Introduction
Determining the concentration of substances is often involved in experiments especially for
diagnosis, biological research and forensic investigations. This includes determining the
protein concentration in the urine and food samples (Kielkopf et al., 2020). Proteins are
considered as the building blocks of life because all organisms contain protein in the form of
tissues, hair, enzymes and etc (Kovacs et al., 2017). One way to measure protein
concentration is by Bradford Assay.
Since the late 1970s, the Bradford assay has been used as a preferred technique in many
laboratories to find the protein concentration in a substance because of its inexpensiveness,
high throughput, accuracy, adaptability, low interference and simplicity to use (Ku et al.,
2013). The Bradford assay is known as a dye-binding method where the Coomassie brilliant
blue G-250 dye-protein interaction causes a colour change. The higher the degree of colour
change, the higher the absorbance at 595nm (Gazzola et al., 2015).
The Bradford assay can be used in two ways, microplate assay (colorimetric) or cuvette
assay (quantitative) (Goldring, 2019). However, in this experiment, the microplate assay of
the Bradford assay will be used.
Similar to other colorimetric Bradford assays, this experiment involves few steps whereby it
is started by the preparation of protein standard solutions containing bovine serum albumin
(BSA) which are then mixed with the Bradford reagent containing Coomassie Brilliant Blue
G-250 dye. At the end, Absorbance of said protein standards will be assessed by a
spectrophotometer at 595 nm (Kielkopf et al., 2020).
In this experiment, the Bradford assay will be used mainly to determine the protein
concentration of unknown samples. Results will show that the higher the amount of dye is
bound, the higher the absorbance value and thus, the higher the protein concentration in the
sample (Goldring, 2019).
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Objectives
1) To determine the net absorbance of BSA standards with distilled water ‘blank’ as the
control.
2) To calculate the protein concentration of unknown samples A, B and C.
3) To practice using Bradford Assay as a method to find protein concentrations of unknown
samples.
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Materials and methods
Materials
-
500ml Bradford reagent (B6916 Sigma)
-
50μl 2.0mg/ml Bovine serum albumin (BSA) standard (A595 standard)
-
Unknown protein samples labelled A, B and C
-
Spectrophotometer reader at 595nm
-
96 well plate
-
Pipettes – 10μl, 200μl and 1000μl
-
Pipettes tips – white, yellow, blue
-
0.5mL microcentrifuge tube (5x)
-
A beaker containing distilled water labelled ‘W’
-
An empty beaker labelled ‘for waste’
Methods
1. Prepare protein standards with different concentrations of BSA standards using serial
dilution which reduces the concentration by half between each successive dilution Mix
well before transferring standard to a different tube e.g. 2, 1.00, 0.50, 0.25 and 0.125
mg/ml (Refer to figure 1).
150μl W
300μl BSA
150μl
Dil. Ratio: 1
Conc. : 2mg/mL
150μl W
150μl
1:2
1mg/mL
150μl
150μl
1:4
0.5mg/mL
150μl W
150μl W
1:8
0.25mg/mL
1:16
0.125mg/mL
Figure 1 Serial Dilution of protein standards BSA.
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2. Prepare 150μl of W. Label this as ‘blank’.
3. Using a micropipette and a white pipette tip, add 5μl of each protein standards prepared
in step 1 into separate wells in the 96 well plate. Add 5μl of ‘blank’ into another separate
well. Add 5μl of unknown samples A, B and C into separate wells. Take note of the the
location of each sample put into 96 well plate (Refer figure 2).
4. Prepare triplicates (3x wells) for each of the protein standard, blank and unknown
samples. Label first attempt as A1, second attempt as A1 and third attempt as A3 (Refer
figure 2).
Figure 2 96 well plate containing protein standards, blank, and
unknown samples A, B and C.
5. To every used well, add 200μl of the Bradford Reagent and carefully mix the solutions for
30 seconds.
6. Leave the samples to incubate for 5 minutes at room temperature.
7. Using a spectrophotometer, measure the absorbance of of the samples at 595nm.
Record absorbance of samples within 60 minutes after step 5.
8. Plot Standard Curve of Net Absorbance at 595nm of protein standards and blank
against protein concentration of protein standards and blank. Then, determine the
trend line equation and R-value of the line.
9. Using the trend line equation and comparing the values of absorbance of unknown
samples A, B and C with the graph, calculate the concentration of unknown samples
(Lee, 2017; Nouroozi et al., 2015).
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Results
Table 1 Concentrations of protein standards and blank and colour observation of the
standards and blank after Bradford reagent is added.
Standards
Concentration (mg/ml)
Colour of Solution
Blank
0.00
Pale purple
Standard 1
2.00
Deep purple
Standard 2
1.00
Purple
Standard 3
0.50
Less purple than standard 2
Standard 4
0.25
Pale purple
Standard 5
0.125
Pale purple
Table 2 Net Absorbance of Samples of Protein Standards, Blank and Unknown Samples at
595 nm and The Average Net Absorbance of Every Sample.
