using UV absorbance scans to 'measure' homogeneity of proteins

Achim Recktenwald, PhD ARecktenwald at StressGen.com
Tue Sep 7 14:30:39 EST 1999


I agree with what Peter wrote.

In case you would like to compare different purification-batches of the same
protein, you might want to look into higher order derivative spectra, e.g.,
2nd or 4th order derivatives. The normal UV/VIS-spectrum of a protein is a
rather unexciting very smooth curve, the derivatives let you discern and
compare some of the finer details of a set of spectra.

But this method is usually only good to compare different batches of the
same protein; it will show you, if there are difference in absorbance, but
it is very difficult  to attribute those differences to distinct compounds
or impurities.

Achim



Prevelige <prevelig at uab.edu> wrote in message
news:37D4E330.6424EBF9 at uab.edu...
> David,
>
> the quality of the answer depends on the quality of the question.
> homogeneous with regard to what? if you are concerned about nucleic
> acid, you can pick up contamination at some level...you can do a quick
> back of the envelope calculation about what fraction of nucleic acid
> (given it spectrum) you can detect. if that level isn't sufficient, you
> will have to try another assay (ie: fluorescence of intercalating
> agents).  if you are concerned about contaminating proteins, then you
> are pretty much out of luck, unless one is say hemeglobin, with a very
> different absorption spectrum. you might want SDS-PAGE or mass
> spectrometry instead.  if you are concerned about homogeneous with
> respect to conformation (same molecule, different structure) you are
> also out of luck.
>
> the game you have to play is to postulate what might be in there, (other
> than your protein) that concerns you  and demonstrate analytically that
> it is or isn't present at a level that you deem worrysome.
>
> Peter
>
>
>
> David wrote:
> >
> > Hi,
> >
> > I've heard that one can tell from the UV absorbance scan of a protein
> > solution, whether it is likely to be homogeneous or not. I'd be grateful
> > for any comments on this method, e.g. what features of the scan one
should
> > look for, and how reliable this method is.
> >
> > Thank you very much in advance,
> >
> > David





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