Dear All, Thank you for the many suggestions. After sending my first message to the BB, I tried exchanging the sample into buffer containing 5 mM EDTA and also into buffer at pH 9.0 (using BICINE). Neither of these appear to have helped the instability--precipitation still occurred within ~1 min of removal of the tube from ice. The theoretical pI is ~6.1, which is far from my working pH, although as Mark indicated the calculated pI may be inaccurate, and I may even need to try a more acidic pH. I will test the ideas provided by everyone over the next week and leave some feedback. It may be that I need to prepare a different truncation, as this domain is excised from a larger covalent assembly. I have purified homologs trimmed at a similar position in the past, but of course that doesn't guarantee good behavior in my current system.
Best, Chris On Fri, Jul 14, 2017 at 10:20 AM, Eugene Osipov <e.m.osi...@gmail.com> wrote: > Hi, try to add 1-5 mM EDTA, because trace amounts of metals cause > precipitation of tagged proteins. > > 14 июля 2017 г. 1:40 пользователь "Chris Fage" <fage...@gmail.com> > написал: > > Dear CCP4BB Community, >> >> This week, I purified a nicely overexpressing protein by Ni-NTA followed >> by gel filtration. In a 4 C centrifuge, I concentrated my gel filtration >> fractions to ~1 mL, transferred the spin filter to ice, and then collected >> 2 uL for measurement on the Nanodrop. Sadly, the protein precipitated >> heavily in the pipet tip before I could dispense it onto the Nanodrop >> pedestal, directly adjacent to my ice box. This effect seems to be abated >> at 4 C, as the protein remained stable in cold room-chilled pipet tips. >> However, the protein also precipitated heavily when overnight at 4 C in 1 >> mL gel filtration buffer (150 mM NaCl, 10 mM HEPES pH 7.5), but not >> overnight at 4 C in 10 mL Ni-NTA buffer (500 mM NaCl, 30 mM HEPES pH 7.5, >> 10% glycerol) prior to gel filtration. Has anyone experienced and resolved >> a similar issue before? Do any useful additives come to mind? >> >> Things I have tried with the gel filtration sample: >> -Exchanging buffer to restore the salt concentration to Ni-NTA levels >> (e.g. 500 mM). >> -Exchanging buffer to add 10% glycerol. >> -Simply diluting the protein in gel filtration buffer to rule out >> concentration dependence. >> >> In each case, the protein precipitates to a milky solution within about a >> minute of removal from ice (I am working with 20-50 uL volumes in PCR >> tubes). >> >> Many thanks for any suggestions! >> >> Best, >> Chris >> >
