I everybody!
I am puryfing thioredoxin (extremely stable protein) and the last step is GF, in Sodium phosphate pH7 50mM.
I will use this protein for Crystallization and spectroscopic experiments.
I usually dyalize it in MOPS pH7 5mM but I was wondering if I can skip this last step and keep it in Sodium phosphate.
Do you think it could work or this buffer is not fitting with my planned experiments?
Thank you!
Hi!
50mM Sodium phosphate sounds reasonable for spectroscopic experiments and you should be happy to have such a simple buffer that keeps your protein stable. I personally prefer potassium phosphate for spectroscopy.
Some people will suggest you to change the phosphate to another buffer prior to crystallization experiments (phosphate salts crystallize very well) but I had good protein crystals from phosphate buffered protein before. So unless you worry a lot about false positives in the crystallization (salt crystals) or have limited access to protein and/or crystallization reagents I would just try the sample as it is in the phosphate buffer.
Hi Silvia
Sodium phosphate will precipitate as well thus it will be difficult to differentiate between protein and phosphate crystals. to be in safe do it as usual especially you had no problem.
Hope this helps
I agree with those mentioned above. Phosphate is generally avoided in crystallization due to high propensity for salt crystals, but it may work well for you if you can tolerate screening out all the salt crystals. Is there a reason you need to have your protein in phosphate buffer for your last GF step? Or could you use the gel filtration step as a buffer exchange into MOPS and, therefore, cut out unnecessary steps?
- Badges
- Science method