Protein Assay Using Bradford Method

Protein Assay Using Bradford Method
Biochemistry Laboratory
Abstract
This experiment is an attempt to exemplify the usage of the Bradford Assay technique by determining
the concentration of a given protein, this method implies the use of the acidic Coomassie dye as a
colouring agent (the solution is red-brown in it’s acidic solution. When protein binds, the pKa of the
dye shifts causing the dye to become blue) and BSA to prepare standards. A standard is the ‘control’ of
this experiment, our standard in this case was created by BSA. Since it is clear that concentration is
directly proportional to absorption, UV-Vis Spectrophotometer was used to measure the absorption of
the analytes. We already know the protein concentration of the analytes; therefore by preparing
samples with known amounts of protein and comparing their absorbances with that of the unknown,
the unknown quantity can be estimated. In a graphical representation of the relationship of absorption
and concentration, a Standard Calibration Curve was developed and then used to infer the unknown
concentration of the sample.
Introduction include many detergents and basic buffers;

The Bradford protein assay was introduced there are modifications of this assay that
to us as one of the methods to determine reduce or eliminate the effects of many
protein concentration in the mixture. The interfering substances.
method is based on the proportional binding
2
of the dye Coomassie to proteins, the more Protein assays are designed to measure the
protein present, the more Coomassie binds total protein in a solution; that is all of the
and it produces a significant change in colour proteins in solution. Protein assays are
of the mixture. And since the assay is quantitative if the protein to be assayed is
colorimetric, we learned that as the protein available in sufficient quantity such that one
concentration increases, the colour of the is able to use it to create a standard curve. If
test sample becomes darker. Coomassie this can not be achieved, then a standard
absorbs at 595 nm. The protein protein, such as albumin, may be used for a
concentration of a test sample is determined standard curve with the understanding that
by comparison to that of a series of protein the results on the unknown protein are
standards known to reproducibly exhibit a semiquantative. 5Because most proteins are
linear absorbance profile when plotted in a not available in large quantities, standard
graph. Although different protein standards curves for protein assays are typically based
can be used, we have chosen the most on the use of either bovine serum albumin
widely used protein as our standard - Bovine (BSA) or bovine gamma globulin (IgG).
Serum Albumin (BSA).
1
The Bradford assay is faster, involves fewer
mixing steps, does not require heating, this
assay has been found to be useful for
peptides and proteins having molecular
weights greater than approximately 3,000-
5,000, depending on the presence of
charged groups. Interfering substances
rest followed in similar fashion. We poured
BSA on 8 test tubes (omitting the first) again
in varied amounts, this time the preceding
test tubes had more BSA than the latter
ones, effectively making them less
concentrated with BSA. Each test tubes was
mixed thoroughly, 1.5ml of Bradford reagent
was then added into all test tubes. The test
tubes stood undisrupted for 5 minutes then
we transferred their contents into 9 cuvettes,
Structural formula of Coomassie dye variant
one additional cuvette contained the sample.
(most likely) used in our Bradford Method
The prepared cuvettes were taken to
Laboratory 17 downstairs and we measured
Coomassie dye. Dr. Marion Bradford was
their absorbance using UV-Vis
the first scientist to ever use Coomassie dye
Spectrophotometer An albumin standard
to determine protein concentration in his
curve was made showing the relationship of
solution. 1In the acidic environment of the
A545 with concentration of each prepared
reagent, protein binds to the coomassie dye.
standard mixtures. Points were connected in
Advantages of using Coomassie dye in
linear regression, in doing so, our group
Bradford Assay: 5Coomassie dye binding
determined the total protein concentration of
assays are the fastest and easiest to perform
the sample.
of all protein assays. 2The assay is performed
at room temperature and no specials
Results
equipment is required. Standard and
unknown samples are added to tubes
containing preformulated Coomassie assay
reagent and the resultant blue color is 0.9
f(x) = 0.06 xAlbumin Standard Curve
measured at 595 nm following short room 0.8 R² = 0.98
temperature incubation. Disadvantages A

0.7
3
include incompatibility with surfactants at b 0.6
s
concentrations routinely used to solubilize o 0.5
r
membrane proteins. 4The presence of a b 0.4
a Linear ()
surfactant in the sample, even at low n 0.3
c
concentrations, causes precipitation of the e 0.2
reagent. In addition, the Coomassie dye
0.1
reagent is highly acidic, so proteins with
0
poor acid-solubility cannot be assayed with 0 2 4 6 8 10 12 14 16
this reagent. Concentration (mg/mL)
Methodology Discussion
After procuring our own Bradford reagent
and BSA aliquot onto our work table, we The Coomassie Blue is a brown liquid that
prepared 9 test tubes and filled each with turns blue when attached to a protein; the
varied amounts of distilled water. The first more intense the blue color, the more
test tubes had slightly more than the latter protein. By setting the Spectrophotometer to
ones, test tubes 2 and 3 for instance, had a 595 nm of wave length, we effectively
difference of approximately 0.5ml, and the
measured how much light has been
absorbed by the sample.
References
1
Krohn, R.I. (2001). The Colorimetric
Determination of Total Protein, Current
Protocols in Food Analytical Chemistry ,
B1.1.1-B1.1.27, John Wiley & Sons, Inc.-
2
Hopkins, (1993) Maton et. Al. Human
Biology and Health. Englewood Cliffs,
Michigan, USA: Prentice Hall
3
Kruger,N.(2003) The Protein Protocols
Handbook B.14-16,
4
www.piercenet.com/browse.cfm?
fldID=876562B0-5056-8A76-4E0C-
B764EAB3A339 Retrieved January 10 2011
5
http://www.tutorvista.com/content/chemi
stry/chemistry-ii/Bradford.htm Retrieved
1/10/2011

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Protein Assay Using Bradford Method

Introduction include many detergents and basic buffers;

temperature incubation. Disadvantages A

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