Fig. 4.

Sample gel of in vitro cleavage assay products. Double-stranded DNA target substrates with an incorporated A647 fluorophore are incubated at 37 °C in the presence of free enzyme released from the yeast surface by DTT. The resulting cleavage products are run on an acrylamide gel and visualized by a fluorescence imager. The sample lanes shown here represent fully cleaved, partially cleaved, and non-cleaved target substrate. The non-tethered nature of this in vitro assay provides an important validation of cleavage activity observed in the flow-cytometric cleavage assay. In this assay, the enzyme must have suffi-cient binding affinity for its DNA substrate to produce observable cleavage products