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QUANTITATIVE ANALYSIS OF PROTEIN

Gabrielli Anne B. Saavedra, Juan Miguel C. Santos, Rea Christine B. Tapel, Kyra Zara N. Tieng, Anthea
Jae O. Ungsod, Jacquelyn Faye S. Usero
Group 7
2G Pharmacy Pharmaceutical Biochemistry Laboratory

ABSTRACT
A series of test tubes were prepared in the experiment with the following reagents: Bovine serum albumin (BSA)
standard (100 g/mL), Bradford reagents, distilled water and an unknown sample. The first tube contained distilled
water only and the tenth test tube contained the unknown sample only. The remaining test tubes were a combination
of distilled water and bovine serum albumin (BSA) standard that would total to 1.5mL of solution. 1.5mL of the
Bradford reagent was added to all of the test tubes. The absorbance at 595nm was read and the data retrieved was
used to construct the albumin standard curve by plotting A 595 against concentration ( g/mL). Also, this was used
to determine the concentration of the unknown sample, which is 19.8 g/mL .

INTRODUCTION
The objective of the experiment is to
quantitatively determine protein concentration in
a given sample though Bradford assay.

EXPERIMENTAL

The Bradford assay is commonly used to


determine the total protein concentration of a
sample. [1] The initial color of the solutions of
the test tubes was red. Upon the addition of the
Bradford reagent, the solution turned blue which
indicates that the solution is acidic and its
absorbance it at 595nm. The results can be seen
in figure 1.

A. Compounds tested (or samples used)


Bradford reagent, Bovine serum albumin
(BSA) standard, distilled water and unknown
sample.

B. Procedure
A series of test tubes was prepared as
follows:
Table 1 Preparation of Test Tubes

Tube #
1
2
3
4
mL
0
0.10
0.15
0.20
standar
d
mL H2O
1.50
1.40
1.35
1.30
5
6
7
8
9
0.25
0.30
0.35
0.40
0.45
1.25
1.20
1.15
1.10
1.05
1.5 mL of unknown sample was placed in
a test tube, which was labeled as 10. 1.5 mL of
Bradford reagent was added to each test tube.
The test tubes were mixed well and left to stand
for 5 minutes. 3 drops from each test tube were
placed in a microplate. The samples on the
microplate
were
placed
in
a
UV-Vis
Spectrophotometer. The data that was retrieved
was used to construct the albumin standard curve
by plotting A595 against concentration ( g/mL)

RESULTS AND DISCUSSION

Figure 1 Test Tube

Ultraviolet-visible spectrophotometry is
the absorption spectroscopy or reflectance
spectroscopy in the ultraviolet-visible spectral
region. The microplate containing few drops of
each of the test tubes underwent ultravioletvisible spectroscopy at absorbance 595nm. The
instrument used was an ultraviolet-visible
spectrophotometer. The data retrieved in figure 2
is row B, which is used to construct the albumin
standard curve.

Table 2 Concentrations of Test Tubes

Test Tube No.

Figure 2 Data from UV-Vis Spectrophotometer

In constructing the albumin standard


curve, the A595 is plotted against concentration (
g/mL). The concentration of each test tube
must be calculated by using this formula:

100 g
=x
mL
x
=concentration
total mLeach tube which (3.0 mL)

mL standard

The first test tube will have no


concentration since there was no mL standard
added to it. The following are the solutions for
the test tubes 2 to 9 respectively below. As for
the concentration of the tenth test tube or the
unknown sample, it is solved using the albumin
standard curve.

0.10

100 g 10 g
=
3.0 mL=3.33 g /mL
mL
mL

0.15

100 g 15 g
=
3.0 mL=5 g /mL
mL
mL

0.20

100 g 20 g
=
3.0 mL=6.67 g/mL
mL
mL

0.25

100 g 25 g
=
3.0 mL=8.33 g/mL
mL
mL

0.30

100 g 30 g
=
3.0 mL=10 g /mL
mL
mL

0.35

100 g 35 g
=
3.0 mL=11.67 g/mL
mL
mL

0.40

100 g 40 g
=
3.0 mL=13.33 g/mL
mL
mL

0.45

100 g 45 g
=
3.0 mL=15 g/mL
mL
mL

Concentration
( g/mL)
0
3.33
5
6.67
8.33
10
11.67
13.33
15

1
2
3
4
5
6
7
8
9
10
x
Before the albumin standard curve can be
made, the A595 of test tubes 2 to 10 are
subtracted to the A595 of test tube 1. The reason is
test tube 1s absorbance should be zero. This will
result to the data shown in Table 2.
Table 3 Absorbance at 595nm

Test Tube No.


1

A595

2
3
4
5
6
7
8
9
10

0.097
0.191
0.133
0.292
0.356
0.364
0.375
0.377
0.622

Table 4 Albumin Standard Curve Data

Test
Tube
No.
1

Concentration
( g/mL)

A595

2
3
4
5
6
7
8
9
10

3.33
5
6.67
8.33
10
11.67
13.33
15

0
0.097
0.191
0.133
0.292
0.356
0.364
0.375
0.377
0.622

Table 4 is just a combined data of table 2


and 3. Also with the data of table 4, it will result
to figure 3.

y=0.056 x0.0271
yb
x=
m
0.622(0.0104)
x=
0.0318
x=19.89 g /mL
If the concentration of the unknown
sample is to be plotted, the albumin standard
curve will look like figure 4

Figure 3 Albumin Standard Curve based on Table


4

For the concentration of the unknown


sample, the slope of the albumin standard curve
(figure 3) must be used. The formula for this is
y=mx +b . It is shown in figure 3 that there are
y=0.0521 x and
two equations which are
y=0.056 x0.0271 . The latter equation is to
x
be used because 0.056 x is closer to 1.
is to be solved in the formula because
concentration is plotted at the x-axis which is the
unknown. The derived formula is below:

y=mx +b
y b mx
=
m
m
yb
x=
m
The solution to solve for the concentration
of the unknown sample is

Figure 4 Final Albumin Standard Curve

Based on figure 4, the unknown sample


did not meet the range of absorbance based on
the data and the plot graph. This can be due to
inaccuracy in pipetting and contamination of the
reagents used. To be able to meet the range, the
amount of distilled water and the standard should
be adjusted to a certain concentration.

REFERENCES
[1] Crisostomo, Angelica C., et al (2010).
Laboratory Manual in General Biochemistry.
Quezon City: C & E Publishing House. Pg. 25-26