BBL433 (Enzyme Science and Engineering)
Experiment 1
Protein Quantification and Pipette Calibration
Experiment Date: 10 Jan 2025, Friday
Report Submission Date: 24 Jan 2025, Friday
Group Members:
1]Arsh Gupta [2022BB11387]
2]Prashant Kumar [2022BB11409]
Aim: Protein Quantification Using the Bradford Assay
INTRODUCTION: The Bradford assay is a widely used colorimetric method for protein
quantification due to its simplicity and sensitivity. It relies on the binding of Coomass Brilliant Blue G-
250 dye to proteins, causing a measurable color change proportional to protein concentration. This
experiment utilized the Bradford assay to determine the protein concentration of an unknown sample
by generating a standard curve with bovine serum albumin (BSA). Accurate protein quantification is
essential in various biochemic applications
PRINCIPLE:
The Bradford method is a colorimetric assay used to quantify protein concentration based on the binding
of Coomassie Brilliant Blue (CBB) dye to proteins. The principle of the Bradford assay involves the
following steps:
Dye Binding: The Coomassie Brilliant Blue G-250 dye binds to proteins primarily through ionic and
hydrophobic interactions, especially with arginine residues and the aromatic rings of tryptophan,
tyrosine, and phenylalanine.
Color Change: In its unbound form, the dye is reddish-brown. When the dye binds to proteins, it
undergoes a shift in its absorption maximum from 465 nm (reddish-brown) to 595 nm (blue), with the
intensity of the color being directly proportional to the protein concentration.
Measurement: The absorbance of the solution is measured at 595 nm using a spectrophotometer. A
standard curve, created using known concentrations of a protein standard (commonly bovine serum
albumin, BSA), is used to determine the protein concentration in the unknown sample.
The Bradford method is widely used due to its simplicity, rapidity, and sensitivity, particularly for
protein concentrations in the range of 1-100 µg/mL. However, it can be influenced by the presence of
detergents or other compounds in the sample.
MATERIALS & EQUIPMENTS:
1. Spectrophotometer
2. Weighing balance
3. Cuvettes
4. Pipettes and Tips
5. Microcentrifuge tubes (Eppendorfs)
6. Test tubes (Falcons)
REAGENTS
1. Bradford reagent
2. Bovine serum albumin (BSA) – 2mg
3. Standard solutions of protein
4. Distilled water
PROCEDURE
1. Dilute the stock solution of protein (2 mg/mL) in the range of 50 to 1000 µg/mL using
distilled water to prepare the standards. Prepare each stock with a final volume of 100µL.
Note: You can either use the standards of concentration - 62.5, 125, 250, 500, 1000 µg/mL
or prepare your own choice.
2. After preparing the dilutions and blank (water and Bradford reagent) sample, add 1mL of
Bradford reagent to each tube along with the unknown samples. Incubate for 10 minutes at
room temperature away from light.
3. Set the spectrophotometer to 595nm. Zero the instrument using a blank sample. Measure
the absorbance of the standards and unknown samples.
