SWISS GERMAN UNIVERSITY

BIOCHEMISTRY LABORATORY REPORT

Subject : Biochemistry Laboratory

Lecturer : Kholis Abdurachim Audah, M.Sc., PhD
: Ms. Silvya Yusri, S.Si.
Instructor
Ms. Purwanty Rara Azura
Faculty/ Department : LST/ BME
Date of Experiment : Friday, September 2nd, 2016
Date of Lab. Report : Friday, September 9nd, 2016
Semester : 3
Time of Experiment : 08.00 – 11.00 (180 minutes)

- Determination of Protein Concentration Using the

Experiment:
Spectrophotometric Method

GROUP : 3

PARTICIPANTS : 1. Irena Sugiarto 11506014

2. Rakaputra P. 11506006
3. M. Kevin Achsanta 11406009

Swiss German University Tel. +62 21 3045 0045

EduTown BSDCity Fax. +62 21 3045 0001
Tangerang Selatan 15339 [email protected]
INDONESIA www.sgu.ac.id
I. Objective
To determine the protein concentration of a food sample (in this experiment: full cream
milk) via spectrophotometry method.

II. Theoretical background

Spectroscopy is an analytical method, which uses the ability of molecules to absorb
light (Sutanto, 2016). The amount of light absorbed is a function of the molecules its
concentration and the thickness of the light path (Murov, 2004). This relation is
described by Lambert Beers law:
A=bc
I
A = absorbance, log , I is the light intensity before the sample, I after the sample
I0 0
 = Extinction coefficient (depend on the molecule and wavelength of the light)
b = thickness of the light path
c = concentration in mole/l

Proteins are naturally colorless under normal circumstances; however, with the
addition of chromogenic compounds, they can be colored (Audah, 2016). Therefore, the
concentration of a sample protein can be measured using spectrophotometry. This is so
because spectrophotometry involves the amount of light that is absorbed and
transmitted when passed through a solution (Brady, 2015).

The Lowry method is highly sensitive to proteins and often used to calculate their
concentrations. It is used most often to calculate proteins with concentrations of 0.01–
1.0 mg/mL (Henry, 1996). The addition of Folin-Ciocalteau causes the solution to turn
blue due to the coordination reaction between peptide bonds and the copper ions from
biuret reagent and the reduction of Folin-Ciocalteau by tyrosine and tryptophan in
protein (Everette, 2010).

However, it has its weakness; other compounds can react and disturb the measurement
like ammonium sulphate, glycine, and Mercaptan (Audah, 2016). Depending on the type
of protein analyzed, the color formation is different (Brady, 2004). This method is
therefore best used to determine the change of protein concentration rather than the
exact concentration.

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The spectroscopic method used to measure the concentration of protein in a solution is
called the Bradford protein assay (Okutucu, 2007). It is dependent on the amino acid
composition of the measured protein (Aylward, 2008). The Bradford protein assay is
simple and easy to use, it’s also one of the fastest and most efficient way for protein
assay. Furthermore, they can measure protein concentrations from 1 to 20 micrograms.
The dye used is also stable and can be stored in room temperature. However, it’s linear
and very short range, thus requiring sample dilution before analysis, it is also inhibited
by detergents.

Bovine Serum Albumin, or BSA, is a serum albumin taken from cows (Invitrogen, 2012).
BSA is often used to determine an unknown protein concentration of a solution by
comparing the concentration of the BSA solution to the unknown sample. This is so
because of its ability to increase signal in assays, its lack of effect in many biochemical
reactions, and its low cost.

