detection of proteins in FPLC

Mario J. Borgnia mborgnia at welchlink.welch.jhu.edu
Fri Mar 21 00:08:10 EST 1997


> In the referenced article, ita at DNA.CBIOT.UFRGS.BR (ITABAJARA DA SILVA VAZ JUNIOR) writes:
> >Please,
> >Is there a simple method of transform the AU detected by  monitor UV in FPLC 
> >in mg/ml concentration?
> >Thanks, 
> >
> Assuming 280nm detection, 1.00 AU is roughly equivalent to 1.0 mg/ml, so,
> for example, if your sensitivity is set to 0.01, then full scale
> deflection is roughly 10ug/ml.  The total amount of protein in a pooled
> peak is proportional to the area under the peak, but if you don't have an
> integrating chart-recorder, as a rule of thumb, we used to assume that,
> when we pooled a bunch of fractions from an FPLC run, the protein
> concentration of the pool was about half the height of the peak, (since
> most of the fractions in the pool are not the full peak height).  
> Obviously, this starts to break down if you start adding in loads of 
> low-protein fractions from trailing edges of the peak.

Alternatively you can cut and weigh the area bellow the curve (in a chart 
recorder paper or its xerox copy) and compare with a piece of the 
same paper of known area.

Mario J. Borgnia
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