See "Protocol: HEK293 cell culture for co-immunoprecipitation experiments" for preceding culture and transfection. For the present study, HEK cells were transfected with EGFP-tagged bait proteins. Prey proteins were either endogenous or transfected with HA- or SNAP-tags.

Prepare wash buffer:

A	B
Tris-HCl pH 7.5	10 mM
NaCl	150 mM
EDTA	0.5 mM
Triton-X (optional)	0.4%

Prepare lysis buffer. Lysis buffer composition differed for experiments needing lambda phosphatase treatment

Lysis buffer for non-lambda phosphatase experiment:

A	B
Tris-HCl pH 7.5	10 mM
NaCl	150 mM
EDTA	0.5 mM
NP-40	0.5%
PMSF	1 mM
TAME	0.01 mg/mL
Leupeptin	0.01 mg/mL
Pepstatin A	0.001 mg/mL

Lysis buffer compatible with lambda phosphatase experiment:

A	B
1x NEBuffer for Protein MettaloPhosphatases (New England BioLabs)	1x
NP-40	0.5%
Leupeptin	0.01 mg/mL
ddH2O	To desired volume

24 hours after transfection, wash HEK cells twice in ice-cold PBS and lyse in appropriate lysis buffer. Use 600 µL lysis buffer per experimental condition (pooled across the three 10cm dishes).

Clarify lysates at 10 x g at 4 degrees C for 10 minutes

Wash 25 µL GFP-trap beads per experimental condition in 500 µL wash buffer, in low protein binding Eppendorf tubes under rotating agitation.

Equilbrate beads in lysis buffer for 5 min at 4 degrees C under rotating agitation

For lambda phosphatase experiments: add 60 µL of 10 mM MnCl₂ and 24 µL lambda phosphatase (2,400 units; New England BioLabs) for a final reaction volume of 600 µL per experimental condition.

Incubate at 30 degrees C for 30 minutes

Incubate beads with clarified lysate (or lambda phosphatase-treated lysate) for 1 hour at 4 degrees C under rotating agitation

Wash beads three times for 5 min in wash buffer at 4 degrees C under rotating agitation

Resuspend beads in 60 µL denaturing buffer, and boil for 10 minutes to release bound protein