Protein

Quantifying proteins using the Bradford method

The Bradford method is a quantitative protein assay method, based on the binding of a dye, Coomassie
Brilliant Blue, to a protein sample, and comparing this binding to a standard curve generated by the reaction
of known amounts of a standard protein, usually BSA.
For this assay, protein samples should be diluted in an appropriate buffer (generally the same buffer in which
they are dissolved). The BSA standard curve should be prepared using the same buffer.

Protocol 14. Bradford assay procedure Protocol 14

Materials
▼ ▼

BSA standard solution (1 mg/ml*)

Bradford assay dye reagent (available commercially, e.g., Bio-Rad Protein Assay Dye Reagent Concentrate,
cat. no. 500-0006)
Protein dilution buffer
▼

Proteins should be diluted in the buffer in which they are dissolved. Use the same buffer to prepare the
standard curve.

* Concentration of the BSA standard solution should be measured photometrically. A 1 mg/ml solution of BSA should have an A280 of 0.66.

1. Prepare a standard curve by pipetting together carefully the solution volumes listed in Table 3 overleaf
corresponding to 0, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg/ml BSA. The end volume of all samples
should be 200 µl.
2. Dilute an aliquot of the dye reagent concentrate 1:5 with distilled water†. Pipet 20 µl of each BSA dilution
into a plastic cuvette, add 1 ml diluted dye reagent, mix well, and incubate for 5 min at room temperature.
3. Measure the OD at 595 nm for each sample, and plot the standard curve.
4. Prepare a second standard curve by pipetting together carefully the solution volumes listed in Table 3
corresponding to 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg/ml BSA. The end volume of all samples should
be 200 µl.
5. Dilute an aliquot of the dye reagent concentrate 1:5 with distilled water.† Pipet 20 µl of each BSA dilution
into a plastic cuvette, add 1 ml diluted dye reagent, mix well, and incubate for 5 min at room temperature.
6. Measure the OD at 595 nm for each sample, and plot the standard curve. protocol continues overleaf

†
Stored in the dark, diluted dye reagent is stable for 2 weeks. Write the date of preparation on the bottle and cover with aluminum foil.

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Protocol 14. Continued

7. Prepare dilutions of the protein sample of interest and test 20 µl aliquots in duplicate as above. It is
important that the protein samples to be tested are handled in exactly the same manner as the samples
used in generating the standard curves. Calculate the protein concentration of the test samples by
comparing the OD595 with the standard curve(s), taking into account dilution factors (Figure 7).

Determining Protein Concentration From a Standard Curve

1.2

0.8
OD595

0.6
Sample OD595
0.4
Read off protein
0.2 concentration

0
0 0.2 0.4 0.6 0.8 1 1.2
BSA (mg/ml)

Figure 7. Standard curve generated using the Bradford method.

Table 3. Standard curve samples for Bradford protein assay References

1. Ausubel, F.M., et al. (1999)
BSA conc. (mg/ml) Volume of BSA standard solution (µl) Volume of protein dilution buffer*(µl) Current Protocols in Molecular
Biology. New York: John Wiley
0 – 200 and Sons.
2. Sambrook, J. and Russell, D.
0.05 10 190
(2001) Molecular Cloning: A
0.1 20 180 Laboratory Manual. 3rd ed.
Cold Spring Harbor, NY: Cold
0.2 40 160 Spring Harbor Laboratory.
0.3 60 140 3. Kaufmann S.H., Ewing, C.M.,
and Shaper, J.H. (1987) The
0.4 80 120
erasable Western blot. Anal.
0.5 100 100 Biochem. 161, 89.

0.6 120 80
0.8 160 40
1.0 200 –

* Use the same buffer as that used to dilute the protein of interest.

QIAGEN offers a wide range of products and resources for the expression, purification, detection, and assay
of proteins. The QIAexpressionist is a comprehensive manual that provides background information and
protocols for the cloning, expression, and purification of 6xHis-tagged proteins expressed using QIAGEN pQE
vectors. The QIAexpress Detection and Assay Handbook provides detailed information and protocols for the
detection and assay of 6xHis-tagged proteins. Many of the protocols in these manuals can be adapted to
general use. For further information about QIAGEN products and literature please refer to the QIAGEN Product
Guide, visit us online at www.qiagen.com, or contact QIAGEN Technical Services or your local distributor.

Protein 87