Assay Method Information

Assay Name:  Multiplexed dose response screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bfl-1.
Description:  The multiplex is constructed by using beads for each protein target that have been labeled with varying intensities of red color, so that each assay is built on a unique bead set, and each bead set is associated with a unique optical address. Beads are first washed in buffer for 20 minutes before adding the appropriate GST-Bcl fusion protein. The HTS assay was conducted in 384-well microplates in a total assay volume per well of 10.1 microliters (5 microliters of bead mixture, 0.1 microliters of test compound, and 5 microliters of 100 nM F-Bim in HPSMTB). Test compound concentration was 10 microM. Controls, which contained bead mixture and F-Bim but no test compound, were located in columns 1 and 2 on each plate. Plates were placed horizontal axis on rotators and incubated for 1-2 hours at 4 degrees C. A glutathione-only bead set control (no associated GST-protein) was incorporated into each well as a fluorescence scavenger to determine inherent fluorescent properties (at 530 nm emission) of the test compounds. In the study reported here, the 237 compounds that satisfied the hit selection criterion in the primary screen (change in %Inhibition greater than 40%) were tested in a dose response format to confirm activity and determine potency. Additional compounds, which were actives from other Bcl targets, were also included in the dose response evaluations. Final compound dilutions in DMSO ranged from 1 microM to 10 mM. These dilutions were then diluted 1 to 100 to give an assay concentration range of 10 nanoM to 100 microM. Sample acquisition and preliminary analysis is conducted with the HyperCyt(R) high throughput flow cytometry platform. The HyperCyt system interfaces a flow cytometer and autosampler for high-throughput microliter-volume sampling from 384-well microtiter plates. The stream of particles is excited at 488 nm and 635 nM, and flow cytometric data of light scatter and fluorescence emission at 530 +/- 20 nm (FL1) and emission at 665 +/- 10 nm (FL8) are collected on a Cyan Flow Cytometer (Dako). Compounds with EC50 less than 10 microM and magnitude of response greater than 40% (i.e., Bottom of sigmoidal curve < 0.6 Top of sigmoidal curve, listed as FIT_PERCENT_SPAN) were said to be Active.
Affinity data for this assay
 

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