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Pregled bibliografske jedinice broj: 1112990

Protocol for pH-gradient chromatofocusing of the native and desialylated human apo-transferrin


Friganović, Tomislav; Borko, Valentina; Šeba, Tino; Kerep, Robert; Weitner, Tin
Protocol for pH-gradient chromatofocusing of the native and desialylated human apo-transferrin, 2020. doi:10.5281/zenodo.4006003 (ostalo).


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Naslov
Protocol for pH-gradient chromatofocusing of the native and desialylated human apo-transferrin
(Protocol for pH-gradient chromatofocusing of the native and desialylated human apo-transferrin)

Autori
Friganović, Tomislav ; Borko, Valentina ; Šeba, Tino ; Kerep, Robert ; Weitner, Tin

Vrsta, podvrsta
Ostale vrste radova, ostalo

Godina
2020

Ključne riječi
transferrin, sialoforms, FPLC, chromatofocusing, pISep buffers

Sažetak
This protocol describes the method used for the separation of the native human apo-transferrin from the desialylated apo-transferrin using low-pressure pH gradient ion exchange chromatography. The separation is performed using specialized pH gradient ion exchange chromatography buffers, pISep (CryoBioPhysica, Inc.). The mixture of desialylated apo- transferrin and native apo-transferrin is dissolved in the start buffer (pISep A, pH = 8) and injected onto two HiTrap Q HP (GE Healthcare) anion exchange chromatography column connected in a series. Elution is done by a single step linear gradient (0 – 100 % pIsep B, pH = 4) using ÄKTA Start FPLC system (GE Healthcare). Protein concentration in the eluate is monitored by measuring absorbance at 280 nm and the protein fraction recovery can be calculated by integration over the chromatogram surface area. After separation, the pH value of each fraction containing eluted protein is measured, corresponding to the approximate protein pI value. The observed pI values for the native and desialylated apo-transferrin sialoforms differ significantly and hence can be fully separated.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Farmacija



POVEZANOST RADA


Projekti:
UIP-2017-05-9537 - Glikozilacija serumskog transferina kao faktor u mehanizmu prijenosa željeza (GlyMech) (Weitner, Tin, HRZZ - 2017-05) ( POIROT)

Ustanove:
Farmaceutsko-biokemijski fakultet, Zagreb

Profili:

Avatar Url Valentina Borko (autor)

Avatar Url Tino Šeba (autor)

Avatar Url Robert Kerep (autor)

Avatar Url Tin Weitner (autor)

Avatar Url Tomislav Friganović (autor)

Poveznice na cjeloviti tekst rada:

doi zenodo.org

Citiraj ovu publikaciju:

Friganović, Tomislav; Borko, Valentina; Šeba, Tino; Kerep, Robert; Weitner, Tin
Protocol for pH-gradient chromatofocusing of the native and desialylated human apo-transferrin, 2020. doi:10.5281/zenodo.4006003 (ostalo).
Friganović, T., Borko, V., Šeba, T., Kerep, R. & Weitner, T. (2020) Protocol for pH-gradient chromatofocusing of the native and desialylated human apo-transferrin.. Ostalo doi:10.5281/zenodo.4006003.
@unknown{unknown, author = {Friganovi\'{c}, Tomislav and Borko, Valentina and \v{S}eba, Tino and Kerep, Robert and Weitner, Tin}, year = {2020}, DOI = {10.5281/zenodo.4006003}, keywords = {transferrin, sialoforms, FPLC, chromatofocusing, pISep buffers}, doi = {10.5281/zenodo.4006003}, title = {Protocol for pH-gradient chromatofocusing of the native and desialylated human apo-transferrin}, keyword = {transferrin, sialoforms, FPLC, chromatofocusing, pISep buffers} }
@unknown{unknown, author = {Friganovi\'{c}, Tomislav and Borko, Valentina and \v{S}eba, Tino and Kerep, Robert and Weitner, Tin}, year = {2020}, DOI = {10.5281/zenodo.4006003}, keywords = {transferrin, sialoforms, FPLC, chromatofocusing, pISep buffers}, doi = {10.5281/zenodo.4006003}, title = {Protocol for pH-gradient chromatofocusing of the native and desialylated human apo-transferrin}, keyword = {transferrin, sialoforms, FPLC, chromatofocusing, pISep buffers} }

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