DETERMINATION OF PROTEIN

CONCENTRATION
(BRANDFORD PROTEIN ASSAY)
Experiment 4
Ebru AKHARMAN, Gebze Technical University, Turkey

AIM:
Before examining the enzyme kinetics, determining the protein content of cell fragments, or
trying to determine the binding constant of a solution, it is necessary to start with the
measurement of the protein concentration of the solution. When we know how much protein a
solution contains, we can compare the results of one experiment with another better. We can
better examine the effect of an external effect on a protein. There are three standard methods for
measuring and calculating the protein concentration: 1.) absorbance at 280 nm, 2.) BCA assay
and 3.) Bradford assay. We will use Brandford assay in this experiment.

INTRODUCTION: As might be expected, molecules can not be

Before examining the enzyme kinetics,
determining the protein content of cell
fragments, or trying to determine the
binding constant of a solution, it is
necessary to start with the measurement of
the protein concentration of the solution.
When we know how much protein a
solution contains, we can compare the
results of one experiment with another
better. We can better examine the effect of
an external effect on a protein. There are
many ways to measure and calculate the
protein concentration. Despite the
multitude of methods, there is usually no
way to determine the exact amount of
protein in any sample and give the exact
result we want.
However, in order to optimize the amount of
protein, the method of protein sensitivity
and sensitivity of the assay should be
chosen for the structure of the protein and
other compounds.
counted under a microscope like bacteria
colonies or cells. In order to be able to
measure the amount of molecules such as
proteins, a parameter that is directly
proportional to the concentration of the
molecules present in the medium must be
used. The most frequently used parameter
is light absorption. According to Beer's law,
if a dissolved matter absorbs light at a
certain wavelength, this absorption is
directly proportional to the amount of that
solution in that solution.
This absorption can be measured with the
aid of a spectrophotometer. Usually,
however, the substance to be investigated
can not absorb enough to be analyzed in its
own right. In this case, colored components
that can be connected to the substance to be
examined are connected and measured.
But knowing the absorbance values alone
does not give us much information about
the quantity. A standard is needed to
compare the absorbance to determine the
amount of protein desired. For example, if X
protein gives 0.5 absorbance, we need
another protein to know its concentration
and still give 0.5 absorbance under the same
conditions to find its amount.
Because we know the amount of this
sample, we can assume that the amount of
protein X is the same at 0.5 absorbance.
However, generally there will be protein-
containing solutions in varying
concentrations.
A reference interval is required to
implement the same approach. For this
reason, a solution of a certain concentration
of protein is prepared at regular intervals
(standard) and a standard chart is drawn
based on its absorption. The standard graph
is a plot of the concentration of an unknown
substance in the absorbance values of the
solution containing the known pure protein,
followed by substitution of the absorbance
values of the unknown protein solution on
this standard curve.
But this is not always possible. Because the
protein may not be obtained purely. In these
cases, commercially pure proteins are used
as standard. The most commonly used
protein standard is bovine serum albumin
(BSA).
Determination of total protein content in a
solution takes an important place in the
selection of separation and purification
processes and in the control of protein yield
and purity at certain stages. Particularly in
electrophoretic and chromatographic
separations, quantitation is required for
optimization of experiments.
It is also essential that the total amount of
protein is known when the specific activity
and purification degree of an enzyme being
studied is determined.
Determination of Protein Amount by
Bradford Method:
The Bradford method was developed using
the Coomasie brilliant blue G-250 dye,
which binds to different concentrations of
the proteins and reveals blue color solutions
of varying color intensity. The amino acid
composition of the protein is important for
blue color formation. The dye has been
shown to be particularly prone to binding to
basic amino acids such as arginine and
certain aromatic amino acids. Therefore, in
this method, the structure of the protein
primer is important. The protein attached to
the stain in the method gives maximum
absorbance at a wavelength of 595 nm.
MATERIAL AND METHODS:
Bradford reagent
BSA
Tubes
Micropipets
Spectrophotometer
We have 1 mg/ml stock BSA.
500 ml stock BSA is taken to tube 1. 500 ml
ddwater is added to tube 1. This mixture is
completely mixed with micropipet. 0,5
mg/ml BSA is obtained in this situation.
500 ml is taken from 0,5 mg/ml BSA to tube
2. 500 ml ddwater is added to tube 2. This
mixture is completely mixed with
micropipet. 0,25 mg/ml BSA is obtained in
this situation.
500 ml is taken from 0,25 mg/ml BSA to
tube 3. 500 ml ddwater is added to tube 3.
This mixture is completely mixed with
micropipet. 0,125 mg/ml BSA is obtained in
this situation.
500 ml is taken from 0,125 mg/ml BSA to
tube 4. 500 ml ddwater is added to tube 4.
This mixture is completely mixed with
micropipet. 0,0625 mg/ml BSA is obtained The average of the obtained
in this situation. absorbances is calculated. The average
of blank is decreased from other average
500 ml is taken from 0,0625 mg/ml BSA to of absorbance.
tube 5. 500 ml ddwater is added to tube 5.
This mixture is completely mixed with The concentration of samples of BSA
micropipet. 0,0625 mg/ml BSA is obtained calculated.
in this situation.

