Jonathan Nguyen

Megan Ringling

MCB253

09 February 2024

Background:
The purpose of this experiment is to determine the concentration of the unknown BSA
protein. We will be utilizing the concept of protein-dye binding in a Bradford Assay to carry out
the experiment. The Bradford assay technique “is based on the observation that Coomassie
Brilliant Blue G-250 exists in two different color forms, red and blue. The red form is converted
to the blue form upon binding of the dye to protein. The protein-dye complex has a high
extinction coefficient thus leading to great sensitivity in measurement of the protein. The binding
of the dye to protein is a very rapid process (approximately 2 min)... thus making the procedure
very rapid” (Bradford, 1976, p. 249). The Bradford assay’s ability to measure protein
concentration is based on the shift in absorbance maximum of Coomassie Brilliant Blue G-250
dye from 465 to 595 nm following binding to proteins in solution.

Hypothesis: If I produce a standard curve of concentration vs. absorbance with an R^2 value
greater than 0.95, then the predicted concentration of the unknown BSA protein #1 that we
calculate should be accurate.

Procedure:

1. Make 50 µl of each BSA standard as shown in Table #1

2. 1.2 mL of Bradford dye (coomassie blue) into each cuvette using the P1000 pipette
3. Add 24 μL of your BSA standard or blank PBS using P200 pipette
4. Mix thoroughly with pipette
5. Wait for 10 min at room temperature
6. Read absorbance of each cuvette in spectrophotometer at 595 nm
7. Create a graph of BSA concentration vs. absorbance
a. Make sure to show graph equation and R squared value
8. Calculate concentration of unknown samples using graph equation
9. Repeat steps 2-6 for the 7 unknown dilution samples.
a. 100 μL PBS, 0 μL unknown BSA
b. 1 fold: 50 μL PBS, 100 μL unknown BSA
c. 2 fold: 50 μL PBS, 50 μL of 1 fold dilution
d. 4 fold: 50 μL PBS, 50 μL of 2-fold dilution
e. 8 fold: 50 μL PBS, 50 μL of 4-fold dilution
 f. 16 fold: 50 μL PBS, 50 μL of 8-fold dilution
g. 32 fold: 50 μL PBS, 50 μL of 16-fold dilution

Data/Dilution Charts:

Table #1

[BSA] Standard PBS Buffer (μL) Stock BSA (μL) Absorbance @ Concentration
(mg/mL) (2 mg/mL) 595 nm (mg/mL)

0 50 0

0.25 43.75 6.25

0.50 37.5 12.5

0.75 31.25 18.75

1.0 25 25

1.25 18.75 31.25

1.50 12.5 37.5

Table #2

Serial Dilutions PBS Buffer (μL) Unknown BSA Absorbance @ Concentration

#1 (μL) 595 nm (mg/mL)

0 100 0

1 fold 50 100

2 fold 50 50 unknown

4 fold 50 50 of 2-fold

8 fold 50 50 of 4-fold

16 fold 50 50 of 8-fold

32 fold 50 50 of 16-fold
<Insert standard graph here>

Explanation of Expected Results:

After producing our BSA standard samples, we expect an overall increase in
concentration and absorbance correlated with an increase in BSA standard concentration. We
expect this since concentration is directly related to absorbance and increasing the concentration
of BSA (protein) would increase the absorbance of the solution and consequently, the
concentration. After producing our BSA unknown samples, we expect an overall decrease in
concentration and absorbance after each subsequent dilution since we will be adding a smaller
amount of unknown BSA while maintaining the same volume of PBS buffer (50 μL) for each
serial dilution. After adding our protein, the Coomassie Blue dye should change color from
reddish to blue which indicates the presence of protein. We should produce a standard curve
graph of concentration vs. absorbance of BSA that has a R^2 value greater than 0.95 because this
would allow us to accurately calculate the concentration of unknown protein #1. Some possible
sources of error that may affect the accuracy of our measurement of protein absorbance include
the loading time of our cuvettes in the spectrophotometer, impurities in the cuvette, cuvette
material, and dye temperature.
References:

Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities
of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1–2),
248–254. https://doi.org/10.1006/abio.1976.9999