COMPARISON OF RETENTION AND BINDING BEHAVIOR OF DUTP AND BIOTIN-CONJUGATED DUTP USING AN IMMOBILIZED SILVER ION CHROMATOGRAPHY SUPPORT

A gel filtration chromatographic packing was modified to contain immob ilized silver ions in order to study the retention and binding behavio r of biotin-labeled b-dUTP versus dUTP. The immobilized silver column retains unlabeled dUTP (with the retention time depending on sodium ch loride 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 silve r column, while b-dUTP is fully bound to dhe immobilized silver column . Approximately 90% of b-dUTP is recovered when b-dUTP is applied to t he 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 ana lysis 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.