Journal of the American Chemical Society
Communication
moiety contributes to the affinity of 8 and, consequently, the
affinity of the probe.12
ASSOCIATED CONTENT
* Supporting Information
Synthetic procedures, characterizations, and control experi-
ments are provided. This material is available free of charge via
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S
An important control experiment, for this approach, is to
ensure that the analyte molecules themselves do not alter the
activity of the enzyme (PLE) in any way. Therefore, it is
essential that we routinely carry out a control experiment,
where we assay the activity of the enzyme against its substrate
in the presence of the ligand candidates. At high concentrations
of the analyte molecules, we found that the activity of PLE was
indeed unaffected (Figure S3).
AUTHOR INFORMATION
Corresponding Author
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Notes
The approach outlined here works well in providing an
evaluation of the relative affinities but does not provide direct
quantitative binding affinity measurements. Therefore, for such
an approach to be useful, it is necessary that we can adapt this
strategy for the rapid screening of analytes. For this purpose, we
further tested the versatility of our approach in a 96-well plate
reader setup. In such a setting, the volume of the components
needed to generate a data point would be low and the screening
can be done in a highly parallel fashion. The data generated
from this experiment are shown in Figure 5 (compare with
Figure 3a). The results indeed are consistent and are reliably
reproducible.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank the NIGMS of the NIH (GM-065255) for support.
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dx.doi.org/10.1021/ja301204z | J. Am. Chem. Soc. 2012, 134, 7235−7237