Example 5
The heparin affinity of AAV2-retro and wild-type AAV2 were analyzed as previously described (Jang et al., 2011, Mol. Ther., 19:667-75). Briefly, approximately 1011 purified genomic particles were loaded onto a 1 mL HiTrap heparin column (GE Healthcare Sciences, Piscataway, N.J.) previously equilibrated with 150 mM NaCl and 50 mM Tris at pH 7.5. Elution was then performed by increasing the concentration of NaCl in steps of 50 mM up to a final concentration of 950 mM, followed by a wash with 1M NaCl. A small fraction of each elution was used to infect HEK293T cells, and the percentage of GFP positive cells was quantified 48 hours post-infection using a Guava EasyCyte 6HT flow cytometer (EMD/Millipore).