III. Equipment
 Spectrophotometer VIS and cuvette
 Test Tubes and rack
 Beaker Glass
 Volumetric Flask
 Vortex shaker
 Micro-pipettes and tip

IV. Chemical/ Reagent

 Protein sample: full cream milk
Chemicals MSDS
Bovine Serum Albumine Not a dangerous substance or mixture according to the
(BSA) 1 mg/ml Globally Harmonized System (GHS)
Causes severe skin burns and eye damage. Causes serious
eye damage. Harmful to aquatic life. May cause irritation
Biuret reagent to skin, eyes, respiratory tract, and/or gastrointestinal
tract. May be harmful if swallowed. Inhaled, or absorbed
through skin.
Folin-Ciocalteau reagent Causes skin irritation. May be corrosive to metals.
Aquadest None

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V. Procedure
 Standard BSA Solution Preparations
1. 100 mg of BSA was taken and mixed with 50 ml of aquadest.
2. The mixture was shaken gently and stirred, and then thinned with more aquadest
until the total volume was 100 ml. This results in a solution with a 1.0 mg/ml
concentration.
3. 6 test tubes were then prepared and 6 different concentrations of BSA were
prepared for each tube.
Table 1. Dilution table of standard BSA solution.

Tube BSA conc. (mg/ml) BSA vol. (ml) Aquadest vol. (ml)
1 1.0 2.5 0
2 0.8 2.0 0.5
3 0.6 1.5 1.0
4 0.4 1.0 1.5
5 0.2 0.5 2.0
6 0.1 0.25 2.25

 Sample Preparations
1. 2 ml of the sample (full cream milk) was taken and was diluted by aquadest until
100 ml.
2. 4 test tube was prepared. 3 test tubes were added by 0.6 ml of samples and the
other tube was added by 0.6 ml aquadest to be a blank and then vortexed.
3. All tubes were incubated for 10 minutes in room temperature.
4. After 10 minutes all test tubes was added by 0.15 ml of folin and then vortexed.
5. All tubes were incubated for another 30 minutes in room temperature.
6. After 30 minutes the samples and the blank were moved to cuvettes and checked
by spectrophotometer. The absorbance was noted.

VI. Experiment Data

Table 2. Absorbance and concentration of standard BSA solution.

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 Concentration (M) Absorbance (A)
1.0 0.250
0.8 0.163
0.6 0.131
0.4 0.114
0.2 0.062
0.1 0.020

Table 3. Observed absorbance of full cream milk.

Sample No. Absorbance (A)

1 0.135
2 0.106
3 0.105
Á 0.1205
=
Data from sample 3 (the smallest) was eliminated, to reduce the data range.

VII. Analysis and Discussion

The Making of the Standard Curve
1.0 mg/ml BSA was made with 100 mg of BSA. The reason 50 ml of aquadest was added
and the solution was stirred and let to be settled before another 50 ml was added was to
prevent the formation of bubbles. BSA forms bubbles when mixed with aquadest, and it
can interfere with the precision of the data.

In order to create a standard curve, varying concentrations of BSA had to be made so we

can have the absorbance data for each concentration as a “ladder”. Hence, that can be
used to approximate the amount of concentration of the sample protein.

In order to create the varying BSA concentrations from 1.0 mg/ml solution, refer to
Table 1. The amount of 0.1 mg/ml BSA solution needed in order to create a certain
concentration 2.5 ml solution can be calculated with the following equation, for
instance:
(desired concentration)(volume of solution)
volume of base solution needed =
concentrationof base solution
Thus, for example:
(0.8 mg ml −1 )(2.5 ml)
=2.0 ml
1 mgm l−1

The Preparation of the Sample

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 Figure 1. Full cream milk sample. Figure 2. Diluted sample. Figure 3. Biuret reagent.

Figure 4. 3 tubes of 0.6 ml diluted samples + 1 tube Figure 5. After added by 3 ml biuret each.
of 0.6 ml aquadest as the blank.

Figure 6. After added by 0.15 ml folin each. Figure 7. Final form before being moved into
cuvettes.

The sample was diluted (Figure 1 – 2) because the standard has a very short range (1.0
mg/ml and below), therefore it was diluted so it can fit within the range. The
concentration can be measured and the mass can be calculated (concentration
multiplied by the original volume of the protein solution).

