MadSci Network: Biochemistry

Re: How do you KNOW what wavelengths to use for BSA and casein assays

Date: Tue Mar 30 13:58:30 1999
Posted By: Dick van Wassenaar, Analytical PROTEIN Biochemist, Unilever Research Laboratory
Area of science: Biochemistry
ID: 922805673.Bc

The question is much simpler than the answer to give. The first question 
should be WHAT do you want to measure and then how?
When I assume that you want to use ultraviolet spectroscopy [UV] to measure 
the protein content of casein and BSA in solution then you have the 
following possibilities. 
First of all: all proteins including the caseins, (which is 
actually a group of at least 4 different ones [alpha s1, alpha s2, beta and 
kappa-casein]) are composed of amino acids linked together through the 
peptide bond. A characteristic feature of the peptide bond is UV 
absorption between 200 and 230 nm. It is very common to measure proteins 
using UV-detection at 214 nm. Furthermore most, but not all, proteins 
contain aromatic amino acid residues like Tyrosine and Tryptophan. These 
amino acids are responsible for UV-absorption in the 260-280 nm range 
therefore, apart from using detection at 214, proteins are measured at 280 
nm. As a rule of thumb [so it is dangerous and different for each protein] 
it is said that 1mg/ml has a UV-absorption of 1 Unit at 280 nm.
What to choose? You need to make a series of standard protein solutions and 
measure them at e.g. 280 nm and use that for you sample. Remember to state 
your results 'expressed' as mg reference protein, either BSA or the casein 

If this is not clear enough repeat your question more defined or mail me at

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