GST-tagged protein-protein interactions
Note: This protocol is used to study protein-protein interactions in vitro using GST-tagged proteins mised
with protein lysates (either nuclear or whole cell). GST tagged proteins that have been produced and
purified from e.coli are mixed with protein extracts. After mixing, GST proteins are bound to reduced
glutathione beads and precipitated. The bound proteins are boiled and run on a gel for identification by
western blot or silver stained and identified by mass spectrometry.
Binding Buffer
PBS
1% NP-40
2mM DTT (200ul of 1M DTT/100ml)
Protease inhibitor tabs
PRECLEAR CELL LYSATES
1. Equilibrate glutathione beads (25ul slurry/cell lysate plus one extra for error)
 Wash beads x3 total with 100ul binding buffer/25ul slurry, pellet at 2,000rpm x2
min at 4ºC. Discard supernatant inbetween washes.
2. Thaw protein lysates (use the same ug protein per GST binding reaction, and bring
them up to the same volume in the binding buffer.)
ex/
80ul HeLa ptn lysate is 600ug ptn; add 220ul BB for vol of 300ul
60ul CaSki ptn lysate is 600ug ptn; add 240ul BB for vol of 300ul
3. Resuspend equilibrated beads in 75ul binding buffer/25ul beads, and aliquot 100ul
slurry into each protein lysate for final volume of 400ul.
4. Rotate in cold room for 30 minutes.
5. Pellet beads at 2,000rpm x2 min at 4ºC. (Removes non-specific sticky proteins)
6. Place supernatant in new microcentrifuge tubes. Save 5% vol of pre-cleared protein
for input.
PROTEIN BINDING REACTIONS
7. Thaw GST-tagged proteins and GST-alone vector control protein.
8. Add GST-tagged proteins to each aliquot of precleared lysate.
9. Add Binding Buffer to make all volumes equal in a 0.6ml microcentrifuge tube.
Ratio used: 1-7.5ug GST-tagged protein to 150-200ug protein lysates. Adjust based on experimental
design and proteins of interest.
BINDING REACTIONS (EXAMPLE)
Proteins
NFX1 WT (1ug)
NFX1 N term (7.5ug)
GST (7.5ug)
HeLa (200ug)
CaSki (200ug)
293T (200ug)
Binding Buffer
Total Volume (ul)
1
20
2
20
3
20
133
4
5
6
10
10
10
133
133
347
500
347
500
7
8
9
5
133
5
5
133
133
347
500
357
500
357
500
133
133
357
500
362
500
362
500
133
362
500
10. Rotate for 1 hour in cold room. (protein-protein interactions)
11. Preequilibrate glutathione beads (50ul slurry/cell lysate plus one extra for error)
 Wash beads x3 total with 200ul binding buffer/50ul slurry, pellet at 2,000rpm x2
min at 4ºC. Discard supernatant inbetween washes.
12. Add an equal volume of binding buffer to equilibrated beads, and aliquot 100ul slurry
into each protein lysate.
13. Rotate for 1 hour in cold room (to precipitate GST-proteins)
14. Pellet the beads at 2,000rpm x2 min at 4ºC.
15. Wash the beads 3 times in 500ul binding buffer (flick to mix, pellet, repeat).
16. Boil the beads in sample buffer for western and silver stain gel. May be stored at -20
or -80 degrees until needed.
Reagents used:
Protease inhibitor tablets, Complete tab mini, EDTA-free, Roche, 1183617001
Glutathione sepharose 4B, GE Healthcare, 17-0756-01