BRADFORD PROTEIN ASSAY

The Bradford assay works by the action of Coomassie brilliant blue G-250 dye
(CBBG). This dye specifically binds to proteins at arginine, tryptophan, tyrosine,
histidine and phenylalanine residues. It should be noted that the assay primarily responds
to arginine residues (eight times as much as the other listed residues) so if you have an
arginine rich protein, you may need to find a standard that is arginine rich as well.
CBBG binds to these residues in the anionic form, which has an absorbance maximum at
595 nm (blue). The free dye in solution is in the cationic form, which has an absorbance
maximum at 470 nm (red). The assay is monitored at 595 nm in a spectrophotometer,
and thus measures the CBBG complex with the protein.

Detection Limitations

• Micro assay (1-20 µg): The volume of the assay is completed to 1 ml by

adding 0.8 ml CBBG dye reagent.
• Macro assay (20-200 µg): Total volume of the assay is completed to 5 ml
by adding 4 ml of CBBG dye reagent and 1 ml protein solution or standards.

Advantages

• Fast and inexpensive

• Highly specific for protein
• Very sensitive
• Compatible with a wide range of substances
• Extinction co-efficient for the dye-protein complex is stable over 10 orders
of magnitude (assessed in albumin)
• Dye reagent is complex is stable for approximately one hour

Disadvantages

• Absorbance spectra of the two Coomassie Brilliant Blue G-250 species

partially overlap making the standard curve very important
• Non-linear standard curve over wide ranges
• Response to different proteins can vary widely, choice of standard is very
important
Protocol
Preparation of bovine serum albumin protein assay standards:
In order to measure and plot a standard curve of protein concentration versus absorbance
at 595 nm, a series of dilutions of the BSA protein standard stock solution must be
prepared. Protein standards should be prepared in the same buffer as the samples to be
assayed (extinction coefficient of BSA is 0.667).

Bradford Reagent - Bradford reagent can be made by dissolving 100 mg Coomassie

Blue G-250 in 50 ml 95% ethanol, adding 100 ml 85% (w/v) phosphoric acid to this
solution and diluting the mixture to 1 liter with water.

Procedure: Warm up the spectrophotometer for 15 min. before use.

1. Prepare a 5-fold dilution of a 1 mg/ml BSA sample by adding 200 µl of 1 mg/ml
BSA to 800 µl of distilled water to make 200µg/ml BSA.
2. Generate test samples for the reference cell, blank, BSA standards and the protein
sample to be tested according to Table 1 in disposable cuvettes (Coomassie dye binds
to quartz, so it is advisable to use glass or plastic cuvettes).
3. Note that a dilution of the unknown protein sample may be required for the
resulting absorbance to fall within the linear range of the assay.
4. Allow each sample to incubate at room temperature for 5 minutes.
5. Measure the absorbance of each sample at 595 nm using a UV-visible
spectrophotometer.
6. Use Excel to plot the absorbance of each BSA standard as a function of its
theoretical concentration. Determine the best fit of the data to a straight line in the
form of the equation "y = mx + b" where y = absorbance at 595 nm and x = protein
concentration.
7. Use this equation to calculate the concentration of the protein sample based on the
measured absorbance. The linear range for the assay is 0.2 - 0.8 O.D. units).

Table 1: Preparation of test samples for the Bradford protein assay.

Test Sample Sample vol., Water, Bradford reagent,
µl µl µl
Blank 0 200 800
BSA Standard (4 µg/ml) 20 180 800
BSA Standard (8 µg/ml) 40 160 800
BSA Standard (12 µg/ml) 60 140 800
BSA Standard (16 µg/ml) 80 120 800
BSA Standard (20 µg/ml) 100 100 800
Protein Sample (unknown) 100 100 800