I am working on a protein of 25 kDa with pI of 9.29. I have this protein in phosphate buffer 20 mM pH 7, NaCl 150 mM. The protein behave very well during gel filtration (buffer NaAc 40mM pH5.5, NaCl 150 mM) and elutes with a symmetric peak of a dimer. Surprisingly, it aggregates just after elution. I have no idea why it is happening?
DEar RAhul
it seems to me that the proteins don't like reduction in terms of ph that happened in the SEC. (buffer exchange from 7 to 5.5)
Why did you used tNaAC buffer and not the PBS pH=7 for the SEC?
ciao
Manuele
Dear Manuele,
My protein show max activity at photo 5.5. Therefore I have to go for buffer exchange
..
The pH at which the protein has maximal activity is not necessarily the optimal pH for solubility of the protein.
I agree with Adam’s view and you should use buffer with pH=7 for gel filtration.
Good luck!
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