Rac pulldown assay (with GST-PBD beads)
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| Materials | add fresh | |
|---|---|---|
| Lysis Buffer | Tris pH 7.5, 50 mM, NaCl 150 mM, MgCl2 10 mM, NP40 1%, Glycerol 10% | DTT 1 mM, Leupeptin, Aprotinin, PMSF |
| Wash Buffer | Tris pH 7.5, 25 mM, NaCl 40 mM, MgCl2 30 mM, NP40 1% | |
| Glutathione-Sepharose beads with bound recombinant GST-PAK-binding-domain (GST-PBD) |
Procedure
- Prepare lysis and wash buffer and keep on ice. Thaw beads (-80°C) on ice.
- Stimulate cells to activate Rac (e.g. phagocytosis, cell spreading) For small cells (e.g. HL60) use 1-2 x 107 cells/pulldown, for larger cells less
- Lyse cells on ice in 600 ul complete lysisbuffer and incubate 20 min on ice
- Centrifuge lysates for 10 min at 14k rpm at 4°C
- Transfer 500 ul supernatant to new pre-chilled tube on ice
- Keep the remaining supernatant as “input” samples (60 ul sup + 20 ul 4x SDS- LB)
- Add 20 ul PBD-beads (use cut-off yellow tip) to each 500 ul supernatant
- Incubate pulldowns for 45-60 min on rotating wheel in the cold room (4°C)
- Centrifuge the lysates for 2 min at 2000 x g
- Remove the supernatant carefully (don’t disturb the pelleted beads)
- Wash each bead pellet 3 times with 500 ul ice-cold washing buffer
- Resuspend the beads in 30 ul 2x SDS-LB and boil in a shaking heat block (7 min at 99°C, 1000rpm)
- Centrifuge the beads after boiling for 5 min at 14k rpm
- Transfer the supernatant to a new tube (try NOT to transfer any beads)
- Run samples on a 12.5% SDS-PAGE gel with large slots. Load the complete pulldown sample (30-40 ul) and 30 ul of the input samples.
- Transfer to PVDF (Western Blot) and use an anti-Rac antibody to detect GTP- Rac (pulldown samples) and total Rac (input samples)