Noninvasive method for determination of immobilized protein A
| Autor: | Rok Mravljak, Metka Stantič, Ožbej Bizjak, Aleš Podgornik |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2022 |
| Předmět: |
Chromatography
udc:66 udc:66.08:577.112 metoda pH prehoda Sepharose Organic Chemistry CNBr-Sepharose poliHIPE direct noninvasive immobilized protein CNBr-sefaroza General Medicine protein A Hydrogen-Ion Concentration Enzymes Immobilized direktna neinvazivna imobilizacija proteina kvantifikacija protein A metoda pH prehoda poliHIPE CNBr-Sefaroza Biochemistry quantification Analytical Chemistry pH transition method direct noninvasive immobilized protein quantification protein A pH transition method polyHIPE CNBr-Sepharose direktna neinvazivna imobilizacija proteina polyHIPE kvantifikacija Lactates Staphylococcal Protein A |
| Zdroj: | Journal of chromatography, str. 1-7 : Ilustr., Vol. 1671, 24 May 2022 COBISS-ID: 6398471 Journal of chromatography. A, vol. 1671, no. 462976, 2022. |
| ISSN: | 0021-9673 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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