Table 2.

AF4-AF9 Binding Assay Protocol

Step	Parameter	Value	Description
1	Water/0.01% Tween-20	4 μL	Add to 384-well plate
2	Compound in DMSO	50 nL	Pintool transfer compound into water
3	Biotin-AF4 27-mer peptide	3 μL	Tip transfer on JANUS
4	AF9-FLAG	3 μL	Tip transfer on JANUS
5	Incubation time	90 min	At room temperature
6	Anti-FLAG beadsStreptavidin beads	2 μL	BioTek reagent dispenser or tip transfer on Janus
7	Incubation time	60 min	Plates maintained in low light at room temperature
8	AlphaScreen readout	cps	Perkin Elmer EnVision® Multilabel Reader with standard AlphaScreen option (Em=570 nm)

Step Notes

1. Water with 0.01% Tween-20 (or 750 nM unlabeled AF4 27-mer for low-control wells) added by BioTek reagent dispenser during the MicroSource collection screen and added by tip transfer on the Janus during the TimTec screen. The switch from BioTek to Janus reagent dispensing resulted in improved assay statistics (Table 3).

3. Added 8 nM biotin-AF4 27-mer peptide in 2.3×PBS buffer. Final concentration 2.4 nM in 10 μL assay volume before bead addition.

4. Added 8 nM FLAG-AF9 in 1× PBS buffer. Final concentration 2.4 nM in 10 μL assay volume before bead addition. AF9 is a highly disorganized protein prone to aggregation. It is added last because it is stabilized by binding to AF4.

5. Plates were sealed during the incubation period.

6. Beads were premixed and added simultaneously in 1× buffer under subdued lighting. For the MicroSource collection screen, beads (120 μg/mL each, final concentration 20 μg/mL in 12 μL) were added by reagent dispenser. For the TimTec screen, beads (48 μg/mL each, final concentration 8 μg/mL in 12 μL) were added by tip transfer on the Janus.

7. Plates were sealed during the incubation period.

DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline.