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Univerziteta u Beogradu
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A highly optimized protocol for the isolation and purification of transferrin from human serum
Četić, Danilo
Miljuš, Goran
Stevanović, Jovana
Dobrijević, Zorana
Šunderić, Miloš
Nedić, Olgica
Gligorijević, Nikola
Vilotić, Aleksandra
Borko, Valentina
Gabričević, Mario
Weitner, Tin
Penezić, Ana
Different methods for the isolation and purification of human transferrin (Tf) from human plasma and se rum have been described in the literature. However, none provide a highly pure final product in a time-ef ficient manner, whereas the method described in this work accomplishes both. It consists of two separate steps—a sample preparation and an anion-exchange chromatography step. Our initial methodology used rivanol for protein precipitation in the first step and activated charcoal for the removal of excess rivanol from the remaining supernate. This provided Tf of >99% purity while retaining Tf function, analyzed by immunofluorescent staining for transferrin-transferrin receptor 1 in teraction and ferrozine method for iron binding capacity, and structure, confirmed by recording and comparing fluorescent spectra of purified Tf and commercially available pure Tf. However, the method produced a relatively low Tf yield. The initial method was then modified to use potassium bromide for excess rivanol removal, increasing yield by approximately 82% compared to the initial protocol. We then tested if saturating Tf with iron(III) ions before precipitation with rivanol would increase yield. This increased yield by a further 20% compared to potassium bromide rivanol removal, and a final 119% compared to the initially proposed method. The improved method was additionally evaluated for the purification of Tf from the serum of patients with different iron-associated pathologies, showing its applicability on different types of samples.
engleski
2025
Ovo delo je licencirano pod uslovima licence
Creative Commons CC BY-NC-ND 4.0 - Creative Commons Autorstvo - Nekomercijalno - Bez prerada 4.0 International License.
http://creativecommons.org/licenses/by-nc-nd/4.0/legalcode
transferrin, anion-exchange chromatography, protein purification