The addition of the biuret reagent (Figure 3) into the protein solution creates a chemical
reaction between the copper ions of the reagent and the protein complex, creating a
purple coloration (Figure 4 – 5, Figure 8). The addition of Folin-Ciocalteu's reagent
cause the Cu+ to be oxidized back to Cu2+ by MoVI, which creates molybdenum blue
(MoIV), which explains the blue coloration upon the addition of the reagent (Figure 6,
Figure 9). The solution needs to be incubated for 30 minutes because it needs time to
react (Lowry, 1951). The solution after the incubation period is shown on Figure 7.

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 Figure 8. Biuret reaction.

Figure 9. The reaction of protein with biuret reagent and Folin-Ciocalteu reagent.

Calculation
Based on Table 2, a standard curve can be made. The wavelength used was 650 nm.

Figure 10. Standard curve of BSA solution.

Based on the standard curve on Figure 10, we can draw a linear regression line with an
equation as shown on the graph. The basic linear equation is
y = mx + c, so we get m=0.2247 and c=0.0073. Therefore,
we can calculate the protein concentration of the sample
(full cream milk), with the y (absorbance) from Table 3.
sample absorbance−c
[ sample ] = [ m ]
× dilution factor

[ sample ] = 0.1205−0.0073 × 50=25.19 mg

[ ]
0.2247 ml

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Figure 11. Nutrition Facts as

written on the milk box.
m protein ∈200 ml sample=[ sample ] ×200 ml
m protein ∈200 ml sample=6297 mg=6.297 g

The full cream milk contains 6.297 g of protein per 250 ml of milk based on our
experiment. Compared to the nutrition facts written on the box (7.5 g) (Figure 11), the
error was 16% which is slightly big. Our assumption, this error happens because of the
inaccuracy of the spectrophotometer, not so clean cuvette, and maybe the written value
on the nutrition facts was not an exact number (rounding). There might be protein or
non-protein complexes that absorbs UV light, causing an interfere to the absorbance
when it is analyzed using spectrophotometer

VIII. Conclusion
Spectrophotometry method can be used to determine the protein concentration from a
food sample. The procedure consists of matching the absorbance of the sample to the
standard curve that has been made before based on known concentration grading.
The full cream milk from the experiment contains 6.297 g of protein per 250 ml of milk,
with 16% error compared to the nutrition facts.

IX. Reference
Audah, Kholis. (2016). Chemistry 3 Laboratory Manual. BSD: Swiss German University.
Aylward, G., Findlay, T. (2008). SI Chemical Data, sixth edition. Australia: John Wiley.
Brady, J. E., Jespersen, N. D., Hyslop, Alyson. (2015). Chemistry, Seventh Edition.
Singapore: John Wiley and Sons.
Brady, J. E., & Senese, Fred. (2004). Chemistry Matter and Its Changes Fourth Edition.
Singapore: John Wiley and Sons.
Everette, J. D. (2010). Thorough study of reactivity of various compound classes toward
the Folin-Ciocalteu reagent. PubMed.gov. DOI: 10.1021/jf1005935.
Henry, J. B. (1996). Clinical Diagnosis and Management by Laboratory Methods. USA:
Saunders.
Invitrogen. (2012). BSA FAQ (online, https://www.neb.com/products/b9000-bsa-
molecular-biology-grade#pd-faqs, retrieved on 8 September 2016). New England
Biolabs.
Murov, S. L. (2004). Experiments in General Chemistry. Thomson Bookstore.
Okutucu, B. (2007). Comparison of five methods for determination of total plasma protein
concentration. Journal of Biochemical and Biophysical Methods. 70 (5): 709–711.
doi:10.1016/j.jbbm.
Sutanto, Hery. (2016). Chemistry 2 Laboratory Manual. BSD: Swiss German University.
http://sciencelab.com/, retrieved on 4 September 2016.

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