| 1 |
Compounds and controls (20
nL) were predispensed into 1536-well plates. |
Compound transfer was performed
using an ECHO 650 acoustic dispenser (LabCyte). Compounds in doses
were dispensed to columns 5–48, and controls were dispensedto
columns 1 using 1536-well plates (catalog no. EWB010000A, Aurora Microplates). |
| 2 |
Dispensed 3 μL of
biotin-BSA acceptor beads to all wells, except column 2. |
2× biotin-BSA acceptor
beads (0.02 mg/mL, TruHits kit catalog no. 6760627, PerkinElmer) in
PBS (catalog no. 10010–031, ThermoFisher) + 0.05 mg/mL BSA
(catalog no. BP1600-1, Fisher Scientific) |
| 3 |
Incubated at RT for 30 min
and 200 rpm shaking. |
incubation of Biotin-BSA
acceptor beads with test compounds |
| 4 |
Dispensed 3 μL of
streptavidin donor beads to wells, except column 3. |
2× streptavidin donor
beads (0.02 mg/mL, TruHits kit catalog no. 6760627, PerkinElmer) in
PBS (catalog no. 10010–031, ThermoFisher) in PBS + 0.05 mg/mL
BSA |
| 5 |
Incubated at RT for 30 min
and 200 rpm shaking. |
Streptavidin–biotin
complex formation. Final assay conditions: 10 μg/mL streptavidin
donor beads, 10 μg/mL Biotin-BSA acceptor beads in PBS + 0.05
mg/mL BSA |
| 6 |
Read on PHERAstar
FSX (BMG
Labtech). |
Fastest
read settings, AlphaLISA
Module (680 nm excitation, 615 nm emission) (catalog no. 1802H1, BMG
Labtech) |
