MadSci Network: Biochemistry

Re: How can we do quantitative tests for determining protein and carbohydrates?

Date: Thu Jun 18 09:12:32 1998
Posted By: Terry Hebert, Faculty, Universite de Montreal, Biochemistry, Montréal Heart Institute
Area of science: Biochemistry
ID: 897652814.Bc

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,

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