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Rapid Method Development With the BioLogic DuoFlow Chromatography System for the Purification of His-Tagged Proteins

0 mM imidazole)
B7: elution buffer 5 (loading buffer + 750 mM imidazole)

Experimental
A 1 ml Econo-Column chromatography column (0.5 cm ID x 5 cm) was packed manually with Profinity IMAC Ni2+-charged resin (60 m). Five separate runs were conducted, with either 75, 100, 250, 500, or 750 mM imidazole in the elution buffer. Following elution and a subsequent wash step, the column was regenerated using the loading and charging buffers listed under Setup. These five successive runs were linked through the buffer scouting function of the BioLogic DuoFlow software. For each run, the column was preequilibrated with 10 column volumes (CV) of loading buffer at a flow rate of 1.5 ml/min. Crude E. coli lysate (250 l) containing a 75 kD His-tagged protein was loaded into a 3 ml injection loop and injected into the column through an AVR7-3 valve. Host protein contaminants, which were not retained by the Profinity IMAC resin (Figure 2), were removed by washing the column with 10 CV of the loading buffer. The 75 kD His-tagged protein was then eluted with 5 CV of imidazole elution buffer, followed by an additional wash with 10 CV of the loading buffer to eliminate residual proteins from the column. The column was then regenerated as detailed in the following steps using 10 CV of each buffer: metal removal with the discharging buffer; washing with the cleaning buffer; recharging of the metal ion with the charging buffer; removal of excess metal ions with the cleaning buffer; and finally, reequilibration with the loading buffer.

In order to confirm the separation of the target protein from other E. coli proteins in the crude sample, the peak fractions we
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