I want to evaluate the percentage of native Interferon beta-1b after refolding process.
As a NMR spectroscopist, I would recommend you to compare the CD or NMR spectra recorded on you refolded sample with that of the native protein (if available). For sure, this will allow you to check if the refolding process was complete. If not, I don't think it will be easy to quantify (even roughly) the amount of refolded protein unless you have only 2 states in solution (the native state and the fully unfolded state).
Other methods are cited in this discussion:
https://www.researchgate.net/post/Should_I_refold_my_his-tagged_protein_after_Triton-X100_digestion
Good luck!
Ludovic idea is perfect, but you can have the problem to find the NMR/CD lab. 20 kDa I guess is your protein mass? Could be just to big for NMR, will be very suitable for size-exclusion. S-E will give you the info about the aggregates level and will result with purified fraction of the native protein. FPLC would be be perfect, but simple gravity flow column should work as well. In every lab you have a lot of old sizing resins (sephadex), just pick up with the MWCO in the range of 25-100 kDa. Don't be afraid to use 30-years old one, just check if it is dry powder without any smell. The day before take one table spoon per 200ml of your native buffer (the resin volume will dramatically increase). Welcome to ask about details if you will find it:)
I know a very good method to do this, and that is hplc with c4 column. Unfortunately in our country, only some special centers have this and they do not allow university students to use them! So i want to find a cheap and omnipresent method to do this.
I would send you this resin, if I’m not afraid of customers. If they will find an white powder nicely packed they would go crazy…
Thank you Jan for this cheaper and more reasonable suggestion! Sometimes I forget that a NMR or CD spectrometer is very expensive and not available in all universities (I actually don't think my laboratory belongs to a "rich" university, but this is not the subject of the discussion...).
More seriously, Jan's idea is interesting. The unfolded states of your protein should have distinct retention times on a Sephadex column in comparison with the native state. By comparing peak volumes, you should be able to quantify the amount of refolded protein. I've never done it by myself but it makes sense. I'm more skeptical with HPLC and C4 columns because acetonitrile or other organic solvent is likely to unfold (at least partly) your protein which will lead to a bias in the quantification. This is only a feeling, you may have found such method in the literature.
A last point: smoking is not good for your health Jan! :-)))))
Only problem with gravity flow column is you spent half a day counting the drops, and collecting the fractions. The column you can do almost by yourself out of tube of 1-2 cm diameter, 20 cm long and transparent. Aggregates would appear first, than the native form. Princeton University page is refering to http://en.wikipedia.org/wiki/Size-exclusion_chromatography ;)
The calibration you should do with any available protein similar in shape and size to your one.
Dear Ludovic, I'm very grateful you take care of me, I also worry about you, since your picture is taken in the place, let me guess they sell alcohol. -:))))))))))))))
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