determ. of prot. conc. in 8M urea

[Warren Wakarchuk]

In article <1994Feb25.141039.940 at cathy.ijs.si> Robert.Kuhelj at ijs.si (Robert Kuhelj) writes:
>From: Robert.Kuhelj at ijs.si (Robert Kuhelj)
>Subject: determ. of prot. conc. in 8M urea
>Date: 25 Feb 94 14:10:39 +0100

>Any idea how to quickly determine protein concentration in 8M urea solution?


>SDS PAGE is too slow and you can only estimate the amount of protein.
>Bradford or Lowry are probably strongly influenced by such a high urea
>concentration, and the calibration curve must be prepared using the 8M urea
>solutions of the standard.


>WHAT ABOUT SPECTROPHOTOMETRY?

>What wavelength to use (a slight red shift of the spectrum occurs at high
>denaturant conc.!)?

>What happens to the molar absorbance coefficient of the protein 
>absorbance of aromatic amino acids increase substantially at elevated
>urea conc.!)?

>Any experiences?

>Robert                               robert.kuhelj at ijs.si

  The classical determination of protein concentrations by A 280 works just 
fine denaturants such as urea or guanidine-HCL.  There is almost no effect 
on the extinction coefficent for trp and tyr (what you see at 280 nm) in 
these denaturants.