Yes, I once spent quite a bit of time engineering mutations into my target to improve solubility to exclude detergent from the purification to improve crystal growth and diffraction: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2374227/
If you think buffer conditions hold the key to your solubility woes over other factors, you can look into Hampton's (or other vendors) Solubility and Stability Kit: https://hamptonresearch.com/documents/product/hr008095_binder1.pdf Or the pH Slice Kit: https://hamptonresearch.com/product_detail.aspx?cid=30&sid=200&pid=616 Best, Debanu > On Mar 30, 2017, at 4:47 AM, Antonio Ariza <antonio.ar...@path.ox.ac.uk> > wrote: > > 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 > Tel: 00 +44 1865 285655 > > Links to my public profiles: > ResearchGate > LinkedIn > GoogleScholar > Twitter > > Check out my latest paper!!! > The toxin-antitoxin system DarTG catalyzes reversible ADP-ribosylation of DNA > 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.
