using UV absorbance scans to 'measure' homogeneity of proteins

Achim Recktenwald achimr at home.com
Thu Sep 9 22:27:11 EST 1999


David <david at cryst.bioc.cam.ac.uk> wrote in message
news:david-0809991507400001 at mac355.nmus.pwf.cam.ac.uk...
> Thank you to everyone for your replies. It is much appreciated.
>
> I'm sorry for not making it clear, but the kind of homogeneity I was
> thinking about was with respect to proteins rather than nucleic acids, and
> I guess in particular, whether a protein that appears pure on, e.g. a gel
> or gel filtration columnm, has higher-order oligomers or aggregates
> present.

[snip]


If you are looking for non-covalent aggregates, i.e., no intermolecular
bonds like SS-bridges, you can in many cases check  your protein by simple
UV280nm measurement.

Make several dilutions of your protein over a wide range, e.g., 5fold up to
500 or 1000fold,  then measure your absorbance. If your total absorbance
(absorbance x dil.factor) does not stay (more or less) constant for all
dilutions but increases,  you have a good indication for protein-aggregates
in your original solution - assuming you made sure their is no other
particular matter in your protein or buffer solution.
Often you can observe that the total absorbance increases up to a certain
dilution factor and then stays constant.

The aggregates tend to break up upon higher dilution, and you 'find' more
protein.

The other methods mentioned above, like gel filtration, are more accurate
and also quantitative, but I usually try this method first, if I suspect
aggregates - it's faster.

Cheers,

Achim





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