500 ml is taken from 0,0625 mg/ml BSA to According to datas, absorbance -

tube 6. 500 ml ddwater is added to tube 6. concentration graph is created and slope
This mixture is completely mixed with is found.
micropipet. 0,0313 mg/ml BSA is obtained BSA Bradford 2. absorbans BSA
in this situation. BSA Yzdesi
A 1 mg/ml 200 l 10 l 0,836 100
Thus, the dilution process ends. Then, stock
and diluted solutions are placed in 96 well B 0,5 mg/ml 200 l 10 l 0,741 50,5
plate. Each diluted solutions are placed as
C 0,25 200 l 10 l 0,563 25,74
Replica1 and Replica2.
mg/ml
200 micro liter Bradford reagent is added to D 0,125 200 l 10 l 0,372 13,37
mg/ml
each diluted solutions in the 96 well
E 0,0625 200 l 10 l 0,218 6,18
plate.10 micro liter BSA is added to each mg/ml
well.Then, absorbance of these solutions is F 0,0313 200 l 10 l 0,121 4,09
measurement with spectrophotometer. mg/ml
Datas are recorded. G 0,0 mg/ml 200 l 10 l 0 0,99

RESULTS: Table.2: Showing Values Table

REP1 REP2 REP1andREp2 avr.- This information is used to create graphics.

avr. blank The graphical curve is the standard curve.
avr. The slope of this standard curve is
A 1,029 1,25 1,14 0,836
important. We can calculate the
concentration of unknown protein
B 0,938 1,151 1,045 0,741
according to the formula below.
C 0,895 0,839 0,867 0,563

D 0,684 0,668 0,676 0,372

E 0,518 0,525 0,522 0,218

F 0,407 0,443 0,425 0,121

blank G 0,304 0,304 0,304 0

unknown H 0,494 0,494 0,494 0,19

Table.1: The Table of Absorbance Image 1: Formula image

If you want to show this data graphically:

Graph 1: Concentration of unknown protein garph

= ,

= ,

,
=
,

The concentration of unknown protein. = ,

DISCUSSION: been made in the experiment. As a personal

mistake, BSA may not be mixed. Used
It is important to know the amount of used materials may be damaged.
substances in biochemical experiments. In
this experiment, an unknown protein RESOURCES:
concentration was calculated.A protein with
https://www.researchgate.net/post/How_d
a known concentration is used. We used
oes_one_calculate_protein_concentration_us
bovine serum albumin (BSA). Absorbances
ing_formula
of prepared BSA at different concentrations
were obtained by https://info.gbiosciences.com/blog/bid/16
spectrophotometer.Absorbance - 4578/bradford-protein-assay-calculation-
Concentration graph was created and the of-an-unknown-standard
slope of this graph was used. In this way the
concentration of unknown protein was
calculated. According to this procedure, the
unknown protein concentration was found
to be 17, 93. Various mistakes may have