the NGC column switching valve. A single method was generated in ChromLab software specifying identical buffer conditions and flow rate for all three columns. Selecting the column variable in the ChromLab software scout feature llowed us to assess protein binding under identical sample and chromatography conditions. An automated queue of the three runs was made possible by using the NGC sample pump to directly inject the sample onto the column during the sample application phase and by automatically assigning collected fractions during elution to the appropriate column scouting run.
Even though all three resins are anion exchange resins, marked differences in their ability to bind prancer purple were observed, most likely due to differences in bead chemistries. Looking at the relative area percentages (Table 1), prancer purple bound most efficiently to Foresight Nuvia Q resin, less efficiently to Bio-Scale Mini UNOsphere Q resin, and did not bind to Bio-Scale Mini Macro-Prep High Q resin (Figure 2). The calculated purity quotient difference for prancer purple (PQDPP) and SDS-PAGE data supported the conclusion that Foresight Nuvia Q was the best resin choice for the first step of the prancer purple purification workflow.
pH Scouting Identifies Optimal Binding pH
With the best column resin chosen, we next optimised the method’s buffer pH. Since pH affects the ionisation status of proteins, it is a particularly crucial factor in ion exchange chromatography. Utilising the buffer blending valve and the pH scouting option in the ChromLab software scout feature, we tested binding of prancer purple to Foresight Nuvia Q resin over a pH range from 7.0–8.5 in an automated queue of four runs. Although prancer purple bound to the Foresight Nuvia Q resin at every tested pH, at higher pH more contaminants bound to the columns (Figure 3). To minimise contaminants, a buffer pH of 7.5 was chosen.
LAB ASIA - MARCH/APRIL 2014
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