GST Pulldown Assay

Materials

• 2x HNG (100mL). Combine 100mM HEPES (10mL), 300mM NaCl (7.5mL), 20% glycerol (20mL)
• Wash Buffer (50mL). 1x HNG (25mL) and 25 mL water.
• Lysis Buffer (10mL). Combine 5mL 1x HNG, 1 PI tablet, 1mL of 10% NP40 (or 0.5 mL of 20% Triton X-100) and any other cofactors as needed. Bring up to 10 mL in water. This buffer is just a starting point and may need to be modified for particular interactions.
• Glutathione sepharose beads

Immobilization of Protein onto Glutathione Beads

1. Combine 1 mL PBS (or other buffer if needed) with 200 ug of protein and 40 uL of resuspended gluthione beads.
2. Incubate 30min-1h end over end at 4C to bind to beads.
3. Centrifuge beads for 30s on high. Aspirate supernatant and add 1 mL PBS (or other buffer)
4. Repeat wash step 2x.
5. Resuspend beads in ~200 uL final volume of buffer and make 20 uL aliquots (each will contain ~10 ug of protein and will be enough for ~5 pull down assays)
6. Freeze these aliquots at -80

Protocol

1. Prepare lysis buffer with detergent (10 mL) and without detergent (50 mL)
2. Prepare and treat cells as needed
3. Wash cells 2x with ice cold PBS (10 mL for a 10cm dish, 25 mL for a 15cm dish)
4. Add 1 mL lysis buffer and scrape cells with cell scraper. Transfer to 1.5 mL eppendorf tube
5. Incubate end over end for 15-30 min at 4C to lyse.
6. Centrifuge at 4°C for 10 minutes.
7. While waiting for the previous two steps, prepare the affinity beads:
   1. Thaw one aliquot of immobilized protein and an aliquot of GST.
   2. Calculate volume of lysate to be used per pulldown. For example if you have >1 mL lysate and you want to do 3 pull downs then you can use up to 333 uL of lysate per pull down. Alternatively you can do a bradford assay and determine the volume of lysate to be used based on the amount of protein. Record how much lysate will be used per pulldown
   3. Calculate how much buffer to resuspend the beads in. Typically we use one volume of beads per volume of lysate, so if you want to use 300 uL of lysate you would resuspend the beads in 300 uL of buffer. Calculate which combinations of lysate and beads you want to use. Based on the entire tube containing 10 ug, record how much protein is being used for each pulldown. Remember to use the same amount of immobilized protein for each pulldown.
   4. If necessary aliquot beads into different tubes so that you have one tube for each bead/lysate combination.
8. Once lysate is done with centrifugation, transfer the supernatant to a fresh tube.
9. Take out a 50 uL aliquot of each lysate sample and add 50 uL of 2x sample buffer as a lysate control.
10. Add the appropriate amount of lysate to each set of beads
11. Place tubes end over end for 30 min-4h at 4°C. Use 30 min for time sensitive interactions (ie GST-GTPase Pull Down Assay) and longer time for more stable interactions.
12. Add 50µL of glutathione sepharose beads to each tube to increase the bed volume of beads and to limit accidental aspiration of beads
13. Wash each tube with 1x HNG five times at 4°C.
14. Add 20-40 uL of 2X SDS Loading Dye.
15. Boil samples for loading on a gel.
16. Calculate what 0.5% Lysate Volume is (remembering it is diluted 2x in sample buffer).
17. Load 0.5% Lysate plus the entire volume of the pulldown. If you need to do several blots of the pulldown, decrease both volumes accordingly.