Yes, I'm also surprised why people run gradients for the capturing step ? How often is your desired protein spread out throughout the whole gradient ? So what's the point the of the gradient if in the end you merge the whole peak again ? Maybe for a pilot study to identify how much imidazole could I add to my binding buffer I can see the point of the gradient, but otherwise batch binding and step elution is just fine (and faster).
We do batch binding in the presence of some imidazole 10 -25 mM then do a step elution with 250 - 500 (depending on His6 or His12) if we use imidazole for elution. Alternatively we do low-pH elution e.g. if your protein requires a divalent ion or elution with EDTA at a more physiological pH if no divalent ion is required by our protein. The cleanup is a Resource Q or Resource S followed by size exclusion or other steps in between. If the captured protein is not >50% in the first step then we try to enrich e.g. by HIC or simply reducing the amount of resin used in the first capturing step. My lab is a big fan of Talon Celltru lately. And yes, they really should send me some free samples for making this advertisement :-) Jürgen On Aug 22, 2013, at 9:37 AM, David Mueller wrote: I've never done the experiment, but it is easy to determine the absorption spectra of imidazole at different pH values and see if there is an isosbestic point - and then plot (A280 minus the Abs at the isobestic point), vs pH, and see if the pKa is about 7.0. Imidazole is aromatic as the free base but not as the protonated species, so I expect a change in the UV spectrum vs pH. Qiagen used to recommend not doing a gradient of imidazole to elute the protein but simply stepping the protein off with a high concentration of imidazole - it works for me. On Thu, Aug 22, 2013 at 8:03 AM, Gloria Borgstahl <gborgst...@gmail.com<mailto:gborgst...@gmail.com>> wrote: 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 <epozh...@umaryland.edu<mailto:epozh...@umaryland.edu>> 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<http://Jan.Abendroth_at_gmail.com/> > > work: JAbendroth_at_embios.com > > http://www.emeraldbiostructures.com<http://www.emeraldbiostructures.com/> > > > > On Aug 21, 2013, at 7:33 AM, Bernhard Rupp > > <hofkristall...@gmail.com<mailto:hofkristall...@gmail.com> > > <mailto:hofkristall...@gmail.com<mailto:hofkristall...@gmail.com>>> 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<tel:%2B43%20%28676%29%20571-0536> > >> bernhard.r...@i-med.ac.at<mailto:bernhard.r...@i-med.ac.at> > >> <mailto:bernhard.r...@i-med.ac.at<mailto:bernhard.r...@i-med.ac.at>> > >> ---------------------------------------------------------------------------------------- > >> Dept. of Forensic Crystallography > >> k.-k. Hofkristallamt > >> Vista, CA 92084 > >> 001 (925) 209-7429<tel:%28925%29%20209-7429> > >> b...@ruppweb.org<mailto:b...@ruppweb.org> > >> <mailto:b...@ruppweb.org<mailto:b...@ruppweb.org>> > >> b...@hofkristallamt.org<mailto:b...@hofkristallamt.org> > >> <mailto:b...@hofkristallamt.org<mailto:b...@hofkristallamt.org>> > >> 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 / -- David M. Mueller Biochemistry and Molecular Biology The Chicago Medical School Rosalind Franklin University of Medicine and Science 3333 Green Bay Road North Chicago, IL 60064 david.muel...@rosalindfranklin.edu<http://david.muel...@rosalindfranklin.edu/> http://lmb-il.org/index.html Office: 847-578-8606 FAX: 847-578-3240 ...................... Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
