The Bradford assay is very fast and uses about the same amount of protein as the Lowry assay. It is fairly accurate and samples that are out of range can be retested within minutes. The Bradford is recommended for general use, especially for determining protein content of cell fractions and assesing protein concentrations for gel electrophoresis.

The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change. The assay is useful since the extinction coefficient of a dye-albumin complex solution is constant over a 10-fold concentration range.

In addition to standard liquid handling supplies a visible light spectrophotometer is needed, with maximum transmission in the region of 595 nm, on the border of the visible spectrum (no special lamp or filter usually needed). Glass or polystyrene (cheap) cuvettes may be used, however the color reagent stains both. Disposable cuvettes are recommended.

1. Bradford reagent: Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol, add 100 ml 85% (w/v) phosphoric acid. Dilute to 1 liter when the dye has completely dissolved, and filter through Whatman #1 paper just before use.
2. (Optional) 1 M NaOH (to be used if samples are not readily soluble in the color reagent).

The Bradford reagent should be a light brown in color. Filtration may have to be repeated to rid the reagent of blue components. The Bio-Rad concentrate is expensive, but the lots of dye used have apparently been screened for maximum effectiveness. “Homemade” reagent works quite well but is usually not as sensitive as the Bio-Rad product.

1. Warm up the spectrophotometer before use.
2. Dilute unknowns if necessary to obtain between 5 and 100 µg protein in at least one assay tube containing 100 µl sample
3. If desirred, add an equal volume of 1 M NaOH to each sample and vortex (see Comments below). Add NaOH to standards as well if this option is used.
4. Prepare standards containing a range of 5 to 100 micrograms protein (albumin or gamma globulin are recommended) in 100 µl volume. See how to set up an assay for suggestions as to setting up the standards.
5. Add 5 ml dye reagent and incubate 5 min.
6. Measure the absorbance at 595 nm.

Prepare a standard curve of absorbance versus micrograms protein and determine amounts from the curve. Determine concentrations of original samples from the amount protein, volume/sample, and dilution factor, if any.

The dye reagent reacts primarily with arginine residues and less so with histidine, lysine, tyrosine, tryptophan, and phenylalanine residues. Obviously, the assay is less accurate for basic or acidic proteins. The Bradford assay is rather sensitive to bovine serum albumin, more so than “average” proteins, by about a factor of two. Immunoglogin G (IgG – gamma globulin) is the preferred protein standard. The addition of 1 M NaOH was suggested by Stoscheck (1990) to allow the solubilization of membrane proteins and reduce the protein-to-protein variation in color yield.

• Bradford, MM. A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. 1976.
• Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).

The principle of the biuret assay is similar to that of the Lowry, however it involves a single incubation of 20 min. There are very few interfering agents (ammonium salts being one such agent), and Layne (1957) reported fewer deviations than with the Lowry or ultraviolet absorption methods. However, the biuret assay consumes much more material. The biuret is a good general protein assay for batches of material for which yield is not a problem. The Bradford assay is faster and more sensitive.

Under alkaline conditions substances containing two or more peptide bonds form a purple complex with copper salts in the reagent.

In addition to standard liquid handling supplies a visible light spectrophotometer is needed, with maximum transmission in the region of 450 nm. Glass or polystyrene (cheap) cuvettes may be used.

1. Volumes sample, reagent can be scaled up/down and/or volume ratios varied, as with any assay.
2. Warm up the spectrophotometer 15 min. before use.
3. Prepare standards from bovine serum albumin, preferably calibrated using absorbance at 280 nm and the extinction coefficient. Using 5 ml color reagent to 1 ml sample a recommended range is 0.5 to 20 mg protein.
4. Prepare a reference tube with 1 ml buffer.
5. If possible, dilute unknowns to an estimated 1 to 10 mg/ml with buffer; a range of dilutions should be used if the actual concentration cannot be estimated.
6. Use 1 ml sample per assay tube
7. Add 9 ml Biuret reagent to each tube, vortex immediately, and let stand 20 min.
8. Read at 550 nm.

Prepare a standard curve of absorbance versus micrograms protein (or vice versa), and determine amounts from the curve. Determine concentrations of original samples from the amount protein, volume/sample, and dilution factor, if any.

The color is stable, but all readings should be taken within 10 min. of each other. As with most assays, the Biuret can be scaled down for smaller cuvette sizes, consuming less protein. Proteins with an abnormally high or low percentage of amino acids with aromatic side groups will give high or low readings, respectively.

For Bovine serum albumin we typically obtain a linear relationship between absorbance and amount protein over a range of 0.5 to 20 mg protein. The assay has not been reliable for amounts below 0.5 mg, however the actual sensitive range may extend beyond the upper limit.

• Gornall, AG, CS Bardawill, and MM David. J. Biol. Chem. 177: 751. 1949.
• Layne, E. Spectrophotometric and Turbidimetric Methods for Measuring Proteins. Methods in Enzymology 10: 447-455. 1957.
• Robinson, HW and CG Hogden. J. Biol. Chem. 135: 707. 1940.
• Slater, RJ (ed.). Experiments in Molecular Biology. Clifton, New Jersey: Humana Press, 1986. P. 269.
• Weichselbaum, TE. Am. J. Clin. Pathol. Suppl. 10: 40. 1946.