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

ProtocolPreparation 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.,

µlWater,

µlBradford reagent,

µlBlank 0 200 800BSA 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