A gel filtration Chromatographic packing was modified to contain immobilized silver ions in order to study the retention and binding behavior of biotin-labeled b-dUTP versus dUTP. The immobilized silver column retains unlabeled dUTP (with the retention time depending on sodium chloride concentration in the mobile phase), but no affinity binding is evident with dUTP. In the absence of sodium chloride, dUTP was seen to have a retention time of 66 minutes using a 10.3-mL immobilized silver column, while b-dUTP is fully bound to the immobilized silver column. Approximately 90% of b-dUTP is recovered when b-dUTP is applied to the immobilized silver column using 10-3 M PBS and eluted using 0.2 M NaCl in the mobile phase. These results demonstrate the potential for using silver ions in immobilized soft metal affinity chromatography (ISMAC) in order to selectively target biotin labeled molecules. An analysis of the data yielding mathematical models with specific focus on the interaction between chloride and silver ions is provided in order to guide method development for other biotin-labeled oligonucleotides.
ASJC Scopus subject areas
- Chemical Engineering(all)
- Process Chemistry and Technology
- Filtration and Separation