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Methodology:
Results:
TUBE NO.
mL
STANDAR
D
mL H2O
0
mL
0.10
mL
0.15
mL
0.20
mL
0.25
mL
0.30
mL
0.35
mL
0.40
mL
0.45
mL
1.50
mL
0.04
5
0
1.40
mL
0.48
3
3.33
1.35
mL
0.51
8
5
1.30
mL
0.59
8
6.67
1.25
mL
0.65
1
8.33
1.20
mL
0.68
4
10
1.15
mL
0.69
1
11.6
7
1.10
mL
0.70
7
13.3
3
1.05
mL
0.79
4
15
ABSORBA
NCE (A595)
Concentra
tion
(ug/mL)
(Add 1.5 mL Bradford Reagent to all the tubes to make 3mL)
10
(Unkno
wn)
1.25 mL
-------1.644
50
Methodology
Protein Assay Using the Bradford Method
A series of test tubes were prepared as follows:
TUBE
NO.
mL
STANDA
RD
mL H2O
1
(Bla
nk)
0 mL
0.10
mL
0.15
mL
0.20
mL
0.25
mL
0.30
mL
0.35
mL
0.40
mL
0.45
mL
1.50
mL
1.40
mL
1.35
mL
1.30
mL
1.25
mL
1.20
mL
1.15
mL
1.10
mL
1.05
mL
10
(Unkno
wn)
1.25 mL
--------
1.5 mL of Bradford Reagent was added to each test tube. After 5 minutes
standing, 5 drops of each sample were put into a microwell plate. The
microwell was subjected to an absorbance reading machine. The absorbance
was read at 595 nm. An albumin standard curve was constructed with a plot
of concentration (ug/mL) against absorbance (A595). Linear regression
analysis was also used to determine the concentration of the samples.
Results and Discussion:
The Bradford assay is commonly used to determine the total protein
concentration of a sample. The method is based on the binding of Coomassie
dye to proteins in acidic solution leading to an increased absorbance of the
sample at 595 nm. [1] The assay is sensitive to about 20 to 200 microgram
protein.
The dye involved in the process is the Coomassie Brilliant Blue G-250. Under
acidic conditions the red form of the dye is converted into its bluer form,
binding to the protein being assayed. During the formation of this complex,
two types of bond interaction take place: the red form of Coomassie dye first
donates its free electron to the ionizable groups on the protein, which causes
a disruption of the protein's native state, consequently exposing its
hydrophobic pockets. These pockets in the protein's tertiary structure bind
non-covalently to the non-polar region of the dye via van der Waals forces,
positioning the positive amine groups in proximity with the negative charge
of the dye. [2] The bond is further strengthened by the ionic interaction
between the two. The binding of the protein stabilizes the blue form of the
Coomassie dye; thus the amount of the complex present in solution is a
measure for the protein concentration, and can be estimated by use of an
absorbance reading. [3] The result for the absorbance reading and the
computed amount of concentration can be seen on Table 2.
TUBE NO.
ABSORBA
NCE (A595)
Concentra
tion
(ug/mL)
1
(Bla
nk)
0.045
0
0.48
3
3.33
0.51
8
5
0.59
8
6.67
0.65
1
8.33
0.68
4
10
0.69
1
11.6
7
0.70
7
13.3
3
0.79
4
15
10
(Unkno
wn)
1.644
?????
References
[1] http://www.ruf.rice.edu/~bioslabs/methods/protein/bradford.html
[2] http://www.sigmaaldrich.com/life-science/proteomics/proteinquantitation/bradford-reagent.html
[3] http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4110065A.pdf
[4] https://en.wikipedia.org/wiki/Bradford_protein_assay