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The biotinylated Tat proteins were purified on a column of soft release avidin resin, cleaved from the fusion protein using factor Xa and eluted from the column followed by desalting with a PD10 column.

To the biotin moieties on the solid surface, biotinylated adenoviral vectors were attached by using avidin as a molecular bridge between the biotin moieties on the solid surface and the viral surface.

CMV-LacZ, which had been biotinylated by treatment with 15 µg/ml sulfo-NHS-LC-biotin as above, was applied to the wells and allowed to bind to unoccupied biotin-binding sites of Neutralite avidin that had been bound to biotin moieties on the solid surface.

Unbound Neutralite avidin was removed by washing the wells with PBST.

Concurrently, we surface labelled zona-free eggs with biotin and performed 2D avidin blotting to attempt to identify surface proteins.

Biotinylation and avidin blotting identifies surface-labeled egg proteins Separation of surface-labeled biotinylated egg proteins followed by avidin blotting was utilized to identify surface-labeled proteins.

Two proteins in the unlabeled eggs bound streptavidin, corresponding to endogenous non-specific avidin binding proteins not included in the repertoire of surface-labeled proteins (data not shown, see figures in Coonrod et al.

For multi-BAC (two-color) FISH, biotinylated probes were detected with FITC avidin and digoxigenin-labeled probes were detected with Rhodamine anti-digoxigenin (Roche).

After incubation with avidin or anti-digoxigenin, slides were washed in coplin jars on a rotating shaker for three 5-min washes in 4x SSC/0.


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