Dear all, I will give my advice once again and hopefully you will discuss on it the next 2 days :P Chris might be worth trying a 2ndary or 3ary structure prediction tool to ensure that you do not have f.i. flexible ends. Disordered regions may lead to this kind of issues, as well. Personally I love HHPRED. Previous experience on closely homologous proteins may give you a good idea on how to treat your protein.
Good luck! Vicky On Fri, Jul 14, 2017 at 9:21 AM, Mark J van Raaij <mjvanra...@cnb.csic.es> wrote: > 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 <+34%20915%2085%2046%2016> > http://wwwuser.cnb.csic.es/~mjvanraaij > > On 14 Jul 2017, at 08:14, Debanu Das <debanu....@gmail.com> 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 > <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 > > >
