Comparison of the Lowry and Bradford Protein Assays

Session date: Name:

Submission date: Partner:

Group: Demonstrator:

Aim:
 Estimating the concentration of two unknown protein solutions containing either
Bovine Serum Albumin (BSA) or Bovine Gamma Globulin (IgG) by the both Lowry
and Bradford assays and spectrophotometry, using a standard curve generated by
using a standard solution of Bovine Serum Albumin (BSA).
 Comparing the sensitivity and specificity of both Lowry and Bradford assays with the
help of the obtained results.
Method:
Refer to Biochemistry and Molecular Biology 1 Lab Manual, session 2 pages 29-33 in
year 2020 semester 1. A moderation was done to step 1 part (iii), where instead of
vortex mixer, solutions were mixed manually using paraffin papers.

Results / Calculations / Discussion:

The Lowry assay

Table 1. Absorbance and total protein concentration values for tubes 1-12 for Lowry
assay.

Tube 1 2 3 4 5 6
Standard BSA (50µg/mL),mL 0 0 0.1 0.1 0.2 0.2
H2O, mL 0.5 0.5 0.4 0.4 0.3 0.3
Final volume/tube, mL 0.5 0.5 0.5 0.5 0.5 0.5
Final [BSA] (µg/mL) 0 0 10 10 20 20
Absorbance (690 nm) 0.0520 0.0520 0.0840 0.0830 0.1100 0.1090
Absorbance - blank 0.0000 0.0000 0.0320 0.0310 0.0580 0.0570

Tube 7 8 9 10 11 12
Standard BSA (50µg/mL),mL 0.3 0.3 0.4 0.4 0.5 0.5
H2O, mL 0.2 0.2 0.1 0.1 0 0
Final volume/tube, mL 0.5 0.5 0.5 0.5 0.5 0.5
Final [BSA] (µg/mL) 30 30 40 40 50 50
Absorbance (690 nm) 0.1280 0.1350 0.1630 0.1670 0.1940 0.1900
Absorbance - blank 0.0760 0.0830 0.1110 0.1150 0.1420 0.1380

Table 2. Absorbance and unknown protein concentrations for tubes 13-24 for Lowry Method.
Tube 13 14 15 16 17 18

BSA unknown (undiluted), mL 0.5 0.5

BSA unknown ( 1/2 ), mL 0.5 0.5

BSA unknown ( 1/4 ), mL 0.5 0.5

Absorbance (690 nm) 0.211 0.203 0.133 0.121 0.096 0.095

Absorbance - blank 0.159 0.151 0.081 0.069 0.044 0.043

Unknown protein concentration

- - 28.9 24.5 15.4 15.1
(µg/mL)

Mean Protein Concentration - 26.7 15.3

Tube 19 20 21 22 23 24

IgG unknown (undiluted), mL 0.5 0.5

IgG unknown ( 1/2 ), mL 0.5 0.5

IgG unknown ( 1/4 ), mL 0.5 0.5

Absorbance (690 nm) 0.202 0.193 0.125 0.116 0.088 0.098

Absorbance - blank 0.150 0.141 0.073 0.064 0.036 0.046

Unknown protein concentration

22.8 25.9 12.4 16.2
(µg/mL)

Mean Protein Concentration 24.4 14.3

The Bradford assay

Table 3. Absorbance and total protein concentration values for tubes 1-12 for Bradford
assay.

Tube 1 2 3 4 5 6
Standard BSA (50µg/mL),mL 0 0 0.1 0.1 0.2 0.2
H2O, mL 0.5 0.5 0.4 0.4 0.3 0.3
Final volume/tube, mL 0.5 0.5 0.5 0.5 0.5 0.5
Final [BSA] (µg/mL) 0 0 10 10 20 20
Absorbance (595 nm) 0.3720 03780 0.5030 0.5010 0.5760 0.6100
Absorbance - blank 0.000 0.000 0.128 0.126 0.201 0.235

Tube 7 8 9 10 11 12
Standard BSA (50µg/mL),mL 0.3 0.3 0.4 0.4 0.5 0.5
H2O, mL 0.2 0.2 0.1 0.1 0 0
Final volume/tube, mL 0.5 0.5 0.5 0.5 0.5 0.5
Final [BSA] (µg/mL) 30 30 40 40 50 50
Absorbance (595 nm) 0.6930 0.6890 0.7650 0.7320 0.8120 0.8190
Absorbance - blank 0.318 0.314 0.390 0.357 0.455 0.444

Table 4. Absorbance and unknown protein concentrations for tubes 13-24 for Bradford
Method.
Tube 13 14 15 16 17 18

BSA unknown (undiluted), mL 0.5 0.5

BSA unknown ( 1/2 ), mL 0.5 0.5

BSA unknown ( 1/4 ), mL 0.5 0.5

Absorbance (595 nm) 0.868 0.888 0.657 0.643 0.553 0.539

Absorbance - blank 0.493 0.513 0.282 0.268 0.178 0.164

Unknown protein concentration

28.9 27.3 16.8 18
(µg/mL)

Mean Protein Concentration 28.1 17.4

Table 4. Absorbance and unknown protein concentrations for tubes 13-24 for Bradford
Method. (continued)

Tube 19 20 21 22 23 24

IgG unknown (undiluted), mL 0.5 0.5

IgG unknown ( 1/2 ), mL 0.5 0.5

IgG unknown ( 1/4 ), mL 0.5 0.5

Absorbance (595 nm) 0.599 0.609 0.486 0.505 0.399 0.381

Absorbance - blank 0.224 0.234 0.111 0.130 0.024 0.006

Unknown protein concentration

23 23.9 10.9 13.1 - -
(µg/mL)

Mean Protein Concentration 23.5 12 -

On the same graph, plot both the Bradford and Lowry standard curves for BSA. Label this
Figure 1 and give an appropriate title. Don't forget to use suitable labels for the axes.

