International University - Vietnam National University, HCMC
School of Biotechnology - Department of Applied chemistry
REPORT 2
QUANTITATIVE DETERMINATION OF PROTEIN
CONCENTRATION
Course: Methods in Biochemistry Laboratory
Semester 2 (2023 – 2024)
Instructor: MSc. Le Tran Hong Ngoc
TA: Trinh Thi Xuan
Group number: 4
Full name Student ID
Nguyễn Võ Minh Ngọc BTBCIU21082
Lâm Vân Nghi BTBCIU21079
Nguyễn Trần Thiên Ân BTBCIU21065
Trương Hoàn Mỹ BTBCIU21054
Date of submission: 04/04/2024
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International University - Vietnam National University, HCMC
School of Biotechnology - Department of Applied chemistry
ABSTRACT
Bradford method is one of the most common colorimetric ones for protein quantitation. Thus, it
was also the test that this lab used for the same purpose via 96-well plate assay. For mechanism,
Bradford reagent helped form a complex with protein so that the solution could appear in a visible
blue color. Importantly, a protein standard curve needed constructing first based on one blank,
five standard protein points with different concentrations, which relatively were 0.0625 mg/ml,
0.125 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1.0 mg/ml, and their absorbance at 595 nm wavelength.
Statistically, the graph gave R2 value near one (R2 = 0.9895), which meant the data were ideal for
predicting the concentration of unknown sample B (averagely 0.788 mg/ml) after evaluating the
OD. However, despite the ideality of the standard curve’s statistics, there was a point below the
blank point, which regressed the simple theory about the relationship between concentration and
OD due to unwell sample mixing or careless preparation.
INTRODUCTION
Before undergoing the analysis process, the protein samples need their concentration
quantification. There are many colorimetric methods for protein quantification, each of which
brings advantages and disadvantages. On top of that, the Bradford or Coomassie blue test is
common as it can work well in a small-scale sample (suitable for lab work), is fast and is
compatible with reducing agents in the buffer. In this test, Coomassie blue dye is used to make the
protein samples appear in blue so that the samples can be done the absorbance measurement at
595 nm.
Due to the Bradford test benefits, it is used to construct several assays, such as the Standard 3.1
ml assay, the Micro 2 ml assay, and the 96-well plate assay. To prevent time consumption, this lab
just carried out only 96 Well plate assay as it was convenient for groups in class to perform and
have absorbance estimation altogether. The procedure started with five protein standards (the
concentration range would be from 0 to 1.4 mg/ml) and three sample B (unknown protein
concentration) preparations in a 96-well plate to add the Bradford reagent and absorbance
quantification at 595 nm. Purposely, the protein standards were used to plot a standard curve to
predict the concentration of sample B. The perfect standards would result in a regression value
(R2) close to one and the data arrangement raised along the x-axis, which means the more protein
concentration the sample has, the more absorbance should be recorded theoretically.
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International University - Vietnam National University, HCMC
School of Biotechnology - Department of Applied chemistry
MATERIALS AND METHOD
Materials
Chemicals Equipment
● Buffer ● P10, P100 micropipettes
● Bradford reagents ● Eppendorf
● Stock standard BSA solution (5 ● 96-well plates
mg/ml) ● Spectrophotometer
● Protein sample B ● Vortex
● Marker
Method
Firstly, the protein standards were prepared in five eppendorfs numerically marked from BSA
solution and buffer with different concentrations using serial dilution technique and they were all
mixed well before getting to the next step.
Table 1. The concentration of each eppendorf for serial dilutions.
Eppendorf Protein concentration (mg/ml)
1 0.0625
2 0.125
3 0.25
4 0.5
5 1.0
Next, five μL of each standard, blank, and three times five μL of sample B was added into the 96
well plates along with the addition of Bradford reagents. Afterward, the plate was incubated for
about 5 minutes in advance to absorbance recorded at 595 nm.
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International University - Vietnam National University, HCMC
School of Biotechnology - Department of Applied chemistry
RESULTS
96 Well plate assay Protocol
Table 2: The concentration and absorbance of protein (BSA) standards in serial dilutions.
Eppendorf Concentration (mg/mL) Absorbance
Blank 0 0.431
1 0.0625 0.43
2 0.125 0.437
3 0.25 0.522
4 0.5 0.643
5 1 0.921
Figure 1: Standard curve of protein (BSA) using serial dilution technique.