Avg
Standard
Absorbance T1
Absorbance T2
Absorbance T3
Absorbance
Blank
0
0
0
0
Standard 1
0.406
0.282
0.463
0.384
Standard 2
0.191
0.194
0.179
0.188
Standard 3
0.158
0.084
0.06
0.101
Standard 4
0.037
0.064
-0.001
0.033
Standard 5
0.078
0.029
-0.016
0.030
Sample A
0.093
0.123
0.072
0.096
Sample B
0.206
0.308
0.197
0.237
Sample C
0.243
0.306
0.236
0.262
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Absorbance 595 nm
0.4
y = 0.192x - 0.0013
R² = 0.9973
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0
0.5
1
1.5
2
2.5
BSA standard (mg/ml)
Figure 3
Standard Curve of Net Absorbance of Protein Standards and Blank at 595nm
against The Concentration of BSA Standard.The data is fit with the equation y = 0.192x –
0.0013 and has an R² value of 0.9973. The table presented in Table 2 provides the data used
for creating the standard curve illustrated in figure 3.
Calculations:
For Sample A,
0.096 = 0.192x – 0.0013
x = 0.50677
Sample A has a concentration of 0.507 mg/mL.
For Sample B,
0.237 = 0.192x – 0.0013
x = 1.24115
Sample B has a concentration of 1.24 mg/mL.
For Sample C,
0.262 = 0.192x – 0.0013
X = 1.37135
Sample C has a concentration of 1.37 mg/mL.
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Discussions
As shown in table 1 and figure 3, it can be concluded that as the BSA concentration
increases, the colour saturation of protein standard solutions increases. As a result, the
absorbance value at 595 nm increases. According to figure 3, it can be said that the
absorbance value is directly proportional to the BSA concentration.
After calculating the protein concentrations of unknown samples A, B and C using the trend
line equation, it is clear that concentration of sample A which is 0.507 mg/mL is slightly
higher than that of standard 3. Meanwhile, unknown sample B which contains 1.24 mg/mL of
protein is higher than that of standard 2. Lastly, sample C has a protein concentration higher
than that of standard 2 (1.37 mg/mL).
However, there are slight contradictions in the raw absorbance results as illustrated in table
2. Few of the results do not follow the trend predicted and this is due to the measurement
error that arise when more than one people are involved in micro pipetting the solutions and
also from confusion because of the slight differences in colour that are hard to differentiate
which in turn results in the transferring of the incorrect solution to the 96 well plate.
As a method to reduce errors, each solution is triplicated and average of net absorbance of
each solution is taken. Other than that, only one person should do the transferring in order to
reduce inconsistencies in volume of solution taken up. Prior planning should be done to
avoid confusion.
In conclusion, this experiment supports the hypothesis that as the protein concentration
increases, absorbance also increases. Hence, this proves that Bradford Assay is an
accurate and reliable method to measure the protein concentration in solutions (Kielkopf et
al., 2020; Nouroozi et al., 2015).
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References
Gazzola, D., Vincenzi, S., Pasini, G., Lomolino, G., & Curioni, A. (2015). Advantages of the KDS/BCA
Assay over the Bradford Assay for Protein Quantification in White Wine and Grape Juice.
American Journal of Enology and Viticulture, 66(2), 227–233.
https://doi.org/10.5344/ajev.2014.14076
Goldring, J. P. D. (2019). Measuring Protein Concentration with Absorbance, Lowry, Bradford
Coomassie Blue, or the Smith Bicinchoninic Acid Assay Before Electrophoresis (pp. 31–39).
https://doi.org/10.1007/978-1-4939-8793-1_3
Kielkopf, C. L., Bauer, W., & Urbatsch, I. L. (2020). Bradford Assay for Determining Protein
Concentration. Cold Spring Harbor Protocols, 2020(4), pdb.prot102269.
https://doi.org/10.1101/pdb.prot102269
Kovacs, N. A., Petrov, A. S., Lanier, K. A., & Williams, L. D. (2017). Frozen in Time: The History of
Proteins. Molecular Biology and Evolution, 34(5), 1252–1260.
https://doi.org/10.1093/molbev/msx086
Ku, H.-K., Lim, H.-M., Oh, K.-H., Yang, H.-J., Jeong, J.-S., & Kim, S.-K. (2013). Interpretation of protein
quantitation using the Bradford assay: Comparison with two calculation models. Analytical
Biochemistry, 434(1), 178–180. https://doi.org/10.1016/j.ab.2012.10.045
Lee, A. (2017). Protein Measurement using Bradford Assay and Photopette.
Nouroozi, R. V., Noroozi, M. V., & Ahmadizadeh, M. (2015). Determination of Protein Concentration
Using Bradford Microplate Protein Quantification Assay .
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