I hate to be the Negative Nancy here - but removal of detergent can be a significantly harder task than removal of urea. Here's why:
Urea is a small molecule which diffuses freely and does not normally stick to proteins - so removal of urea is no harder than removal of salt (we ignore the considerations of protein stability here). On the other hand, detergents can be easy or very hard to get rid of, depending on which particular detergent and what particular protein are brought together. Some of the nastier polymeric detergents are quite different to remove 'completely' because they stick to proteins, stick to plastic and/or resin beads, form micelles (thus refusing to dialyze) and so on and so forth. There are numerous acceptable ways to remove sticky detergents, and some of them include treatment with hydrophobic matrices that 'suck up' detergent molecules, exchange with a 'milder' detergent, and so forth. Good luck! Artem > > dear sanjiv > i dont think that the removal of detergent is more difficult than > urea,since u are purifying ur protein over Ni-NTA column,so after a few > wash over there u can remove the detergent completely. > > best > atul kumar > > -----Original Message----- > From: CCP4 bulletin board on behalf of David Cobessi > Sent: Tue 5/5/2009 6:18 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Refolding of Denatured Protein > > Sanjiv Kumar wrote: >> I have not tried purifying this protein either with detergent or with >> /Guanidine Hydrochloride. I can try with both /Guanidine HCl and >> detergent. Since I got good purification with the 8M urea, I was >> thinking if this protein could be refolded and used further. Isn't >> removal of detergent more difficult then Urea? >> Sanjiv Kumar >> Lab. No. 411, >> Functional Genomics Unit, >> Institute of Genomics and Integrative Biology, >> New Delhi-110007 >> India > Is it a transmembrane protein or not? or just a protein associated to > the membrane (with a GPI anchor for example)? > If it is a transmembrane protein, you should try to use detergents for > extraction and during the purification. > David > > -- > David Cobessi > Institut de Biologie Structurale > 41, Rue Jules Horowitz > 38027 Grenoble Cedex-1, France > Tel:33(0)438789613 > 33(0)608164340 > Fax:33(0)438785122 > >
