Table 1.

Ultra-High Throughput Screening Protocol

Step	Parameter	Value	Description
1	Dispense reaction buffer	4.5 μL/well	A mixture containing 14-3-3ζ (10 nM), Dy647-pS136-Bad peptide (20 nM), and anti–His-Eu (1 nM),
2	Controls	4.5 μL/well	A mixture of Dy647-pS136-Bad peptide (20 nM) and anti–His-Eu (1 nM) in one column (32 wells)
3	Library compounds	100 nL/well	21.7 μM final from 1 mM stock in DMSO
4	Incubation time	2 h	Plates were incubated at room temperature for 2 h
5	Assay readout	Ex 337 nm; Em1 615 nm, Em2 665 nm; dual mirror D400/D630	Time-resolved fluorescence; Envision Multilabel plate reader

Step Notes

1. 1536-well black plates (Corning Costar; Cat. No. 3724) were used. The assay component was mixed together in FRET buffer (20 mM Tris, pH 7.0, 0.01% Nonidet P40, and 50 mM NaCl). The MultiDrop Combi (Thermo Scientific) was used for dispensing.

2. One column (32-wells) in a 1536-well plate was used for the background control, which defines the minimal signal.

3. Compounds were added using a Pin-Tool (VP Scientific) integrated with a Beckman NX liquid handler (Beckman Coulter).

4. Plates were stacked with the top plate lidded during incubation.

5. A 50 μs delay was used for the TR-FRET signal measurement.

DMSO, dimethyl sulfoxide; TR-FRET, time-resolved fluorescence resonance energy transfer.