to interfering substances that can shift
the ratios between the three forms of the dye.
The concentration range of standards in the kits cover the
linear range of the Bradford assay. Since the curve
flattens at high concentrations of dye, the amount of
protein in the sample will be underestimated when the
concentration of protein is higher than the range of the
linear portion of the curve, that is, at saturation conditions.
Samples that have protein concentrations higher than the
concentrations in the linear range must therefore be
diluted and re-assayed to obtain a more accurate
estimation of the protein concentration.
The standards used most for the Bradford assay are
bovine serum albumin (BSA) and bovine γ-globulin
(BGG). Ideally, the standard should be the same proteins
in the same ratios as are found in the sample (that is, an
absolute reference standard). However, for most samples
this is not practical or even possible, and relative rather
than absolute concentrations are enough in most
applications.

The basis for the Bradford assay is that in order for the
Coomassie dye to bind stably to protein, it needs to be
doubly protonated. When the dye comes in contact with
protein, the first electron is donated to charged groups on
the protein. This disrupts the structure of the protein,
resulting in exposure of hydrophobic pockets. The dye
binds to these pockets, with the sulfonic acid groups
binding to positive amines. In addition, there is attraction
due to Van der Waals forces. The stably bound
Coomassie G-250 is the blue, unprotonated form.

Protein Dye Binding in Bradford Assays

The change in the color of Coomassie G-250 from red to
blue upon binding protein is measured spectroscopically.
In the absence of protein, when the dye is red, Bradford
reagent has an absorbance maximum (Amax) of 470 nm.
In the presence of protein, the change to the anionic blue
form of the dye shifts the Amax to 595 nm.

Since the amount of the blue anionic form is proportional

to the amount of protein in the sample, the quantity of
protein in a sample can measured directly by measuring
the absorption at 595 nm. The neutral green form of
Coomassie G-250 that has donated an electron but is not
bound to protein does not interfere with measurement in
the Bradford assay since it has an Amax of 650 nm.