Guanosine 5 o 3 thiotriphosphate tetralithium salt gtpγs

GST or GST-Rac1 WT immobilized on Glutathione Sepharose 4B (GE Healthcare, 17-0756-01) were washed using GST lysis buffer followed by nucleotide chelating using 0.5 M EDTA (10 mM) and incubation with Guanosine 5′-diphosphate sodium salt (GDP; 100 μM; Sigma-Aldrich, G7127) or Guanosine 5′-O-(3-thiotriphosphate) tetralithium salt (GTPγS; 1 mM; Sigma-Aldrich, G8634) for 15 min at 30 °C. Termination of the reaction was achieved by adding 1 M MgCl₂ (60 mM). HEK293T cells were lysed in GST lysis buffer and equal protein amounts were incubated with GDP or GTPγS loaded GST and GST-Rac1 WT beads for 2 h at 4 °C. Beads were washed following incubation and eluted using 20 μl 2 × SDS–PAGE sample buffer for western blot analysis.

For each pulldown assay, 150 μg GST fusion proteins were immobilized by incubating with 40ul of 50% (vol/vol) Glutathione-Sepharose 4B beads (GE Healthcare) at 4 °C for 1 h. Beads were washed three times and incubated with 0.1 μM prey protein in binding buffer (25 mM Hepes pH 7.4, 150 mM NaOAc, 0.2% Trition and 1 mM DTT) at 4 °C for 2 h.
For GST-Rab GTPase pulldown assays, the GST-Rab proteins immobilized on Glutathione-Sepharose 4B (GE Healthcare) were incubated in 25 mM Hepes pH 7.4, 1 mM EDTA for 30 min at 25 °C. Beads were washed for three times and incubated with Guanosine 5′-diphosphate sodium salt (GDP; 100 μM; Sigma-Aldrich)or Guanosine 5′-O-(3-thiotriphosphate) tetralithium salt (GTPγS; 100 μM; Sigma-Aldrich) in 25 mM Hepes pH 7.4, 150 mM NaOAc, 25 mM Mg(OAc)2 for 60 min at 25 °C. Beads were washed three times and incubated with 0.1 μM prey protein in binding buffer (25 mM Hepes pH 7.4, 150 mM NaOAc, 25 mM Mg(OAc)2, 0.2% Trition and 1 mM DTT) at 4 °C for 2 h.
After pulldown incubation, beads were washed for 4 times and 40 μl 2x SDS loading buffer were added. After boiling the samples were aspirated from the beads and volumes of samples were finalized to 80 μl. 2% Input and 20% samples were loaded for Western Blot assays.