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The absorbance is due to imidazole, for sure. We do a lot of this type of purification in my lab and we have definitely had some low abundance proteins hide under the imidazole gradient. Here is how we deal with it. We expect the proteins to elute around 100-350 mM imidazole, so we run SDS-PAGE on every other fraction in that area to detect the protein. We do 3-5 clarified lysate loads and low imidazole washes to increase the amount of our desired protein on the column before we start the imidazole gradient. Always have a few CV of baseline absorbance before you start the gradient. That's all I know, G On Wed, Aug 21, 2013 at 10:45 AM, Ed Pozharski <[log in to unmask]>wrote: > According to Qiagen > > > > Since imidazole absorbs UV radiation at 280 nm, an elution profile > > measured at 280 nm while purifying a 6xHis tagged protein by FPLC will > > show an increase in absorbance above the background signal allowing > > quantitation of your protein. The absorbance of imidazole can vary > > depending on its source and purity, but elution buffer containing 250 > > mM imidazole usually has an A280 of 0.2–0.4. > > Cheers, > Ed. > > > On Wed, 2013-08-21 at 11:25 -0400, Edward A. Berry wrote: > > Would you happen to know what the A280 of a 1M (or .1 M) solution is? > > I wonder if this absorbance is really due to impurities or is the tail > of a weak absorbance band of imidazole itself. I > > notice A280 is not one of the properties sigma lists for its imidazole. > > > > Jan wrote: > > > Hi Bernhard, > > > this one works for us: > > > Sigma BioUltra imidazole, >99.5% by GC, 56749 > > > > > > Cheers, > > > Jan > > > -- > > > Jan Abendroth > > > Emerald BioStructures > > > Seattle / Bainbridge Island WA, USA > > > home: Jan.Abendroth_at_gmail.com > > > work: JAbendroth_at_embios.com > > > http://www.emeraldbiostructures.com > > > > > > On Aug 21, 2013, at 7:33 AM, Bernhard Rupp <[log in to unmask]<mailto: > [log in to unmask]>> wrote: > > > > > >> Hi Fellows, > > >> could someone please point me towards the source of a known high > purity imidazole > > >> with low absorbance at 280 nm? I am facing the problem of detecting a > low absorption protein > > >> in high imidazole background after IMAC gradient elution. In the UV > spectra of the > > >> 2 imidazoles I checked there is some contaminant that absorbs at 280… > > >> Thx, BR > > >> > ---------------------------------------------------------------------------------------- > > >> Bernhard Rupp > > >> Marie Curie Incoming International Fellow > > >> Innsbruck Medical University > > >> Schöpfstrasse 41 > > >> A 6020 Innsbruck – Austria > > >> +43 (676) 571-0536 > > >> [log in to unmask] <mailto:[log in to unmask]> > > >> > ---------------------------------------------------------------------------------------- > > >> Dept. of Forensic Crystallography > > >> k.-k. Hofkristallamt > > >> Vista, CA 92084 > > >> 001 (925) 209-7429 > > >> [log in to unmask] <mailto:[log in to unmask]> > > >> [log in to unmask] <mailto:[log in to unmask]> > > >> http://www.ruppweb.org/ > > >> > ----------------------------------------------------------------------- > > > > > -- > Edwin Pozharski, PhD, Assistant Professor > University of Maryland, Baltimore > ---------------------------------------------- > When the Way is forgotten duty and justice appear; > Then knowledge and wisdom are born along with hypocrisy. > When harmonious relationships dissolve then respect and devotion arise; > When a nation falls to chaos then loyalty and patriotism are born. > ------------------------------ / Lao Tse / >