Vibhav Singh Lab Section 004 Protein Assay Discussion The results from Figure 2 demonstrates that melamine

isn't a protein since the assay determined the concentration of protein in melamine is within experimental error of 0 mg/ml. Therefore, the results of the experiment supported the hypothesis that the Bradford Assay would be able to correctly identify melamine as a protein imposter. The results also show that the Bradford Assay is effective at measuring the protein concentration of other substances such as baby formula. The values given by the assay closely matched the values of protein concentration calculated from the food label of these items (Figure 2). The assay values for protein concentration in skim milk were inaccurate in Figure 2 because coomassie dye was not added to the samples. This caused incorrect values for absorbances and then the error was further compounded since absorbance is used to calculate protein concentration from the standard curve. Therefore, the values for the skim milk should actually be much higher and so the values found by Kristina Kalland and Anna Wetherille should be referred to (see their results). The Bradford assay can accurately determine the protein concentration for substances that have a concentration between 0 mg/ml and 2 mg/ml (Figure 1). This limits the efficiency for real world application since the implication is that food has to be diluted to be within this concentration range in order for an accurate measurement. Clearly, diluting every food sample can be cumbersome to do so in the field not to mention the monetary costs and logistical problems associated with the Bradford assay. The Bradford assay can be expensive since the spectrophotometer alone can cost anywhere from hundreds of dollars to thousands.1 The logistical problem is that the spectrophotometer is bulky and cant be easily moved to the area where the food is being tested. Other limitations of the Bradford assay are that its highly sensitive to particular amino acids, isnt effective in solutions rich in lipids, and chelating substances.2 Another potential issue is that well-trained workers would be needed to operate the Bradford assay for the test to be effective.

Melamine came under public scrutiny in 2007 when many household pets in the United States became sick and subsequently died.3 This occurred because of the melamine which was thought to be a protein by traditional detection methods poisoned the pet food. Traditional detection methods such as the Dumas determine the concentration of protein by the presence of nitrogen and since melamine is highly rich in nitrogen these tests conclude that food with melamine have high protein concentration.3 Therefore, the price of these foods are higher which gave some companies a financial incentive to add melamine to the food they produce. In the age of globalization problems with contaminated foods are even more disastrous since the food is distributed more widely throughout the world.4 That's why tests such as the Bradford assay which can detect protein fraud by the addition of melamine are so crucial. However, as noted above the Bradford assay isn't a practical solution due the monetary costs, logistical problems, dilution of samples, ineffectiveness in lipids, and need for skilled workers to operate the Bradford assay. Therefore, further research is needed to find more cost-effective solutions for detecting melamine in foods. One such method has already been developed by Professors Larry Wackett and Michael Sadowsky at the University of Minnesota. Wackett and Sadowsky used an enzyme discovered by Jennifer Saefernick, a research associate, that can be used to detect whether melamine is present in food or not.5 Also research should be focused on a general solution to distinguish between compounds that are rich in nitrogen and proteins since melamine isn't the only substance that can be added to food to artificially raise the protein concentration. References. 1. Anon. UV VIS Spectrophotometers | Unico Series | Spectrophotometer. [cited 2013 February 25]. Available from: http://www.blockscientificstore.com/Spectrophotometers-s/359.htm 2. EMD Millipore. IR-based Protein Quantitation Surpasses Calorimetric Assay Quantitation and Results are Independent of Detergents, Reducing Agents and Analysis Time. [cited 2013 February 25]. Available from:

http://www.millipore.com/publications.nsf/a73664f9f981af8c852569b9005b4eee/94bb0de8c276b9fa85 2579ad0051667e/$FILE/an2222en_EM.pdf 3. Snyder A. Protein Pretense: Scientific American. [cited 2013 February 25]. Available from: http://www.scientificamerican.com/article.cfm?id=protein-pretense 4. 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 5. Anon. University of Minnesota research leads to new technology to protect human health: UMNews: University of Minnesota. [cited 2013 February 25]. Available from: http://www1.umn.edu/news/news-releases/2009/UR_CONTENT_123750.html