Table 1.
Optimized 96-Well High-Throughput Screening Parameters and Assay Statistics.a
| SAMHD1 | 0.5 μM |
| PPase | 5 μM |
| dGTP (activator/substrate) | 0.1 mM (~1/10 KmdGTP)b |
| Total reaction volume and time | 40 μL and 20 min |
| Z′ factor | 0.87 |
| Mean signal (± SD) | |
| Positive controlc | 2.28 ± 0.08 |
| Negative controld | 0.26 ± 0.01 |
| Coefficient of variation of signal, % | |
| Positive control | 3.4 |
| Negative control | 3.3 |
| Signal/backgrounde | 9 |
a
The volumes would be reduced by 50% for a 384-well plate format.
b
See reference 4 for the steady-state kinetic parameters of SAMHD1.
c
The mean measured absorbance for 96 reactions carried out using the indicated conditions.
d
The mean measured absorbance for 96 reactions carried out in the absence of SAMHD1.
e
Calculated from the ratio of the mean signals for the positive and negative controls.