4. Plot the absorbance vs protein concentration graph
OBSERVATIONS
mg
Given The concentration of BSA in the stock solution = 2
mL
We know that from mole balance,
𝑀1𝑉1 = 𝑀2𝑉2
where:
𝑀1 = Molarity or concentration of given Stock solution of BSA
𝑀2 = Concentration of BSA in the solution
𝑉2 = Final Volume of BSA solution in 100 𝜇𝐿 final solution
V1 = Volume of stock to be added = 𝑉2 (𝜇𝐿) * 𝑀2 (𝜇g/m𝐿)
𝑀1 (mg/mL)
V1 = 𝑉2 * 𝑀2 ∗ 10–3
𝜇𝐿
𝑀1
(This equation provides the necessary volume of the stock solution (in 𝑚𝐿) to be added to
achieve the desired concentration 𝑀2 in the final BSA solution during dilution to 100 𝜇𝐿 )
𝑉3 = Volume of water to be added for dilution to 100 𝜇𝐿 final solution
M1 (mg/mL) M2 (𝜇g/mL) 𝑽𝟐(𝝁𝑳) 𝑽𝟏(𝝁𝑳)
2 62.5 100
62.5 ∗ 100 ∗ 10–3 = 3.125 𝜇𝐿
=
2
2 125 100
125 ∗ 100 ∗ 10–3 = 6.25 𝜇𝐿
=
2
2 250 100
250 ∗ 100 ∗ 10–3 = 12.5 𝜇𝐿
=
2
2 500 100
500 ∗ 100 ∗ 10–3 = 25 𝜇𝐿
=
2
2 1000 100
1000 ∗ 100 ∗ 10–3 = 50 𝜇𝐿
=
2
𝑽𝟏(𝝁𝑳) 𝑽𝟑(𝝁𝑳)
3.125 𝜇𝐿 = (100 – 3.125) 𝜇𝐿 = 96.875 𝜇𝐿
6.25 𝜇𝐿 = (100 – 6.25) 𝜇𝐿 = 93.95 𝜇𝐿
12.5 𝜇𝐿 = (100 – 12.5) 𝜇𝐿 = 87.5 𝜇𝐿
25 𝜇𝐿 = (100 − 25) 𝜇𝐿 = 75.0 𝜇𝐿
50 𝜇𝐿 = (100 − 50) 𝜇𝐿 = 50.0 𝜇𝐿
Sample number 𝝁𝒈 Absorbance
𝑴𝟐( )
𝒎𝑳 (at 𝟓𝟗𝟓 𝒏𝒎)
01 62.5 0.143
02 125 0.307
03 250 0.547
04 500 0.834
05 1000 1.216
DF= 2 0.608
T1 MT1 0.895
T2 MT2 0.887
CALCULATIONS:
To calculate the concentration of given unknown proteins, we will use the equation of line
obtained from the graph where we estimated the protein concentrations of known proteins.
Line equation: 𝑦 = 0.0011x + 0.1852
∴ 𝑚 = 0.0011 (Slope of the line)
Since we know the OD of the unknown proteins (𝑦), we can use the line equation to
estimate its concentration.
For T1,
Absorbance (OD) = 𝑦1 = 0.895
𝑦1 = 0.0011𝑥1 + 0.1852
0.895 = 0.0011𝑥1 + 0.1852
μg
∴ 𝑥1 =645.27( ) is the concentration of T1 protein
mL
, where:
μg
𝑥1 = T1 unknown protein concentration in (mL)
𝝁𝒈
∴ 𝒙𝟏 = 645.27 ( ) is the concentration of T1 protein
𝒎𝑳
𝑦1 = Absorbance (OD) observed for undiluted T1 in spectrophotometer
For T2,
Absorbance (OD) = 𝑦2 = 0.887
𝑦2 = 0.0011𝑥2 + 0.1852
0.887 = 0.0011𝑥2 + 0.1852
𝝁𝒈
∴ 𝒙𝟐 = 638 ( ) is the concentration of T2 protein
𝒎𝑳
, where:
μg
𝑥2 = T2 unknown protein concentration in ( )
mL
𝝁𝒈
∴ 𝒙𝟐 = 638 ( ) is the concentration of T2 protein
𝒎𝑳
𝑦2 = Absorbance (OD) observed for undiluted T2 in spectrophotometer
Sample number M2 (𝜇g/mL) Absorbance (at 𝟓𝟗𝟓 𝒏𝒎)
01 62.5 0.143
02 125 0.307
03 250 0.547
04 500 0.834
05 1000 1.216
T1 645.27 0.895
T2 638 0.887
RESULTS
Equation of Line plotted between Absorbance vs Protein Concentration: 𝒚 = 𝟎. 𝟎𝟎11𝒙 +
𝟎. 1852
∴ 𝒎 = 𝟎. 𝟎𝟎11 (Slope of the line): The calibration curve's slope (0.0006) indicates a
relatively low absorbance change per unit concentration change, emphasizing the sensitivity
of the assay.
𝝁𝒈
Estimated Concentration of T1 protein = 645.27 𝒎𝑳)
(
𝝁𝒈
Estimated Concentration of T2 protein = 638 ( )
𝒎𝑳
DISCUSSION & SOURCES OF ERROR
Assumptions in Calibration Curve: The accuracy of the estimated concentrations relies on
the assumption that the relationship between absorbance and concentration is linear within
the tested concentration range.
