Figure 5.

Inhibition and stimulation of cleavage activity by mAbs generated against TaqNP. (Top) D16 substrate (5 nM) and gap 4 primer (0.1 μM) were incubated at 55°C for 15 min with 0.8 ng of TaqNP in 10 μl of 10 mM Tris⋅HCl, pH 7.8/50 mM KCl/1 mM MgCl₂/0.2% Tween 20/100 μg/ml BSA in the presence of 150 ng of TaqNP-specific mAb. The products, separated from uncleaved substrate by PAGE, are shown. (Middle) The D16 substrate, gap 4 primers, and antibodies were incubated with 10 ng of TaqN nuclease as described above at 55°C for 40 min. (Bottom) The D6 substrate, gap 4 primers, and antibodies were incubated at 55°C for 15 min with 0.8 ng of TaqNP, as above. In all of these experiments, reaction samples containing all components except MgCl₂ were preincubated at room temperature for 10 min, heated to 55°C, and reactions were started by addition of MgCl₂. A total of 22 different mAbs were assayed and representative products of cleavage are shown; the results are summarized in Table 1.