If I remember correctly, Triton X-100 (or any other surfactant for that matter) is a bad idea for protein intended for crystallography. I can't remember the paper, but I'm sure I read that somebody showed it's basically impossible to remove all of the surfactant molecules from the protein no matter how you dialyse it. These large floppy molecules stick to your protein molecules and interfere with methods such as mass spec and can hinder the crystallisation process.
I'm not sure this is your case, but it might help. If your protein has a very high (or low) PI, it will probably need a strongly ionic environment to be stable. I work with nucleic acid binding proteins and in general I find they do better in buffers with concentrations > 500 mM NaCl until all contaminating proteins have been removed. Only after SEC do I lower the NaCl concentration to between 50 and 150 mM NaCl (you'll need to test which final concentration works best for your protein). In any case, some precipitation is normal and you can easily remove it by centrifugation. An ultracentrifuge works best, but even a benchtop centrifuge for eppendorfs can remove most of the precipitate from your dialysed solution. Best Regards, Tony ------------------------------------------------------ Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE e-mail: antonio.ar...@path.ox.ac.uk<mailto:antonio.ar...@path.ox.ac.uk> Tel: 00 +44 1865 285655 Links to my public profiles: ResearchGate<https://www.researchgate.net/profile/Antonio_Ariza> LinkedIn<https://www.linkedin.com/in/antonioariza1> GoogleScholar<https://scholar.google.co.uk/citations?user=9pAIKV0AAAAJ&hl=en> Twitter<https://twitter.com/DrAntonioAriza?lang=en> Check out my latest paper!!! The toxin-antitoxin system DarTG catalyzes reversible ADP-ribosylation of DNA<http://www.cell.com/molecular-cell/abstract/S1097-2765(16)30722-5> ________________________________ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Akilandeswari Gopalan [akilaibt2...@gmail.com] Sent: 30 March 2017 07:02 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] protein precipitation reg Dear all, I have used the following buffers for purification and dialysis. this is fyi. Lysis buffer: 25mM Tris pH 7 or 7.5 or 8 100-500mM NaCl (increase in salt concentration increased precipitation of the protein in the column itself) 5mM Beta mercaptoethanol 0.5% Triton X 100 I have tried with other buffers also. a. HEPES buffer pH7.5 b. Phosphate buffer pH 7.8 c. MOPS buffer pH 8 Wash and Elution Buffer: 25mM Tris pH 7 or 7.5 or 8 100-500mM NaCl 20 and 30mM Imidazole for wash 300mM for elution Dialysis Buffer: 1. Tris 25mM pH 7 2. Tris 25mM pH 7.5 3. Tris 25mM pH 8 4. Tris 25mM pH 7.5, 5% glycerol 5. Tris 25mM pH 7.5, 10% glycerol 6. Tris 25mM pH 7.5, 20% glycerol 7. Tris 25mM pH7.5, 50mM NaCl 8. Tris 25mM pH7.5, 100mM NaCl 9. Tris 25mM pH7.5, 1mM MgCl2 10. Tris 25mM pH7.5, 50mM NaCl, 50mM L-Arg, 50mM L-Glu In all these cases the protein precipitates. i have tried to do buffer exchange also. i can see precipitate sticking on the walls of the tube during the process.
