Noninvasive method for determination of immobilized protein A

Mravljak, Rok (avtor), Stantič, Metka (avtor), Bizjak, Ožbej (avtor), Podgornik, Aleš (avtor)

The pH transition method, developed for the determination of the ion-exchange group density on chromatographic stationary phase, was used for the quantification of immobilized protein A. Monolithic epoxy polyHIPE and particulate CNBr-Sepharose supports were used for immobilization. A lactate buffer was selected, having a buffer capacity peak approximately 0.5 pH units below the maximum buffer capacity of protein A. The pH transition measurements were performed at pH 4.3, where protein A exhibits maximum buffer capacity, with a lactate buffer concentration of 1 mM for protein A immobilized on polyHIPE monoliths and of 5 mM for protein A immobilized on CNBr-Sepharose. The pH transition height and full width at half maximum for the particulate support and the height for the polyHIPE matrix, showed a linear correlation with the amount of immobilized protein A determined from the absorbance difference before and after immobilization for both supports. The developed method allows a simple, non-invasive on-line determination of immobilized protein A using biological buffers, even for chromatographic columns with an amount of immobilized protein A as low as 0.25 mg. In addition, its sensitivity and duration can be easily adjusted by varying the buffer concentration and pH.

• Jezik: Angleški jezik
• Ključne besede: direct noninvasive immobilized protein, quantification, protein A, pH transition method, polyHIPE, CNBr-Sepharose
• Vrsta gradiva: Članek v reviji
• Tipologija: 1.01 - Izvirni znanstveni članek
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Status publikacije: Objavljeno
• Različica publikacije: Objavljena publikacija
• Leto izida: 2022
• Št. strani: 7 str.
• Številčenje: Vol. 1671, art. 462976
• PID: 20.500.12556/RUL-137650
• UDK: 66.08:577.112
• ISSN pri članku: 0021-9673
• DOI: 10.1016/j.chroma.2022.462976
• COBISS.SI-ID: 103441667
• Datum objave v RUL: 24.06.2022

Gradivo je del revije

• Naslov: Journal of chromatography. A
• Skrajšan naslov: J. chromatogr., A
• Založnik: Elsevier
• ISSN: 0021-9673
• COBISS.SI-ID: 6398471

Licence

• Licenca: CC BY-NC-ND 4.0, Creative Commons Priznanje avtorstva-Nekomercialno-Brez predelav 4.0 Mednarodna
• Povezava: http://creativecommons.org/licenses/by-nc-nd/4.0/deed.sl
• Opis: Najbolj omejujoča licenca Creative Commons. Uporabniki lahko prenesejo in delijo delo v nekomercialne namene in ga ne smejo uporabiti za nobene druge namene.

Sekundarni jezik

• Jezik: Slovenski jezik
• Ključne besede: direktna neinvazivna imobilizacija proteina, kvantifikacija, protein A, metoda pH prehoda, poliHIPE, CNBr-sefaroza

Projekti

• Financer: ARRS - Agencija za raziskovalno dejavnost Republike Slovenije
• Številka projekta: J2-9440
• Naslov: Karakterizacija fraktalnih struktur in povečevalni kriteriji njihove sinteze

• Financer: ARRS - Agencija za raziskovalno dejavnost Republike Slovenije
• Številka projekta: P1-0153
• Naslov: Raziskave in razvoj analiznih metod in postopkov