I am trying to solubilize and refold an inclusion body of NKG2D with theoretical isoelectric point (PI) value of 6.2. I usually purify my solubilized protein with Nickel column (Elution buffer pH 7.4) before refolding. I would be grateful if you could offer me some advice on the suitable pH I could use for the refolding buffer. The solubilization and the purification are very successful but I don't seem to get the refolding step right. I think the difficulty is coming from the pH, nevertheless I would be grateful if you could give me your opinion on this. Thank you
You can screen refolding pH between 8.5 to 10. Normally > 9 is require for thiol suffeling and for proper disulfide bridging/bond formation with properly choosed Redox couple system. You can also improve the refolding yield by different types of additives.
Key :
Design refolding buffer properly.
And choose right refolding technique ( there are different type of refolding techniques possible like In dillution , on column , by dialysis etc.
Best luck.
Thank you very much Yamak, your suggestions are well apprecited
Thank you very much Nese for the education, your suggestions are also well noted.
@ Dr Yilmaz why did you also use the buffer with pH towards the neutral one? If yea, then what were the results? Can it change the aggregation problem if I try the buffer with acidic pH? I have a protein with pI 5.2 and i tried buffers (phosphate, Tris etc) with pH 7.2 & 8 and everytime it aggregates. After reading your comment, 'm thinking to try pH rather acidic, would be pleased to have your opinion. Thanks in advance
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques