Protein quantification with Ammoniumsulfate present?!

Dennison dennison at unpsun1.cc.unp.ac.za
Tue Jun 2 12:50:53 EST 1998


> I am doing hydrophobic interaction chromatography with ammoniumsulfate
> and need to quantify the protein content of the fractions (down to 20
> microgram/ml) which contain 0-3M ammoniumsulfate. Bradford gives
> unreliable results, no matter what I use as a blank. BCA and BioRads
> DC-Assay do not work at all with AS present. Protein precipitation
> with TCA prior to Bradford gives some sort of linear standard curve
> but only with a high standard deviation and not very reproducible. I
> am currently trying UV 260nm and SDS-PAGE scanning, but both have
> severe drawbacks (sensitivity and time/labour intensity, resp.).
> 
> Does anybody have any proposals about how to get a reliable protein
> concentration in samples containing ammoniumsulfate from 0 to 3 M?

A suggestion.  If you add t-butanol, in the presence of 3 M ammonium
sulfate, the proteins should come out of solution into a third layer. 
You could retrieve them from there for quantitation, perhaps.  See Prot.
Expression and Purification (1997) 11, 149-161
Cheers
Clive



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