In fact, I agree with Artem that detergent can be very difficult to get rid of. It totally depend on the property of detergent. In your case that the protein has no transmembrane region. You probably dont need much detergent in your solution to keep your protein happy. I dont think having detergent in solution would cause any problem in crystalization. Frankly, I have never heard of any body get success trying to purify membrane associated protein without detergent. Even if your protein get refolded correctly (this is a very big if here), how can you know that your protein behave nicely after refolding in your buffer solution. After the process, It can aggregate also.
According to you, the protein is in the cell pellet, Is it still membrane associated or it express as an inclusion body? Are you sure that it is correctly folded at the first place or it is already misfolded even before you start purifying it? To my understanding, Refolding is a very difficult process and it is so difficult to control so it is better to avoid the process especially if you want to work with protein structure. You will never know how heterogeneous your sample is or how well did it get refolded. You can only check if the refolding can regain any activity or not. My suggestion is that if you want to crystalize this protein, you should find the way to avoid refolding if possible. If protein express as an inclusion body then changing host might be a better way to go. Good luck, Puey On Wed, May 6, 2009 at 5:27 AM, <ar...@xtals.org> wrote: > 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 > > > > >
