hello everyone,
I made refolding buffer with cystine and cysteine and cystine was solved successfully in the buffer. today after 12h I took out my refolding buffer and added protein from +4, I saw that cystine had dissolved and it is at the bottom of my flask. I do not know if the sample works like before in FPLC. can someone help me with this?
L-cystine has poor solubility, making stock solutions is aided by basic pH, see this article https://www.jbc.org/article/S0021-9258(18)31298-5/fulltext
I had very good luck refolding the HIV antigenic determinant region fusion protein into a fully soluble form with a mix just like this, Good luck!
If Cystein/Cystine is not required for a good reason I would suggest using Glutathion (reduced/oxidize) in your refolding buffer. Usually a 3:1 ratio at 2-4 mM GSH/GSSG is recommended because of similarity to Redox conditions in the ER or periplasm.
Kimia Kaffashi Consider using L-Cysteine/Cystamine-2HCL as your redox system at concentrations between 0-4mM at different ratios between 1-4. Alternatively, GSH/GSSG can be used and it is protein dependent which redox system is preferred. AS mentioned above, L-Cysteine is dissolved better at basic pH of 8-8.5 at a concentration of 100mM. I have developed two commercial scale purification starting from inclusion bodies and gained a lot of experience in creating an efficient refolding buffer of up to 87% refolding recovery.
I hope this information helps, Good luck.
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