Biomedical Engineering Reference
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was developed that cross-reacts against both receptorswith lownanomolar affinity [38].
Secondary assays used the calcium response of the cell lines to distinguish receptor
agonists andantagonists. Probe reports havebeenpreparedusing this peptide to identify
selective antagonists for each receptor that are posted at the MLPCN Web site [39].
Lessons Learned : In cell-based multiplex assays, it is desirable to use the same
detection reagent for all the populations in the multiplex.
4.4.3 Cycle 2. ATP Hydrolysis-Dependent Disassembly of the 26S
Proteasome: 1X01MH077613-01 (Bead-Based Singleplex)
The proteasome assay was developed by D. Skowyra who had reported a cycle of
proteasome degradation earlier [40, 41]. The proteasome assay uses FLAG-tagged
proteasome for capture and display on streptavidin beads with biotinylated anti-
FLAG antibody. Since GFP is associated with the end caps of the proteasome, a
change in the fluorescent signal results when the caps fall off. Under physiological
conditions, caps are proposed to fall off and reassemble during each degradative
cycle. The assay identifies inhibitors of the proteasome degradation cycle as indicated
through inhibition of the release of the caps. Several small molecules with low
micromolar activity dose-response characteristics were identified. During follow-up
dose-response curves, we eliminated fluorescent molecules that interfered with the
assay using uncapped proteasome on beads to measure background fluorescence. We
used a novel secondary cell-based flow cytometry assay as a follow-up to the primary
bead-based screen for the ATP hydrolysis-dependent disassembly of the proteasome
in the presence of copurified endogenous substrates from yeast. The secondary assay
monitors proteasomal degradation of the yeast Sic1 protein, whichwe have previously
linked to the disassembly of the proteasome via in vitro analysis. Sic1 is an inhibitor of
the S-phase cyclin-dependent kinase (CDK) and the prototype substrate of the Cdc34/
SCF
Cdc4
ubiquitination pathway. Sic1 accumulates at the end of mitosis as a stable
protein and is rapidly degraded in response to its phosphorylation at the G1/S
transition. Two SMR molecules defined as active in the bead-based primary assay
were active in the secondary assay by delaying degradation.
Lessons Learned : Although thebeadassay typicallyyieldedasignal/backgroundof 4/
1 or higher, the fluorescence emitted from the complex was often obscured by
fluorescent compounds in the test wells. The secondary assays turned out to be
qualitative in the sense that delayed degradation rather than inhibition was observed.
4.4.4 Assay for Ligands of GPR30 and Classical Estrogen Receptors:
1X01MH077627-01 (Permeabilized Cell Assay)
We have characterized a novel estrogen receptor (ER) and a novel probe (G1) in
Science and Nature publications [42-44]. Although targets have been accepted by the
MLSCN in cycle 2, HTS on a permeabilized whole-cell binding assay has remained
problematic. This assay requires daily transient transfection of GPR30 or ER. The
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