I would like to thank all of you who promptly replied to my posting with so many ideas and suggestions (18 answers so far). I will post a summary soon.
best wishes to all Savvas >> >> >> On Apr 19, 2011, at 12:48 PM, Savvas Savvides wrote: >> >>> Dear colleagues >>> >>> We are working on a large bacterial protein (featuring a large number of >>> repeats) that appears to copurify with a lot of other proteins after >>> Ni-affinity chromatography and gel-filtration. We have tried adjusting the >>> ionic strength of these runs and have gone to as high as 5M NaCl but only >>> saw marginal improvements. It appears that the protein likes to stick to a >>> lot of stuff, and in fact the number of repeats in a given construct >>> appears to correlate with the extent of contaminants in our purification >>> steps. We have admittedly never seen anything like this among the so many >>> different, and often challenging, proteins, we have worked on in our group >>> over the last few years. >>> >>> We are now thinking of trying detergents in the buffers (at non-micellar >>> concentrations), in conjunction with playing a bit with the pH to see if >>> such an approach provides a 'stripping' effect. Interestingly, the protein >>> has a calculated pI of 3.5 ! >>> >>> As the options for handling this protein are indeed quite numerous, we >>> would be grateful for any additional input and possible tips/tricks. >>> >>> I will prompty post a summary of the thread. >>> >>> Best regards >>> Savvas et al. >>> >>> >>> ---- >>> Savvas Savvides >>> Unit for Structural Biology @ L-ProBE >>> Ghent University >>> K.L. Ledeganckstraat 35, 9000 Ghent, Belgium >>> Tel/SMS/texting +32 (0)472 928 519 >>> Skype: savvas.savvides_skype >>> http://www.LProBE.ugent.be/xray.html >>>
