How can I remove unreacted/unused Transferrin and polymer after a bioconjugation reaction using EDC?

03 March 2019 6 9K Report

I am attempting to conjugate the Pluronic F127 polymer with the glycoprotein Transferrin (Tf). I oxidized the Pluronic's terminal -OH groups using Jones oxidation and succinic anhydride, separately.

For the Tf-conjugation, I used EDC and NHS, but I am not sure how to remove the unreacted Tf. Tf is around 80kDa, and the polymer is 12.6kDa. Other studies have used dialysis and gel chromatography. For dialysis, what would be the appropriate MWCO to have the unconjugated Tf eliminated? For chromatography, what gel could I use and how can I monitor the unreacted protein and polymer getting eliminated?

I dialyzed my product using a membrane with MWCO 7kDa, but that would only have removed the extra NHS and EDC.

I centrifuged the sample at 14,000rpm for 30 minute at 4*C, per few papers, and saw some pelleting, but that's an extremely small amount of product (if it is even my desired product).

I used Bradford assay to determine the protein concentration in my un-dialyzed and un-centrifuged product, and got a concentration of 0.24mg/mL. How much of it could have been unreacted Tf? Does/can Tf dimerize or denature in during such reactions?

Thank you!

Shamsher S. Kanwar Popular answer

Yes, as suggested by Mahendra the size exclusion chromatography will work fine. Transferrin has a Mr 80 kDa. The Mr of Pluronic F127 shall add to this after conjugation. thus if Pluronic is 12.5 kDa, the conjugate shall be of ~92 kDa. on a Sephadex G-100 column it shall be the 1st to elute, followed by free transferrin (80 kDa), and P-127. You can try this technique.