Hi Chris, What is the theoretical pI of your protein? If it is around pH 7.5, you might try gel filtering your protein into a different buffer/pH combination. Try changing by at least 1 pH unit in either direction.
If the pI isn't a problem, then you might try try solubility screening as outlined... http://scripts.iucr.org/<http://scripts.iucr.org/cgi-bin/paper?dz5020>cgi<http://scripts.iucr.org/cgi-bin/paper?dz5020>-bin/paper?dz5020<http://scripts.iucr.org/cgi-bin/paper?dz5020> HTH, Dave -- Dr David C Briggs Hohenester Lab Department of Life Sciences Imperial College London UK http://about.me/david_briggs ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Chris Fage <fage...@gmail.com> Sent: Thursday, July 13, 2017 11:40:34 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Protein rapidly precipitates when off ice 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
