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 doesnt 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 doesnt 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. 1. Anon. Melamine Death Toll Passes 8,000 Pets - FDA says health risk for humans unlikely. [cited 2013 March 4]. Available from: http://www.newser.com/story/1927/melamine-death-tollpasses-8000-pets.html 2. Snyder A. Protein Pretense: Scientific American. [cited 2013 February 25]. Available from: http://www.scientificamerican.com/article.cfm?id=protein-pretense 3. Ingelfinder, R. J. Melamine and the Global Implications of Food Contamination NEJM. [cited 2013 February 25]. Available from:

http://www.nejm.org/doi/full/10.1056/NEJMp0808410 4. Nutrition C for FS and A. Consumers - Melamine in Tableware: Questions and Answers. [cited 2013March4].Availablefrom:

http://www.fda.gov/Food/ResourcesForYou/Consumers/ucm199525.htm

5. Field A, Field J. Melamine and Cyanuric Acid do not interfere with Bradford and Ninhydrin assays for protein determination. Food chemistry [Internet]. 2010 August 1 [cited 2013 February 28];121(3):912917. Available from:

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2836520&tool=pmcentrez&renderty pe=abstract