using UV absorbance scans to 'measure' homogeneity of proteins

Frank Fürst ffrank at rz.uni-potsdam.de
Fri Sep 10 08:48:32 EST 1999


Peter Prevelige wrote:
> 
> Achim Recktenwald wrote:
> 
> > [...] 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
> 
[...]
> furthermore, dilution will dissociate aggregates, which will decrease
> scattering, particularly at 280 nm (given the relatively steep
> wavelength dependence of scattering) so it seems likely that dilution
> may actually disproportionatly decrease rather than increase the
> absorbance if there are aggregates, at least in the general case.
> 
Anyway, if aggregation and hence scattering is big enough to have an
effect upon dilution, it will also change the baseline above 320 nm.
Thus measuring one spectrum, which is easier than diluting several
times, will tell you at least the same.

BTW I would never only measure the absorption at 280 nm for
determination of protein concentration. Besides aggregation or
contamination, the cuvette may have some unusual absorption, the
instrument may be miscalibrated and so on. We always run a fast
spectrum first; only for time-dependent measurements we stay at one
wavelength.


Yours, Frank

-- 
Frank Fuerst, Institute of Biochemistry of Potsdam University
Im Biotechnologiepark, D-14943 Luckenwalde
Tel.: +49-3371-681334;   Fax: +49-3371-681339
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