Biuret Protein Assay

The principle of the Biuret assay is similar to that of the Lowry. However, it involves a single incubation of 20 minutes. There are very few interfering agents (ammonium salts being one), and Layne (1957) reported fewer deviations than with the Lowry or ultraviolet absorption methods. However, the Biuret 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.

Principle
Under alkaline conditions, substances containing 2 or more peptide bonds form a purple complex with copper salts in the reagent.

Equipment
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.

Materials

• Biuret reagent
• Bovine serum albumin (BSA)
  
• Spectrophotometer and tubes

Procedure

1. Prepare standard dilutions of BSA containing 1, 2.5, 5.0, 7.5, and 10 mg/mL protein. Prepare serial dilutions of the unknown samples.
2. Add 1.0 mL of each of the standards, each sample, and 1.0 mL of distilled water to separate tubes. Add 4.0 mL of Biuret reagent to each tube. Mix by vortex.
3. Incubate all of the tubes at 37°C for 20 minutes.
4. Turn on and adjust a spectrophotometer to read at a wavelength of 540 nm.
5. Cool the tubes from step 3, blank the spectrophotometer, and read all of the standards and samples at 540 nm.
6. Plot the absorbance of the standards vs their concentration. Compute the extinction coefficient and calculate the concentrations of the unknown samples.

Analysis
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 of protein, volume/sample, and dilution factor, if any.

Comments
The color is stable, but all readings should be taken within 10 minutes 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 produce high or low readings, respectively.