1. Are there any significant discrepancies between the two standard curves?

Standard curves for both Lowry and Bradford assays are Linear.
However, Standard curve for Bradford assay is steeper than the Lowry assay. Steeper the
curve more accurately you can read the values for concentration.

2. Using the standard curves drawn, calculate the final protein concentration of the
BSA unknown as read from each of the standard curves. Which dilution of the
unknown did you use in your calculations above, and why?

Lowry Assay
Unknown protein concentration = 15.3µg/ml × 4 = 61.2 µg/ml
To obtain an accurate value for the protein concentration, tube with least dilution
should use as it will cause less errors. Tubes 13, 14, 15 & 16 are not used as the
absorbance values did not fall within the range of the assay’s standard curve for tubes
and 13 and 14. In case of 15 and 16 data points in the standard curve lies far apart
which can lead to an error. As a result, tubes 17 & 18 are used for calculations.

Bradford Assay
Unknown protein concentration = 28.1 µg/ml × 2 = 56.2 µg/ml
 To obtain an accurate value for the protein concentration, tube with least dilution
should use as it will cause less errors. Tubes 13, 14 are not used as the absorbance
values did not fall within the range of the assay’s standard curve. for tubes and 13 and
14. In case of 17 and 18 are the most diluted. As a result, tubes 15 & 16 are used for
calculations.

Assay BSA (g/ml)

Lowry 61.2
Bradford 56.2

3. Repeat the above calculations for the IgG unknown. Which dilution of the unknown
did you use in your calculations above, and why?

Lowry Assay
Unknown protein concentration = 24.4 µg/ml × 2 = 48.8 µg/ml
To obtain an accurate value for the protein concentration, tube with least dilution
should use as it will cause less errors. Tubes 13, 14 are not used as the absorbance
values did not fall within the range of the assay’s standard curve. In case of 17 and 18
are the most diluted tubes. As a result, tubes 15 & 16 are used for calculations.

Bradford Assay
Unknown protein concentration = 23.5 µg/ml
To obtain an accurate value for the protein concentration, tube with least dilution
should use as it will cause less errors. As a result, tubes 13 & 14 are used for
calculations.

Assay IgG (g/ml)

Lowry 48.8
Bradford 23.5

4. Comment on the results obtained.

Assay BSA (g/ml) IgG (/ml)

Lowry 61.2 48.8
Bradford 56.2 23.5

results obtained for Lowry [BSA] and [IgG] is similar. An error has occurred during
preparation of samples or during the calculations.
 results obtained for Bradford [BSA] and [IgG] is similar. An error has occurred during
preparation of samples or during the calculations.

Summary table for the Lowry, Biuret and Bradford protein assays

Complete the following table:

Table 5. Comparison between Biuret, Lowry and Bradford assays.

Biuret Lowry Bradford

(see the results
from Session 1)

Sensitivity
What was the lowest protein 1.97mg/ml 12.4g/ml -1.86g/ml
concentration detected?

Effect of protein species No No, BSA and No, BSA and

IgG have IgG have
Is BSA an appropriate standard
different different
to use universally in all assays?
concentrations concentrations
(compare Absorbances for
same concentration of BSA and
IgG in today’s session)

Linearity Range From 0 mg to 5mg From 0 g to From 0 g to 50g

Over what range of protein 50g
concentrations was the assay
linear?
Practical issues
How long did assay take? 20min 30min 5min

How many reagents were Biuret assay Copper and Bradford assay
needed? Folin reagent

5. If you were required to measure protein solutions in the range of 1.0 – 5.0 µg/mL,
which protein assay would you use? Why?

Bradford or Lowry assays are more sensitive and specific to find protein solutions between 1.0 to
5.0g /ml. But the Biuret assay is useful to find all protein concentration as it binds to peptide bonds.
6. Which assay gave different absorbances for two different proteins of the same
concentration? Explain the results obtained and comment on why this occurred.

Both assays gave different absorbances for the two different proteins in the same
concentration (refer to tables 2 and 4).
There may be errors that can account for the different absorbances with the unknown
concentration of the BSA and IgG proteins such as pipetting issues (i.e. air bubbles) or dirty
microplates may have cause errors in the absorbance readings of the protein.

7. What would you need to do in the laboratory to confidently estimate the protein
concentration of the IgG unknown using the Bradford method?

In order to confidently estimate the unknown IgG concentration, use IgG standard to perform the
assay.

Conclusion

Aim of this experiment is to estimate the concentration of two unknown protein solutions
containing either Bovine Serum Albumin (BSA) or Bovine Gamma Globulin (IgG) by the both
Lowry and Bradford assays and spectrophotometry, using a standard curve generated by
using a standard solution of Bovine Serum Albumin (BSA). And compare the sensitivity and
specificity of both Lowry and Bradford assays with the help of the obtained results.
Unknown protein concentration (BSA) in Lowry assay is 48.8 µg/ml and for Bradford it’s 56.2
µg/ml. IgG Unknown protein concentration for Lowry assay is 48.8 µg/ml and for Bradford
assay it’s 23.5 µg/ml. In conclusion, the experiment to find the unknown concentrations of
IgG and BSA using the Bradford and Lowry assays was successful but had flaws such as
practical issues (pipetting accurately and efficiently) and calculation errors.