- The document describes a methodology for using spectrophotometry and the Bradford assay to determine the protein concentration of various food samples. - Standard curves were constructed using bovine gamma globulin standards of known concentrations. Absorbance readings of diluted food samples were then used to calculate protein concentrations based on the standard curve. - Calculated protein concentrations from the experiment differed from labeled values, possibly due to errors in dilution or pipetting techniques. The highest measured concentration did not match the expected highest protein food.

1. FACULTY OF INFORMATION SCIENCE AND
TECHNOLOGY
Practical 3
Quantitative determination of protein concentration using
spectrophotometry
HBC 1011
Biochemistry I
Trimester 1 2018/2019
Name: MOHAMED REDA
ID: 1151303266

2. Introduction
Accurate protein quantitation is essential to all experiments related to proteins in a
multitude of research topics. Different methods have been developed to quantitate proteins in a
given assay for total protein content and for a single protein. Total protein quantitation methods
comprise traditional methods such as the measurement of UV absorbance at 280 nm,
Bicinchoninic acid (BCA) and Bradford assays, as well as alternative methods like Lowry assay.
In this practical, the method of Bradford is used to determine the protein concentration. The
Bradford protein assay is a spectroscopic analytical procedure used to measure the concentration
of protein in a solution. It is based on the amino acid composition of the measured protein. The
protein will form a complex with the coomassie blue dye. The protein concentration can be
evaluated by determining the amount of dye in the blue ionic form and by measuring the
absorbance of the solution at 595 nm using a spectrophotometer. While using the Bradford assay,
detergent containing buffer must be avoided as it will disrupt the coomasie dye and produce an
inaccurate result.

3. Methodology
Part A: Construction of linear graph from known protein standards
1) Seven Eppendorf tubes were prepared and labeled as blank and from 1 to 6.
2) An amount of 1ml of dye reagent and 20µl of PBS was pipetted into an Eppendorf tube
labeled blank and was mix by inverting few times.
3) Appropriately labeled Eppendorf tubes were added with 20µl of pre-diluted standard and 1ml
of dye reagent by using a micropipette.
4) The spectrophotometer was set to 595nm and the instrument was set to zero on a 1.5ml
polystyrene cuvette fill with solution from Eppendorf tube labeled blank.
5) The solution was poured back to the Eppendorf tube and the next set of solution was added to
the empty cuvette.
6) The absorbance of all the standard were measured.
7) A linear standard curve was constructed by plotting A595 values of the known standards
against the concentration of standard protein.
Part B: Quantitative determination of protein in food samples
1) A food sample is prepared for dilution.
2) The food sample was diluted with dilution factor of 10x, 50x and 100x and was pipetted in to
the Eppendorf tubes labeled with corresponding dilutions.
3) An amount of 1ml of dye reagent and 20µl of PBS was pipetted into an Eppendorf tube
labeled blank and was mix by inverting few times.
4) An amount of 20µl of each diluted samples and 1ml of dye reagent were pipetted into the
new Eppendorf tubes labeled with corresponding dilutions.
5) The spectrophotometer was set to 595nm and the instrument was set to zero on a 1.5ml
polystyrene cuvette fill with solution from Eppendorf tube labeled blank.
6) The solution was poured back to the Eppendorf tube and the next set of solution was added to
the empty cuvette.
7) The absorbance of each diluted food samples were recorded.

4. 8) Food sample with dilution factor 50x was chosen to be replicated to obtain the average
protein concentration for a more accurate result.
9) Final concentration of the food sample is calculated by multiplying the protein concentration
obtained from the graph with the dilution factor to get the actual concentration.
10) The final concentration of food sample and the actual protein content information was
tabulated and the standard deviation was calculated.

5. Result and Discussion
Table 1: Standard Curve Absorbance Values
Standard Diluent volume
(PBS -µl)
Source of
Standard
(mg/ml)
Standard
volume (µl)
Final
volume (µl)
Final
concentration of
BGG (mg/ml)
1 0 2 100 100 2.000
2 50 2 150 200 1.500
3 10 2 100 200 1.000
4 100 1.5 100 200 0.750
5 100 1 100 200 0.500
6 100 0.5 100 200 0.250
By using the formula of M1V1 = M2V2 the amount of standard volume to be used for each
standard are calculated. The remaining volume was then top up with the diluent which is the PBS
solution to obtain the exact final volume and the final concentration.
Table 2: Protein Standard Absorbance
Standard Final concentration of BGG
(mg/ml)
A595 Average A595
A B C
1 2.000 1.344 1.340 1.143 1.276
2 1.500 1.012 0.937 0.944 0.964
3 1.000 0.711 0.702 0.680 0.698
4 0.750 0.558 0.624 0.531 0.571
5 0.500 0.459 0.424 0.358 0.414
6 0.250 0.175 0.177 0.191 0.181
The reason for obtaining duplicate result of the same standard is to ensure that the result
will be accurate since the pipetting skill of each individual was different. Some of them might

6. have done error during the pipetting process. There are several ways to reduce the error in
pipetting. For example, if you need to dispense 15 µl of solution, a 1ml pipette would be the
wrong choice, whereas a 20 µl pipette would be ideal.
Table 3: Dilution of protein samples
(Soybean Sample)
Dilution Protein Sample (µl) Diluent Volume (PBS
- µl)
Final volume (µl)
10x 20µl of original
protein sample
180 200
50x 40µl of 10x protein
sample
160 200
100x 100µl of 50x protein
sample
100 200
For diluting the protein samples given, dilution factor method was applied to obtain the
desired concentration. Firstly, final volume to be obtain was set. To dilute the protein sample into
10x, 20µl of the original sample was used and the remaining volume was top up with the diluent,
PBS solution. Then obtained 40µl of solution from the 10x diluted sample and the remaining
volume was top up with diluent to create a 50x diluted sample. Lastly, 100µl of 50x diluted
protein sample was obtained and mixed with 100µl of PBS solution to get a final volume of
200µl and 100x dilution.

