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 1 2 3 4 5 6 7 8 9 NFX1 WT (1ug) 20 20 20 NFX1 N term (7.5ug) 10 10 10 GST (7.5ug) 5 5 5 HeLa (200ug) 133 133 133 CaSki (200ug) 133 133 133 293T (200ug) 133 133 133 Binding Buffer 347 347 347 357 357 357 362 362 362 Total Volume (ul) 500 500 500 500 500 500 500 500 500

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