Competition Binding Assay to Study Competing GTPase-Binding Protein Partners

1. Purification of GST-tagged GTPase

1. Culture an E. coli strain such as BL21 transformed with pGEX-Rac1 O/N at 37 °C, shaking at 220 rpm, in 500 ml of autoinduction media (25 mM Na₂HPO₄, 25 mM KH₂PO₄, 50 mM NH₄Cl, 5 mM Na₂SO₄, 2 mM MgSO₄, 2 mM CaCl₂, 0.5% Glycerol, 0.05% Glucose, 0.2% Lactose, 5 g Tryptone, 2.5 g Yeast Extract, 100 µg/ml Ampicillin).
2. Harvest bacteria by centrifugation for 10 min at 10,000 x g, 4 °C.
3. Resuspend bacterial pellet in 20 ml protein extraction reagent, 1x protease inhibitor, and incubate for 20 min at RT with inversion.
4. Clarify the lysate by centrifugation at 40,000 x g for 30 min.
5. Add 2 ml glutathione magnetic beads, washed with phosphate-buffered saline (PBS: 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl).
6. Incubate for 2 hr, mixing by inversion at 4 °C.
7. Wash protein-loaded beads four times with 10 ml PBS, using a magnetic particle sorter to precipitate the beads at each step.
8. Resuspend protein-loaded beads in 2 ml PBS and store at -80 °C in 100 µl aliquots until needed.

2. Expression of GTPase-binding proteins

1. The day before the experiment, transfect plasmids encoding the green fluorescent protein (GFP)-tagged versions of each GTPase-binding protein into a separate 75-cm² flask of HEK293T as follows. For validation of nucleotide loading, transfect GFP-tagged TrioD1 into a third 75-cm² flask of HEK293T.
   1. Dilute polyethylamine to 1 mg/ml in 100 µl sterile 150 mM NaCl.
   2. Add 27 µl diluted polyethylamine to 223 µl reduced serum media.
   3. Add 12 µg plasmid DNA to 250 µl reduced serum media.
   4. Incubate each tube for 2 min at RT.
   5. Combine the polyethylamine and DNA mixes in a single tube and vortex for 2 min.
   6. Incubate for 15-20 min at RT.
   7. Replace the growth media (Dulbecco's Modified Eagle Media, 10% fetal bovine serum, 2 mM L-glutamine, no antibiotics) on 90% confluent HEK293T with 5 ml fresh growth media.
   8. Add the combined polyethylamine/DNA mix to the flask and incubate O/N at 37 °C, 5% CO₂.

3. Purification of GTPase-binding proteins

1. Rinse flasks of transfected cells in PBS and drain the flask for 5 min, aspirating free liquid.
2. Scrape off cells in 500 µl lysis buffer (50 mM Tris-HCl (pH 7.8), 1% Nonidet P-40, 1x protease inhibitor) into a microfuge tube.
3. Lyse cells by mixing by inversion at 4 °C for 30 min.
4. During lysis, wash two lots of 40 µl GFP-Trap beads three times with fresh lysis buffer, sedimenting beads at 2,700 x g for 2 min between washes.
5. Clarify lysates by centrifugation at 21,000 x g for 10 min.
6. Transfer clarified lysate of each of the competitor proteins to separate washed GFP-trap beads and allow GFP-fusion proteins to bind for 2 hr, mixing by inversion at 4 °C. Keep lysate from GFP-TrioD1 cells on ice.
7. Wash loaded GFP-Trap beads twice in 50 mM Tris-HCl (pH 7.8), 50 mM NaCl, 0.7% (w/v) Nonidet P-40 and twice in 50 mM Tris-HCl (pH 7.6), 20 mM MgCl₂, sedimenting beads at 2,700 x g for 2 min between washes.
8. Elute GFP-fusion proteins by adding 40 µl 0.2 M glycine (pH 2.5) and pipetting up and down for 30 sec. Immediately sediment beads at 21,000 x g for 60 s and transfer liquid to a new microfuge tube containing 4 µl 1 M Tris-HCl (pH 10.4). Do this quickly to limit damage to the purified protein.
9. Analyze 1 µl of each purified protein by Western blot and probe with an anti-GFP antibody to establish relative yield using a quantitative blotting system according to the manufacturer's protocol. Alternatively, determine protein concentrations by bicinchoninic acid (BCA) assay but this introduces errors if the proteins do not react with the assay in an identical fashion or if there are contaminant proteins.
10. Equalize molar protein concentration by the addition of 50 mM Tris-HCl (pH 7.6) and 20 mM MgCl₂.

