I recently followed a protocol from Stephen Sligar's lab for the purification of his "nanodisc" protein, which has strong hydrophobic character as it associates with phospholipids. His protocol includes washes with 1% Triton X-100 and then with 50 mM cholate (both at pH 8 in the presence of 300 mM NaCl). Worked great, and I saw stuff coming off the column in both washes. The reference is:
Bayburt et al. (2002) Nano Letters, vol. 2, pp 853-856. http://pubs.acs.org/doi/abs/10.1021/nl025623k Good luck, Arthur Arthur Glasfeld Department of Chemistry Reed College 3203 SE Woodstock Blvd. Portland, OR 97202 USA 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 > > > > > > >
