Measuring Protein Concentration using Bradford Assay

Ever since the melamine contamination of pet food which resulted in the deaths of over

8,000 pet cats and dogs, there has been a need for a way to detect melamine. The traditional

methods of determining how much protein a sample of food has rely on measuring the nitrogen

content. Two such techniques are Kjeldahl method and the Dumas test. In the Kjeldahl method,

measures how much ammonia is present in a sample and since ammonia is a byproduct of

nitrogen this technique also indirectly measures the protein concentration. The Dumas test is

similar, but protein content is measured directly by burning the food sample and measuring the

amount of nitrogen released.

Unfortunately, the drawback of both these techniques is that they rely on nitrogen to

determine the protein concentration. This limitation is precisely why the melamine outbreak

occurred. Melamine is composed of roughly 66% nitrogen and therefore, food samples

contaminated with melamine would show high protein concentrations when tested via the

Kjeldahl method or the Dumas test. However, melamine is not a protein but rather due to the

reliance on nitrogen based protein measurements, melamine has been used as a protein imposter.

High levels of melamine can cause kidney failure, kidney stones, and even death.

Fortunately, other methods of protein concentration can accurately determine the protein

concentration of a food sample containing melamine without being misled by the high nitrogen

content. One such test is the Bradford Assay which uses coomassie dye, which melamine doesn’t

have an effect on, to determine the presence of protein. However, the Bradford Assay does have

limitations of its own namely that samples have to be diluted to extremely small concentrations

before an accurate measurement will be given and that the test doesn’t perform well in samples

that contain detergents or lipids.

The purpose of this experiment was to confirm whether the Bradford Assay does indicate

that no protein is present in food samples that contain melamine. In order to do this a

spectrophotometer was used to measure the absorbance of various food samples and then those

absorbance values were plugged into a standard curve equation that determined the protein

concentration. The hypothesis was that melamine would show a protein concentration of about 0

mg/ml and that milk, egg whites, and baby formula would have a more or less similar protein

concentration as that of their food labels.

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artid=2836520&tool=pmcentrez&rendertype=abstract