Essay, Pages 4 (753 words)
The appearance of blue color showed the present of protein in the BSA dilutions. The more diluted the solution was, the less blue it was. The R2 value of the standard curve of BSA dilution was obtained to be 0.9972, which is close to 1. The closer to 1 the R2 value was, the more accurate the linear portion was.
The error percentage of each unknown was large: 25.9% for skim milk, 95% for soy sauce, and 64.7% for egg white. The vast difference between the theoretical protein concentration and experimental protein concentration of the unknowns showed that Bradford Protein Assay must have limitations.
The Coomassie dye only interacts with certain amino acids such as: arginine, histidine, lysine, tyrosine, tryptophan and phenylalanine. However, each amino acid has different structure from each other; therefore the Coomassie dye will interact differently with each amino acid. The Coomassie dye molecules are bound to proteins by elctronstatic attraction enhanced by hydrophobic bonding (Tal et al. 1984). Besides the interaction between Coomassie dye and amino acids, some compounds can interfere the result of the Bradford assay such as: salt, fat, and detergent.
Another factor that could influence on the Bradford assay is the protein sample must fall within the linear range of standard curve.
Another possible explanation for the difference between theoretical protein concentration and experimental protein concentration is human factor. The bottom part of the cuvettes was not supposed to be touched by because that was a region in which the beam of light goes through. The absorbance values at 595 nm are part of the variable of the standard formula that was used to calculate the experimental protein concentration of the unknowns.
Therefore, the adjustments in these absorbance values would effectively affect on the experiment protein concentration values. Pipetting could be another error source because it was difficult to pipette exactly 20 l Coomassie dye. Moreover, as recording the absorbance by using the spectrophotometer, different results were obtained within one sample. That means there were certain minor factors that influenced on the instrument.
Bradford protein assay was used to determine protein concentration in several samples. Moreover, Bradford protein assay was not able to detect the presence of melamine in the food because the experimental protein concentration was obtained to be -0.005 mg/ml, which is invalid. Bradford protein assay didn’t interact with melamine even though it contains so many amino groups (Field and Field 2010). Melamine is not protein because it doesn’t have either carboxyl groups or functional groups, which are components of an amino acid. At high level, melamine can be toxic by combining with cyanuric acid to form insoluble crystals, which lead to the formation of kidney stones (Eufic, n.d.). Melamine amounts make the amount of protein look higher than they really are when tests that are used to detect nitrogen are used (Science Daily, 2009). Even though melamine has been notified to be toxic, there are still numbers of melamine-contaminated food in the market because the melamine tests are way to expensive to apply on every single product and take to much time to proceed.
In future, to test the accuracy of the Bradford protein assay, more trials will be conducted with different proteins such as: protein shake, chicken broth and 2% whole milk. Since the disability of detecting of the Bradford protein assay, some other methods should be conducted to see whether they could detect the presence of melamine in the food. The University of Minnesota’s BioTechnology Institute developed an enzyme that is used in Bio Scientific’s new MaxDiscoveryTM Melamine Test kit, which simplifies the detection of melamine contamination in food (World Health Organization, n.d.). The melamine deaminase enzyme is created to break one of the C-N bonds in melamine to release ammonia.
Clearly, the Bradford Protein Assay still has some limitations to detect melamine in the food. Therefore, to provide safety food, a better and inexpensive protein method needs to be created.
- Anjarie F., Jeffrey F. Melamine and cyanuric acid do not interfere with Bradford and Nyhydrin assays for protein determination. Food Chemistry 121(2010): 912–917
- Mosche T., Aaron S., Elizabeth N. 1984. Why does Coomassie Brilliant Blue R interact differently with different proteins?. Journal of Biological Chemistry 260(18): 9976-9980
- European Food Information Council (EUFIC). N.D. Frequently Asked Questions. <http://www.eufic.org/page/en/page/faq/faqid/what-is-melamine/> Accessed February 22, 2013.
- Science Daily. 2009. Simple, Inexpensive Method to Detect Melamine Poison in Food. Last updated July 22, 2009. <http://www.who.int/csr/media/faq/QAmelamine/en/index.html>. Accessed February 22, 2013.
- World Health Organization. N.D. Global Alert and Response (GAR). < http://www.who.int/csr/media/faq/QAmelamine/en/index.html >. Accessed February 22, 2013.