|MadSci Network: Biochemistry|
Hi guys, First I'm sorry this is slightly beyond your deadline- I just received the request on Tuesday, June 16th. Actually the Biuret test is quantitative, it just is not very sensitive. The perfect protein assay should be fast, accurate, precise, easy to use and free from interference. Unfortunately, reality is often far from the ideal and many of these assays have an inherent variability beacuse all protein molecules are different. I would suggest that you use the Lowry assay or the Bradford assay. You (or your teachers) can obtain these reagents from Pierce Chemicals or BioRad (both of which should have suppliers in Norway). Unfortunately, both require the use of a spectrophotometer for quantitation. If possible, you should also purchase a standard protein (such as bovine serum albumin) to control for your experimental samples. Then you could compare your samples to different soultions of your standards and thus get an estimate of the concentration without having to use the spec. For your carbohydrate assay, Benedict's test will not detect polysaccharides but only reducing sugars such as glucose. An iodine test can be used to detect polysaccharides such as starch. You need to make an 5 mM solution of potassium iodine. Again you should make a standard with different concentrations of starch etc. For each sample you should acidify it with dilute HCl and then add two drops of your iodine solution. Ideally, to measure both mono- and polysaccharides at the same time you could use the Anthrone reagent (available from most chemical suppliers). Be careful with this reagent because it is a strong acid. Make a solution of 2 g/L in concentrated sulphuric acid and add 4 mL of this solution to 1 mL of your test sample. Mix rapidly and then boil for 10 min and read the samples in a spec at 620 nm. In terms of your project, a comparison of the protein and complex carbohydrate concentrations will give information about the nutritional value of different vegetables which I think would be interesting. Good luck, Terry
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