The elution profile during each run was monitored at 280 nm, and a series of 1 ml fractions was collected using a BioFrac fraction collector.
The resulting chromatograms obtained with the 75, 100, 250, 500, and 750 mM imidazole elution buffers were superimposed using the Trace Compare feature provided within the BioLogic DuoFlow software; these are illustrated in Figure 3.
The data in Figures 2 and 3 indicate that optimal results were obtained with an elution buffer consisting of 250 mM imidazole, 50 mM potassium phosphate, 300 mM NaCl, pH 8.0. This elution buffer provided the most effective separation and removal of the impurities (Figure 2, lanes 5 and 6, respectively).
The development of rapid and efficient methods for the chromatographic isolation and characterization of biomolecules requires a chromatography system that is capable of advanced automated operation with minimal user intervention. This study demonstrates that the sophisticated valve configuration, high degree of flexibility, and versatile software of the BioLogic DuoFlow system make it ideal for meeting the demands of a modern method development laboratory.
Coomassie is a trademark of BASF Aktiengesellschaft.
Information in this tech note was current as of the date of writing (2004) and not neccessarily the date this version (Rev A, 2005) was published.
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