I've been trying to dialyze a 1.5ml His-tag protein which has been purified using 250mM imidazole. The dialysis buffer's pH is 7.6 (1X TBS, 0.1% Titon, 5% glycerol) and the pI of my protein is 6.3. The dialysis is carried out overnight at 4 deg. C. How to avoid its precipitation?
Hi Sucheta. What I can suggest you is that try to dilute your protein. I mean if its highly concentrated it would result in precipitation. Go for 1mg/ml of protein concentration. Hope this could help u some way or the other. Good luck.
1x TBS typically has a salt concentration around 150mM.
Do you have scope to increase :
1) [] NaCl (ie, 250mM, 500mM)
2) glycerol % (ie 10%)
3) pH - ie 8.5 instead of 7.6
or B) is your protein saturating in concentration, can you dilute the concentration to reduce likelihood of precipitation?
Please feel free to discuss this further,
best wishes, Robin
Hi Robin,
Thank you for your suggestion. I would be changing the conditions in my next dialysis keeping them in mind. The protein concentration is also high, so I might have to dilute it as well.
- Badges
- Science method