Figure 1: Separation of DARPin-displaying from DARPin-deficient vector particles.
From: Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors
(a) Schematic drawing of the two-step procedure including density gradient centrifugation followed by IMAC, which separates DARPin-displaying AAV particles carrying a hexa-histidine-tag at the N terminus of the DARPin from DARPin-deficient particles. (b) Example of chromatograms obtained after IMAC of a Her2-AAV vector stock. Left: Loading profile of iodixanol gradient-purified Her2-AAV vector preparations. Minutes 20–40 represent the loading of AAV particles, minutes 40–100 the column washing. Right: Elution profile of Her2-AAV particles. The absorbance at 280 nm is depicted. The loading and elution fraction numbers are indicated. The dashed line represents the imidazole concentration used during washing and elution. (c) 96 fractions collected during column loading (1–11), washing (13–49) and elution (65–72) were analysed by dot blot using the capsid protein-specific antibody B1. Fractions were loaded in alternating order from top left to bottom left as indicated by arrows. (d) Twenty μl of each fraction were used to transduce SK-OV-3 cells. The percentage of GFP-positive cells was quantified 60 h post transduction and is expressed relative to the highest transduction rate observed. Column loading and elution fractions are indicated.
