Did anyone suggest adding any known ligand? If your protein happens to bind some compound/peptide/DNA/RNA/whatever, including that other partner could dramatically change it's solution properties and be an easy fix for your handling/formulation issues. Good luck!
On Fri, Jul 14, 2017 at 6:33 AM, Chris Fage <[email protected]> wrote: > 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 <[email protected]> > 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" <[email protected]> >> 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 >>> >> > -- [This e-mail message may contain privileged, confidential and/or proprietary information of H3 Biomedicine. If you believe that it has been sent to you in error, please contact the sender immediately and delete the message including any attachments, without copying, using, or distributing any of the information contained therein. This e-mail message should not be interpreted to include a digital or electronic signature that can be used to authenticate an agreement, contract or other legal document, nor to reflect an intention to be bound to any legally-binding agreement or contract.]
