Hi, I was in Sung-Hou Kim's group when this work below was performed and published and I also tried it out on many occasions. Elegant piece of work and certainly worth trying.
Aside from the suggestions of trying different pH and related optimum solubility screening and if higher salt and glycerol are not helping when off ice, you can consider the following: a) Try not concentrating the protein and/or reducing the expression levels. Maybe you do not need to have so much protein if it leads to relatively rapid precipitation. b) Set up some crystallization screens with the protein before concentration, especially if the protein is clean enough after Ni-NTA. We crystallized many proteins with Ni-NTA followed by tag cleavage, and second IMAC c) Do a high speed spin of the precipitated sample to remove the precipitate, and run on a gel to verify sample, estimate concentration and set up crystallization screens on that. This is related to (a) to remove excess protein. d) set up crystallization screens at 4C immediately or over a few hours if stabilized by higher salt/glycerol and maybe the chemicals in the crystallization reagents can stabilize the protein. e) If it is a nucleic acid binding protein, try complexes with nucleic acid added during purification or right after at 4C. Or try protein partners or other ligands. f) is the protein Cys rich? Can you anticipate/estimate or model surface exposed Cys or S-S bonds? Do you have adequate reducing agent in the sample? g) Lastly, more esoteric stuff like construct and vector optimization, mutations, etc. I am sure there may be a few other things you can try and there may be more suggestions here. Best, Debanu -- Debanu Das On Thu, Jul 13, 2017 at 10:46 PM, Briggs, David C <david.bri...@imperial.ac.uk> wrote: > 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/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
