FIG. 7.

Accessibility of the FLAG peptide in the context of intact Ad5F_HIFLAG virions. Virions of Ad5F_HIFLAG purified on a CsCl gradient were dialyzed, immunoprecipitated with anti-FLAG M2-affinity gel as described in Materials and Methods, and eluted from the gel with free FLAG peptide. Recombinant adenovirus vector Ad5CMVLuc containing unmodified fiber was used as a negative control for binding. Aliquots of all the fractions collected throughout the purification procedure were treated with DNase I to digest traces of the cellular DNA and then treated with SDS, EDTA, and proteinase K to release adenovirus DNA from the virions. The samples obtained were analyzed on a 0.8% agarose gel, and DNA was detected by ethidium bromide staining. Lanes 1 through 3, AdCMVLuc in the supernatant containing unbound material, buffer wash, and FLAG-eluate, respectively; lanes 4 through 6, Ad5F_HIFLAG in the supernatant, buffer wash, and FLAG-eluate, respectively; lanes M, DNA molecular weight standards (the bands corresponding to marker fragments ranging from 3 to 12 kb are seen on the gel).