Professional Documents
Culture Documents
Determination of the Protein Concentration of Myoglobin from Minced Beef Using the
Bradford Assay
Chua, K.M.D., Cocjin, C.M.PH.R, Dizon, J.A.H., Donato, L.P.G., Dumaplin, R.A.L, Francisco,
R.A.2B-PH, Group 3, Department of Pharmacy, Faculty of Pharmacy, University of Santo
Tomas, Espaa Boulevard, 1015 Manila, Philippines
ABSTRACT
The protein, myoglobin, is found in muscle cells of animals [1].The Bradford assay is a common
method used to determine the protein concentration of a given sample [2]. This experiment was
conducted to successfully isolate the protein myoglobin from minced beef and to quantitatively
determine the protein concentration of myoglobin that was extracted from minced beef by saltinduced precipitation. The Bradford method is based on the binding of acidic Coomasie dye, a
coloring reagent, to proteins in which protein concentration is directly proportional to the dye
[3]. The standard protein that was used in this experiment is Bovine Serum Albumin (BSA)
which the Bradford assay is sensitive to than most common proteins [4]. Seven analytes were
prepared using assigned volumes of distilled water and Bovine Serum Albumin while one test
tube was left blank. The UV-Vis Spectrophotometer was used to measure the absorption of the
analytes. Apart from the nine test tubes that were prepared, another was prepared and it
contained the unknown protein. Since known concentrations of protein were prepared, the
unknown protein can be estimated and can be compared to their absorbance. To compute for the
protein concentration, the linear regression equation was used. To graphically represent the
relation of concentration and absorption, a Standard Calibration Curve was devised. It was then
used to deduce the protein concentration of the unknown sample.
INTRODUCTION
Proteins are large, complex molecule that
are composed of numerous chains of amino
acids in a specific order [5]. There are 20
amino acids that are commonly found in
proteins [2]. They are required for the
structure, function, and regulation of the
cells, tissues, and organs of the body [5].
They are classified according to their
functions. A few examples of proteins are
actin, immunoglobulin, and myoglobin [6].
Myoglobin is a protein that can be found in
the muscle cells of animals. It functions as
an oxygen-storage unit that provides oxygen
to working muscles [1].
Proteins can be isolated from a crude
sample. The most common way to
METHODOLOGY
10
mL
distilled
H2O
mL
Standar
d BSA
2.
5
2.0
2.0
1.5
1.5
1.0
1.0
0.5
0.5
2.
0
0.5
0.5
1.0
1.0
1.5
1.5
2.0
2.0
0.
5
Total protein
concentration
(mg/mL)
A595
Trial 1
A595
Trial 2
A595
Average
Blank
Standard
1
Standard
2
Standard
3
Standard
4
Unknow
n
0.050
0.2485
0.2265
0.2375
0.040
0.2245
0.2036
0.2141
0.030
0.1864
0.1910
0.1887
0.020
0.1529
0.1512
0.1521
0.1421
Sources
[1] Editors of Encylopedia Britannica.
(n.d.).Myoglobin. Retrieved on March 27,
2016
from
http://www.britannica.com/science/myoglob
in
[2] Crisostomo, A., Daya, M., Farrow, F.,
Gabona, M., Liu, M., Pena, G. . . (2010).
Isolation and Characterization of Proteins.
Laboratory
Manual
in
General
Biochemistry. (pp. 17, 20, 25-26). Quezon
City. C & E Publishing
lmgren/Glossary/Definitions/DefP/protein.html
[6] What are proteins and what do they do?
(2016). Retrieved March 27, 2016 from
https://ghr.nlm.nih.gov/handbook/howgenes
work/protein
[7] Salt-Induced Precipitation of Proteins
(n.d.). Retrieved March 27, 2016 from
http://www.rpi.edu/dept/chem-eng/BiotechEnviron/PRECIP/precpsalt.html