4. Nucleotide loading of GTPase

1. Thaw one aliquot of GST-Rac1 magnetic beads, prepared in step 1.
2. Take 90 µl of GST-Rac1 beads and wash three times with 20 mM Tris-HCl (pH 7.6), 25 mM NaCl, 0.1 mM DTT, and 4 mM EDTA, using a magnetic particle sorter to precipitate the beads at each step.
3. Aspirate buffer from beads and add 100 µl 20 mM Tris-HCl (pH 7.6), 25 mM NaCl, 0.1 mM DTT, and 4 mM EDTA.
4. According to whether GDP, GTP, or no nucleotide loading is required for the competition experiment, add 12 µl 100 mM GDP, 12 µl 10 mM guanosine 5'-[γ-thio]triphosphate (GTPγS) or no nucleotide to 60 µl GST-Rac1 beads.
5. For the nucleotide-loading controls, split the remaining beads into three 10-µl aliquots and add 2 µl 100 mM GDP, 2 µl 10 mM GTPγS or no nucleotide to each tube.
6. Incubate bead mixes for 30 min at 30 °C with agitation.
7. Stabilize nucleotide-bound Rac1 by addition of 1 M MgCl₂: 3 µl to the experimental mix (step 4.4), and 0.5 µl to each of the control mixes (step 4.5).

5. Competition binding

1. Set up 6 microfuge tubes, each containing:
   200 µl 50 mM Tris-HCl (pH 7.6), 20 mM MgCl₂
   10 µl experimental nucleotide-loaded Rac1 beads (from step 4.7)
   5 µl Rac1-binding protein A (constant binding protein)
2. To each tube, add 0, 1, 2.5, 5, 10, or 20 µl Rac1-binding protein B (variable binding protein). These volumes assume approximately equal stock concentrations of the constant and variable binding proteins and may need to be adjusted.
   1. Adjust volumes of binding proteins A and B if there are large differences in the binding affinities of the two proteins and this should be determined empirically through the experimental repeats. Make up the total volume of the binding mixture to 235 µl by the addition of 50 mM Tris-HCl (pH 7.6), and 20 mM MgCl₂.
3. Set up a microfuge tube containing:
   200 µl 50 mM Tris-HCl (pH 7.6), 20 mM MgCl₂
   10 µl experimental nucleotide-loaded Rac1 beads (from step 4.7)
   10 µl Rac1-binding protein A (constant binding protein)
4. Set up the GDP, GTPγS, and no nucleotide control tubes:
   200 µl 50 mM Tris-HCl (pH 7.6), 20 mM MgCl₂
   10 µl control Rac1 beads loaded in step 4.5 with GDP, GTPγS, or no nucleotide and stabilized in step 4.7.
   180 µl HEK293T GFP-TrioD1 lysate, prepared as in step 3.6
   4 µl 1 M MgCl₂
5. Incubate the mixture for 2 hr, mixing by inversion at 4 °C.
6. Wash the beads three times with 50 mM Tris-HCl (pH 7.6), and 20 mM MgCl₂.
7. Elute bound proteins in 20 µl reducing sample buffer (50 mM Tris-HCl (pH 7), 5% SDS, 20% glycerol, 0.02 mg/ml bromophenol blue, 5% β-mercaptoethanol).