The observed deviation from linearity in the standard curve for the Bradford assay maybe
because of several factors. Potential sources of non-linearity include,
i) Pipetting errors during the preparation of standard solutions
ii) Incomplete mixing of the Bradford reagent and samples leading to uneven colour
development
iii) Inconsistencies in the incubation time
iv) Bradford reagent concentration and issues related to detergent interference or
the presence of substances outside the linear range of the assay may contribute
to non-linearity.
v) Proper blank correction and calibration of the spectrophotometer at the selected
wavelength are essential for accurate absorbance readings.
vi) Re-evaluating these experimental aspects and addressing potential sources of
error can enhance the reliability and linearity of the standard curve, leading to
more accurate protein concentration determinations.
vii) Additionally, the Bradford assay is known to be sensitive to the composition of
the proteins being assayed, and different proteins may exhibit varying responses
to the dye used in the assay.
PRECAUTIONS:
• Used calibrated and accurate pipettes to ensure precise measurement of
reagents and sample volumes.
• Handled the Coomassie dye with care, and protected it from exposure to light.
Kept the dye stock solution in a dark container by covering it with aluminum foil.
• Maintained consistent acidic conditions during the assay. pH fluctuations can
affect the binding of the dye to proteins.
• Wear gloves and goggles .
• In case of contact with eyes, rinse immediately with plenty of water and seek
medical advice.
• In case of accident or if you feel unwell, seek medical advice immediately.
Attachment – photos for reading taken during lab
Part (B) : Pipette Calibration
Aim: To estimate the error percentage in pipette using distilled water
Introduction: This experiment aims to estimate the error percentage in a pipette by analyzing
the volume of distilled water it dispenses. Using distilled water's consistent density (1 g/mL),
the dispensed volume is determined gravimetrically and compared to the expected volume.
The calculated error percentage helps assess the pipette's accuracy, ensuring it meets
laboratory standards for reliable experimental results.
Principle: Under a constant temperature and atmospheric pressure, the density of distilled water is
constant. The volume of water can be determined by weighting dispensed water. Pipetting Error
happens usually due to human inaccuracies or device inefficiencies. This error can be erased by
calibrating it. The calibration of pipette is carried out by gravimetric method. When determining the
volume of water, the accuracy of measurements is effected by ambient temperature, atmospheric
pressure and relative humidity.Then the calculated volume of water is compared with the theoretical
volume to determine the accuracy and precision of the pipette.
Equipment’s Required: analytical balance with milligram precision, Pipette, tips, MCTs and
distilled water
Procedure:
1) Measured the weight of 1 μl and 0.1 μl volumes of water using weighing machine and
respective pipettes.
2) Used the density of water to calculate expected weight of water in the first pipette
3) Calculated the difference in weight and percentage error for the first pipet
4) Repeated the same procedure for the other pipette
Pipette 1 Pipette 2
(100-1000 μl) (20-200 μl)
R1(at 1000 μl & 100 μl) 1)1.009gm 1)0.097 gm
2)0.999gm 2)0.101gm
3)1.003gm 3)0.100gm
Average 1.004gm 0.099 gm
Expected 1 gm 0.1 gm
Percentage Error 0.4 % 1%
R2(at 500 μl & 20 μl) 1)0.514 gm 1)0.023gm
2)0.503gm 2)0.027gm
3)0.506gm 3)0.018gm
Average 0.508gm 0.023 gm
Expected 0.5 gm 0.0 gm
Percentage Error 0.8 % 0.3 %
Discussion and Conclusion: Poor precision and accuracy affect the quality control results.
Precision and accuracy were calculated for each pipette, the accuracy, standard deviation and CV
failed pre calibration and became less than standard value that shown above. After calibration for
precision and accuracy then compared with standard precision and total allowable error for each test
and found that results were affected after calibration. And before calibration, the results were not
reliable and silent failure occurred. After Calibration, we got idea of error for the device that can be
improved for further measurements.
Precautions :
• An apron and goggles should always be worn in the lab
• Never draw liquid into a pipet by mouth. Use a pipet bulb.
• The balances are delicate instruments and should be treated with care.
