Figure 2 shows the 32P quench curve prepared with yellow food coloring as the quench agent. Quench monitoring was performed with tSIS (a spectrum based quench indicating parameter exclusive to the TopCount). Figure 2 shows that tSIS is capable of accurately monitoring quenching due to color in samples that are Cerenkov counted.
Figure 3 shows a plot comparing the Cerenkov counting results from the kinase assay on both the TopCount and LSC. Data was plotted as % of the maximum binding on the respective instrument. As mentioned earlier, specific levels of kinase activity were measured on TopCount using FlexiFilter. The individual filter spots were removed from the FlexiFilter and Cerenkov counted in a Packard Tri-Carb 2500TR LSC for comparison. As seen in Figure 3, both the LSC and TopCount plots are nearly identical which indicates that equivalent final results will be obtained for the assay.
Cerenkov counting is an attractive alternative for various TopCount applications using 32P.
An important advantage of Cerenkov counting on the TopCount is that it provides the ability to measure 32P labels directly in the microplate without cocktail. This makes stripping and reprobing of 32P labeled dot blots possible after counting. In addition, Cerenkov counting provides the ability to perform other molecular biology applications such as PCR product screening and labeling verification in a rapid and easy manner. Since cocktail addition is not required, throughput is increased because samples are counted directly.
For most microplate applications, the TopCount Cerenkov efficiency gives quantitative results and greatly reduces labor compared to Cerenkov co