Protein detection

A number of assays have been developed to quantitate proteins in solution. All are susceptible to interference by other compounds that may be present. The Bradford method is widely used, but the BCA method is more robust. However, the latter is sensitive to interference from reducing sugars. These assays give us an approximation of the quantity of protein present but not whether these proteins are allergens or not. They are, however, useful for the estimation of residual protein in, for example, oils extracted from seeds where the source material is known to be allergenic.

The Bradford Method (Bradford 1976)

This assay makes use of the acidic dye, Coomassie Brilliant Blue G-250, which binds to any basic and aromatic amino acids present on the polypeptide molecule. This changes the colour of the dye from brownish (absorbance at 465 nm) to blue (absorbance at 595 nm). The colour change is recorded using a spectrophotometer at wavelength 595 nm and the results are read from a standard curve generated from a protein of known concentration. A good description of the technique is provided in Rosenberg (1996), and the detection limit of the assay is approximately 200-1400 ^g/ml. Reagents are available from Sigma-Aldrich.

The Bicinhoninic Acid Method (Smith et al. 1985)

When a protein is placed in an alkaline system containing Cu2+, a coloured complex forms between the peptide bonds of the protein and the copper atoms. Bicinhoninic acid forms a complex with cuprous ion (Cux+) in an alkaline environment, resulting in a stable, highly coloured chromophore with an absorbance maximum at 562 nm. The sensitivity of the assay is approximately 0.5-10 ^g/ml. See Rosenberg (1996) for a description of the method.

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