The Bradford Assay

(http://www-class.unl.edu/biochem/protein_assay/bradford_assay.htm) Reference: Bradford, M. M. (1976) Anal. Biochem. 72, 248

Quick Guide
How does it work?

Absorbance shift in Coomassie Brilliant Blue G-250 (CBBG) when bound to arginine and aromatic residues The anionic (bound form) has absorbance maximum at 595 nm whereas the cationic form (unbound form) has and absorbance maximum at 470 nm

Detection Limitations

1-20 g (micro assay) 20-200 g (macro assay)

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

General Considerations

The dye binds to quartz cuvettes so it is usually better to use glass or plastic cuvettes The dye reagent is usually more convenient to purchase than to make, due to the use of phosphoric acid

Micro Assay Procedure

Warm up the spectrophotometer for 15 min. before use

Dilute samples with buffer to an estimated concentration of 1 to 20 micrograms/milliliter Prepare standards containing a range of 1 to 20 micrograms protein (albumin or gamma globulin are recommended) to a volume of 200 l (to a volume of 100 l if you are adding 1 M NaOH) Prepare unknowns to estimated amounts of 1 to 20 micrograms protein per tube to 200 l (100 l if you are using 1 M NaOH) (Optional) Add 100 l 1 M NaOH to each sample and vortex. Add 800 l dye reagent and incubate 5 min. Measure the absorbance at 595 nm.

Macro Assay Procedure

Warm up the spectrophotometer for 15 min. before use. Dilute samples with buffer to an estimated concentration of 20 to 200 micrograms/ml Prepare standards containing a range of 20 to 200 micrograms protein (albumin or gamma globulin are recommended) to a standard volume (generally 1 ml or less). See how to prepare and use a protein standard curve for suggestions as to setting up the standards. Prepare unknowns to estimated amounts of 20 to 200 micrograms protein per tube, same volume as the unknowns. (Optional) Add 0.25 ml 1 M NaOH to each sample and vortex. Add 5 ml dye reagent and incubate 5 min. Measure the absorbance at 595 nm.

Discussion
The Bradford assay is a very popular protein assay method because it is simple, rapid, inexpensive and sensitive. 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.

Chemical Structure of Coommassie Brilliant Blue G-250 It should be emphasized that the absorption spectra of the two forms of the dye overlap. This causes the assay to respond non-linearly in the standard curve. All kit providers of the Bradford assay, and many researchers insist that the assay performs linearly. However, when a standard curve is performed it is noted that a second order curve will fit much better than a linear curve. The assay does perform linearly over short concentration stretches, and this has most likely resulted in the overall conclusion that the assay is linear. Bradford himself even notes that the assay is not linear over the whole range in the original article.

Typical Bradford Assay Standard Curve One crucial part of this assay is the buffer blank. Since the assay responds nonlinearly it is highly important to lock down the zero point. Because this point is so important to the curve fit, it is highly recommended that at least two buffer blanks be

performed. If it is determined that interference is not occurring, as is the usual case, you can proceed to use a water buffer blank. The choice of standard for this assay is very crucial to the success of the assay. Many investigators have noted abnormalities of using various standards with the Bradford assay. BSA was the original standard of choice, and is standard you are most likely to receive with the assay if purchased as a kit. However, it has been noted that BSA has a double than normal response in the assay and may not always be suitable. Several researchers therefore use Imunnoglobulin G (IgG) as the preferred standard for the assay. The CBBG dye used in the assay binds to quartz cuvettes quite strongly. Therefore, glass or plastic cuvettes should be utilized. Since this assay has a general tendency to bind to cuvettes, it is highly recommended to use disposable plastic cuvettes. This is by no means critical to the assay, but it does make cleaning the cuvettes much more convenient. There are two major formats of this assay each with a different detection range. The micro assay format is designed for protein concentrations between 1-20 micrograms and the macro assay is designed for protein concentrations in the range of 20-100 micrograms. It is generally more convenient to use the micro format and dilute your protein down to the concentration range. The micro assay results in less chemical waste and allows the concentrate to last much longer. The assay can also be performed in a microwell plate, which is a very convenient way to process a large number of samples rapidly. One consideration is to use a small amount of sodium hydroxide in the assay to help solubulize your protein. Some proteins, especially hydrophobic, membrane or sticky proteins tend to precipitate in the presence of coomassie dyes. If you observe a precipitate forming when you add the dye reagent to your sample try adding the specified amount of sodium hydroxide.