Competition Binding Assay to Study Competing GTPase-Binding Protein Partners

Abstract

Source: Williamson, R. C., et al.  Comparing the Affinity of GTPase-binding Proteins using Competition Assays. J. Vis. Exp. (2015)

This video demonstrates a competition assay to study GTPase-binding protein partners. Utilizing nucleotide-bound GTPase protein immobilized on magnetic beads, the competitive binding between two interacting protein partners for the same binding site on the GTPase can be studied to assess the binding affinities of the protein partners.

Protocol

1. Purification of GST-tagged GTPase 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). Harvest bacteria …

Materials

Bugbuster	Novagen	70584-3
COMPLETE protease inhibitor	Roche	05 056 489 001
Glutathione magnetic beads	Pierce	88821
Polyethylenimine, branched, average Mw ~ 25,000	Sigma Aldrich	408727-100ML
OPIMEM	Life Technologies	31985-047
Dulbecco's Modified Eagle Media	Sigma Aldrich	D5796
Fetal Bovine Serum	Life Technologies	10270-1-6
L-Glutamine	Life Technologies	25030-024
GFP-Trap_A	Chromotec	gta-20
GDP	Sigma Aldrich	G7127	Highly unstable. Aliquot and store at -80 immediately upon reconstitution
GTPγS	Sigma Aldrich	G8634	Highly unstable. Aliquot and store at -80 immediately upon reconstitution
Blocking Buffer	Sigma Aldrich	B6429
Tween-20	Sigma Aldrich	P9416
Anti-GFP antibody	Living Colors	632592	Use at 1/1000 dilution
DyLight 800 conjugated goat anti-rabbit secondary antibody	Fisher Scientific	10733944
Odyssey SA Infrared Imaging System	Li-cor	9260-11PC