I'd try varying the pH independent of the theoretical pI, sometimes the real pI is very different. (I've worked on a protein with theoretical pI 9.2, real pI determined by iso-electric focussing 7.8).
I'd also try limited proteolysis on the milky sample and see if you can solubilise it while a sufficiently interesting protein fragment remains. Mark J van Raaij Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC calle Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://wwwuser.cnb.csic.es/~mjvanraaij > On 14 Jul 2017, at 08:14, Debanu Das <[email protected]> wrote: > > 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 > <[email protected]> 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 <[email protected]> on behalf of Chris Fage >> <[email protected]> >> Sent: Thursday, July 13, 2017 11:40:34 PM >> To: [email protected] >> 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
