Chemistry Reference
In-Depth Information
quenching assay can be turned into this type of turn-on assay can be found in the
work of QTL Biosystems [
44
]. A turn-on high-throughput commercial (QTL
Lightspeed
) assay variation was developed of the Whitten and co-workers'
kinase detection turn-off scheme described above in Sect.
5.1.3
. The turn-off
assay detected quenching of polymer-Ga
3+
beads by phosphorylation peptide
kinase substrates labeled with quenchers to signal kinase activity (Fig.
12a
)[
88
].
In the commercial form of the assay, a protein substrate phosphorylated by the
kinase competes with a nonsubstrate “probe” peptide labeled with a quencher and a
PO
3
group (Fig.
12b
). As the kinase phosphorylates its substrate, the extent of
quenching by the probe is decreased and the emission of the overall sample
increases.
™
a
-
+
+
+
+
+
+
Kinase
-
+
+
+
+
+
-
+
+
Quencher-Peptide
Quencher-Peptide-PO
-
Beads with emissive polymer
and cations (Ga
3+
)
b
+
+
-
+
-
Kinase
+
+
+
+
-
-
Quencher-linker-PO
-
(competition probe)
Substrate-PO
-
Kinase substrate
+
-
+
+
-
+
+
+
+
OR
+
+
+
+
+
+
-
Low kinase activity:
low emission
High kinase activity:
high or “turn-on” emission
Fig. 12 Cartoon representing two versions of a kinase assay. (a) The“turn-off” version is a direct
assay; the extent of phosphorylation of the substrate is reflected in the binding of the quencher to
the bead and hence to the quenching [
88
]. (b) The “turn-on” version is a competition assay:
increased phosphorylation of the substrate leads to it competing with the quencher in binding to the
beads, and hence to increased emission [
44
]. Emissive polymers are represented by
green lines
;
quenched polymers by
grey lines
Search WWH ::
Custom Search