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Bernard, Ed, Sounds like an impurity, all right. But if so you wold think the chemical companies would be selling special low-A280 grade imidazole for IMAC, and for some price you could get material that doesn't absorb there at all. Unless its a spontaneous decomposition product. Maybe even the pure compound has some absorbance there, and impurities result in the variability mentioned? eab Ed Pozharski 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 / > > >