7. Table 4: Spectrophotometric Data for Protein Samples
Sample A595 Average
A595
Dilution
Factor
Protein
Concentration
(ml/ml)
Average Protein
Concentration
(mg/ml)
X1 0.440
0.435 50x
0.541
0.533X2 0.447 0.553
X3 0.417 0.505
Y1 0.625
0.660 10x
0.856
0.925Y2 0.684 0.972
Y3 0.671 0.947
Z1 0.337
0.331 50x
0.417
0.411Z2 0.324 0.403
Z3 0.332 0.412
The protein concentration of food samples were obtained by referring to the standard
linear graph with the known value of absorbance which was obtained by measuring the
absorbance of protein samples. The average of absorbance and protein concentration was
calculated to get a more precise reading.
Table 5: Comparing measured protein concentrations to the values found on food labels
Protein Samples Bradford Assay (mg/ml) Final
Concentration from Table 4
(mg/ml)
Food Label (mg/ml)
X
(Soybean Milk)
26.65 22
Y
(Prebiotic Fermented Milk)
9.25 11
Z
(Full Cream Milk)
20.55 34

8. Table 6: Standard deviation of average protein concentration from different sample
Protein Samples Average Protein
Concentration (mg/ml)
Standard Deviation of Average
Protein Concentration
X
(Soybean Milk)
0.533
0.269Y
(Prebiotic Fermented Milk)
0.925
Z
(Full Cream Milk)
0.411
Graph 1: The relationship between Concentration of Standard and A595
Based on the standard linear graph, the experimentally actual protein concentration of
soybean milk is 26.65 mg/ml while for prebiotic fermented milk is 9.25mg/ml and lastly full
cream milk having 20.55 mg/ml. Compared to the actual values on the food label, the result
obtained experimentally is more than the actual value. This can be due to errors in preparing the
diluted protein samples. Possibly more protein samples volume are pipetted without notice
during the dilution process. Therefore acquiring an inaccurate result. For prebiotic fermented
milk, the experimental value is quite close to the actual result. For full cream milk, the

9. experimentally obtained protein concentration was far lesser than the actual value. This might be
due to errors in preparing the diluted protein samples where the dilution factor method applied
might be some mistake in it. From the food label we know that the highest protein concentration
food should be full cream milk while the lowest is the prebiotic fermented milk and the
intermediate is soybean milk. Yet the experimental value obtained was quite different whereby
the highest is the soybean milk and the intermediate is the full cream milk. This could be the
result of lacking pipetting skill and errors in calculating for diluting protein samples.
Conclusion
In conclusion, protein concentration of food samples can be determine by using different
type of method correspond to their requirement.

10. Question
1) What are the functions of spectrophotometer?
Spectrophotometer is used to measure the concentration of the solution by determining
the absorbance, identifying the organic compounds by determining the maximum
absorption and used for color determination within the spectral range.
2) Why is it important to know the linear range of protein standard in protein quantitate on
assay? What do you need to do if the absorbance of the protein is out of the linear range?
it is important to know the linear range of protein standard in protein quantitate on assay
because if you have the standard curve you will be able to identify the concentration of
any unknown protein sample. if the absorbance of the protein is out of the linear range
then you should see If the standard curve is leveling off, then you should not use the
points with the higher absorbance.
3) The protein concentration you obtained from the experiment may be different from the
value on the food label. Explain this observation.
This may be due to error in pipetting th perotein solution when performing the dilution
procedure and transferring the solution. Difference of the protein concentration may also
be due to the uncalibrated micropipette.
4) How long can the samples sit before being read? Is it alright if you measure the sample
absorbance after 1 hour? Why?
The readings will be inaccurate if the sample is let to sit for too long. This is because the
protein molecules in the protein sample will eventually sediment on the base of the
container. Thus the absorbance reading will be inaccurate.
5) You are using bovine gamma globulin as the protein standard, but you are measuring
protein concentration of samples from other sources. What is the assumption you make
for determination of the protein concentration of samples from other sources?
We assume that the standard protein has no errors and it produce a perfect result, the best

11. fit linear graph, and we use it as a reference to the protein that are going to be tested to
find the protein concentration based on the known absorbance value.
6) How do you know the protein absorbance is out of the linear range? Should you use these
values when plotting the graph? Why?
The linear range of the protein absorbance is between 0.1 and 1.0. The Other values
lower or higher than that will be consider out of range. We can use the values, to obtain
sufficient points, to plot the graph but when referring to the graph to obtain the protein
concentration with known absorbance, the value in between the linear range is only to be
used. Any value exceeding the range can be inaccurate.
7) You purified a protein which is eluted in a buffer containing detergent. Which protein
concentration determination method will you use? Why?
BCA assay. It is because this method is not sensitive to detergent. An accurate result can
be obtained. Bradford assay is not used because Bradford assay is sensitive to the
detergent it interferes with the coomasie dye.
8) You purified a protein which is eluted in a buffer containing reducing agent. Which
protein concentration determination method will you use? Why?
Bradford assay. It is because Bradford assay is not affected by the presence of reducing
agents. Lowry assay and BCA assay are sensitive to reducing agent which interfere the
protein thus it will affect the result.