I did not follow all the responses to this, but, did you try to use the Ni-NTA buffer (500 mM NaCl, 30 mM HEPES pH 7.5, 10% glycerol) to run the gel filtration?
Yuzhu. From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Chris Fage Sent: Friday, July 14, 2017 3:33 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Protein rapidly precipitates when off ice 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<mailto: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<mailto: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 This electronic message contains information generated by the USDA solely for the intended recipients. Any unauthorized interception of this message or the use or disclosure of the information it contains may violate the law and subject the violator to civil or criminal penalties. If you believe you have received this message in error, please notify the sender and delete the email immediately.
