GST-Pulldown

Materials
GST-Lysis Buffer:	50 mM Tris-HCl (pH 8), 150 mM NaCl, 10% glycerol, 0.05% sodium deoxycholate, 1% Triton- X, 10 µM ZnCl2, 50mM NaF (add protease and phosphatase inhibitors freshly on the day of the experiment)
GST-Pulldown/wash buffer:	50 mM Tris-HCl (pH 8), 150 mM NaCl, 10% glycerol, 0.05% sodium deoxycholate, 1% Triton- X, 10 µM ZnCl2, 50mM NaF (add 1mM DTT freshly on the day of the experiment)
	Purified recombinant proteins
	Glutathione sepharose resin

Procedure

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Equlibration of beads

Glutathione beads are supplied in 20% ethanol. Before use, the resin must be washed and prepared as a slurry consisting of one part beads and two parts GST- Pulldown Buffer as described in step 1-4. Larger amounts can be prepared and stored at 4°C.

• Gently mix the resin by inverting the flask until you obtain a homogenous solution. Use 10 µl of resin per reaction and transfer into a fresh Eppendorf tube
• Collect the beads by centrifugation at 2700 g at 4°C for 2 min (faster and longer spins can fragment the beads). Remove the supernatant by aspirating carefully using a glass Pasteur pipette attached to vacuum line.
• Wash the beads 3 times with 500 µl of GST-Pulldown Buffer. To perform each wash, add Pulldown Buffer, mix gently, centrifuge at 2700 g for 2 min and aspirate the supernatants
• After the last wash add two bead volumes of GST-Pulldown Buffer resulting in a 50% bead slurry and store on ice

Precleaning of lysate

• Lyse cells according to whole cell lysate protocol with the following changes:
  • Use GST-Lysis Buffer for cell lysis
  • After mechanically shearing the DNA add 20 µl Sepharose beads + 20 µl glutathione sepharose beads + 25 µg of GST to cell lysate and incubate for up to 30 min at 4° on head-to-head rotor.
  • Centrifuge at 13.000 rpm at 4° for 5 min
  • Distribute equal amounts of supernatant into fresh Eppendorf tubes. (Two tubes/sample are required at minimum: one will be incubated with the GST-bait protein and a second with control GST protein.)

Pulldown

• Centrifuge the protein extracts at 10.000 g at 4 °C for 20 min. Transfer supernatants to fresh microcentrifuge tubes on ice (This step is necessary to remove precipitated protein which can happen due to multiple freeze thaw cycles).
• Add 20 µl the 50% bead slurry from step 4 and 2-5 µg of GST-protein to the samples. Equimolar ratios of bait and control protein should be used!!
• Incubate for 2h at 4° on head to head rotor