Equilibrate 20µL Glutathione Sepharose 4B or RFP-Trap Agarose beads with 200µL SEC buffer

Incubate equilibrated beads with GST- or mCherry-tagged bait protein for a final concentration of 5micromolar (µM) in SEC buffer for 1h 0m 0s at 4°C with gentle rotation.

Centrifuge beads at 3000 rcf for 0h 2m 0s at 4°C

Remove the supernatant and wash beads with 200µL SEC buffer

Repeat for a total of 2 washes, then discard buffer

Add 20µL SEC buffer to achieve a beads:buffer ratio of 1:1

Pipette prey proteins into the wells of a 384-well glass-bottom microplate (Greiner Bio-One)

Pipette 1µL of bait-coated beads into each well

Incubate the plate for 0h 30m 0s in the dark at Room temperature

Use a microscope configured to detect fluorescent signal (e.g. Zeiss LSM 700 confocal microscope equipped with Plan-Apochromat 20X/0.8 objective)

Acquire fluorescent images in the middle section of the beads and collect more than one image for each well

Also acquire bright field images for each field

Draw several lines across each bead in the fluorescent channel and measure the intensity along the lines

Record the maximum intensity for each bead

For background correction, measure the average intensity of a rectangular ROI that covers an area of each field of view with no beads

Subtract the average intensity of the background ROI from each bead maximum in that field

Calculate the average of the background-corrected maximum intensities of beads for each sample