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International University - Vietnam National University, HCMC
School of Biotechnology - Department of Applied chemistry
Table 3: The absorbance of unknown samples in serial dilutions.
Eppendorf Absorbance
1 0.796
2 0.813
3 0.798
Average (y) 0.8023
Calculation for unknown sample B:
y = 0.5121x + 0.3986 and y = 0.8023
⇔ 0.8023 = 0.5121x + 0.3986
⇔ x = 0.788 (mg/mL)
Therefore, the average concentration of protein in unknown sample B was 0.788 mg/mL.
DISCUSSION
The Bradford assay stands as a cornerstone in protein quantitation methodologies, offering a
straightforward and reliable means of determining protein concentration in solution. Its
mechanism hinges upon the interaction between proteins and the Coomassie Brilliant Blue G dye
present in the Bradford reagent. Upon binding with proteins, the dye undergoes a color change,
resulting in a shift in absorbance from 465 nm to 595 nm, which is proportional to the protein
concentration. However, the assay's sensitivity to detergents poses a challenge, as detergents can
disrupt the protein-dye binding process, leading to inaccurate readings. Consequently, meticulous
sample preparation is imperative to mitigate such interferences.
The decision to utilize a serial dilution technique in creating standard protein solutions was
strategically sound because serial dilution enabled the creation of a comprehensive range of
concentrations from a solitary stock solution. This systematic dilution process conducted
incrementally, allowed for the generation of standards covering a wide spectrum of
concentrations efficiently. This broad range was pivotal for constructing a standard curve that
precisely delineated the relationship between absorbance and protein concentration. In contrast,
the separation dilution approach entailed the preparation of standards from multiple independent
stock solutions, which may have proved less expedient and labor-intensive, especially when
aiming to encompass a broad concentration range. Moreover, serial dilution provided enhanced
control over the dilution process, thereby yielding more precise and reproducible outcomes. Each
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International University - Vietnam National University, HCMC
School of Biotechnology - Department of Applied chemistry
dilution step was meticulously executed, allowing exact adjustments in concentration. This
meticulousness was imperative for ensuring the robustness of the standard curve and the accuracy
of protein concentration determinations in unknown samples. Furthermore, serial dilution
mitigated the risk of introducing variability associated with the preparation of multiple stock
solutions. By utilizing a singular stock solution for serial dilution, consistency in the starting
material is maintained, thereby reducing potential disparities between standards attributable to
variations in preparation methodologies or reagent quality. In this experimental outcome, a
discernible issue arose with the eppendorf one, wherein its absorbance registered even lower than
the baseline absorbance of the blank solution (0.43 < 0.431). This discrepancy could be attributed
to an initial error in selecting an inadequately low protein concentration, compounded by
inconsistent agitation before plate addition. Subsequent eppendorfs improved results, indicative
of the assimilation of knowledge gleaned from the initial endeavor. Despite that, the regression
value (0.9895) sounded ideal for any sample prediction. The average concentration of sample B
0.788 mg/ml, which lies between the range that Bradford reagent could work for 96-well plate
assay.
CONCLUSION
Based on the results of our protein concentration determination using the Bradford assay, the
average concentration of unknown sample B was 0.788 mg/mL. The group successfully used the
serial dilution technique to create a standard curve, which allowed us to accurately relate
absorbance values to protein concentrations. This systematic approach enabled us to generate a
wide range of protein standards efficiently, ensuring the reliability and precision of our assay. It
was important to note that careful sample preparation was crucial to avoid any interference,
especially unwell mixing and detergents, which can affect the accuracy of our readings.
In summary, the Bradford assay is a simple and reliable method for quantifying protein in
solution. By using the serial dilution technique to create our standard protein solutions, we were
able to obtain a comprehensive range of concentrations and construct a robust standard curve.
These findings contribute to the understanding and application of protein quantification in the
field of biochemistry.
REFERENCES
[1] Experiment 2 Quantitative determination of protein concentration. Methods in Biochemistry
lab manual.
[2] A. (2022, July 11). Dilution - Dilutions of Solutions - Definition; Meaning, Formula, Methods
&amp; Importance of Dilution. BYJUS.
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International University - Vietnam National University, HCMC
School of Biotechnology - Department of Applied chemistry
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