Figure 1.

Illustration of two-cycle CsCl isopycnic density gradient ultracentrifugation

High- and low-density rAAV2 particles were separated by performing two cycles of CsCl isopycnic gradient ultracentrifugation after affinity chromatography purification. Purified rAAV2-CMV-EGFP vectors were developed using a suspended HEK293T production system. Two full particle bands were observed, denoted as F1 and F2 (left). In both fractions, considerable mixing of the two types of particles was observed after the first cycle; thus, a second cycle of CsCl ultracentrifugation (right) was conducted for F1 and F2, where the two sets of particles with different densities were observed in both fractions, leading to further fractionation into F1.1, F1.2, F2.1, and F2.2. In another preparation, the F1 and F2 sample fractionated in a mixture denoted as “Bulk”.