Hi, Regarding the second publication below on the optimum solubility screen, I was a new postdoc in Sung-Hou Kim's lab when the last parts of the work and manuscript was being prepared, and so did quite a few experiments using the screen in conjunction with DLS.
It is worth trying them out but the main thing is that they were to be used at the crystallization screening step to modulate solubility properties with DLS characterization, not to prevent precipitation during purification. Nevertheless, the general ideas of pH etc from them are applicable and may be even more useful if you can purify some of your protein to completion even under low concentration and/or partial precipitation and then try out the solubility screen. The other thing to consider is your protein may just be soluble and crystallizable at low conc so definitely worth setting up some screens at low conc under which you may reduce precipitation or even with partial precipitation. Best, Debanu > On Dec 25, 2016, at 6:43 PM, mesters <[email protected]> wrote: > > Hi, > > although most widely used, Tris-NaCl buffers may be acceptable for many but > unfortunately by far not all proteins. > > The solubilty of a protein can be modulated by the use of the proper anions > and cations. This all depends on the pH of the solution and the pI of the > protein. > > Consult the following papers: > > - https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1978149/ Crystallization > Optimum Solubility Screening > - https://www.ncbi.nlm.nih.gov/pubmed/15333951 Optimum solubility (OS) > screening > - > https://www.researchgate.net/publication/11105024_Importance_of_the_nature_of_anions_in_lysozyme_crystallisation_correlated_with_protein_net_charge_variation > > The Hofmeister series plays a most important role! > > Jeroen > > > > > On Sat, Dec 24, 2016 at 2:52 AM, Praveen Tripathi > <[email protected]> wrote: >> Dear all, >> I am graduate student working on a functional protein which i have cloned in >> pET-28a vector for recombinant protein production in E.coli expression >> system. >> The expressed protein is purified on Ni-NTA resins with Imidazole gradient. >> Surprisingly, i am getting distinct visible white precipitate in pure >> fractions in eluted fractions itself. >> Please suggest how to make it soluble or how to prevent the precipitation. >> On concentrator the precipitate ration is very much increasing. The protein >> is pure in soluble as well as precipitate. >> Buffer condition- 50mM Tris(7.5), 500mM NaCl, 10% Glycerol. Elution buffer >> has varying concentration of imidazole varying from 10mM to 300mM. >> Any kind of suggestion will be highly appreciated. >> My project requires structure determination. >> >> Thanks in advance. >> >> Regards >> Praveen > > -- > Dr. math. et dis. nat. Jeroen R. Mesters > Deputy, Senior Researcher & Lecturer > Program Coordinator Infection Biology > > Institute of Biochemistry, University of Lübeck > Ratzeburger Allee 160, 23538 Lübeck, Germany > phone: +49-451-31013105 (secretariate -31013101) > fax: +49-451-31013104 > > <logo_10564.jpg> > > http://www.biochem.uni-luebeck.de > http://www.eine-stadt-sieht-gelb.de > http://www.uni-luebeck.de/studium/studiengaenge/infection-biology > http://www.iobcr.org > > -- > If you can look into the seeds of time and tell which grain will grow and > which will not, speak then to me who neither beg nor fear (Shakespeare's > Macbeth, Act I, Scene 3) > -- > Only two things are infinite, the universe and human stupidity, and I'm not > sure about the former (Albert Einstein) > -- > Disclaimer > * This message contains confidential information and is intended only for the > individual named. If you are not the named addressee you should not > disseminate, distribute or copy this e-mail. Please notify the sender > immediately by e-mail if you have received this e-mail by mistake and delete > this e-mail from your system. > * E-mail transmission cannot be guaranteed to be secure or error-free as > information could be intercepted, corrupted, lost, destroyed, arrive late or > incomplete, or contain viruses. The sender therefore does not accept > liability for any errors or omissions in the contents of this message, which > arise as a result of e-mail transmission. If verification is required please > request a hard-copy version. Please send us by fax any message containing > deadlines as incoming e-mails are not screened for response deadlines. > * Employees of the Institute are expressly required not to make defamatory > statements and not to infringe or authorize any infringement of copyright or > any other legal right by email communications. Any such communication is > contrary to Institute policy and outside the scope of the employment of the > individual concerned. The Institute will not accept any liability in respect > of such communication, and the employee responsible will be personally liable > for any damages or other liability arising. Employees who receive such an > email must notify their supervisor immediately. > --
