Competition Binding Assay to Study Competing GTPase-Binding Protein Partners

Published: June 29, 2023

Abstract

Source: Williamson, R. C., et alComparing 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 …

Divulgaciones

The authors have nothing to disclose.

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

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Competition Binding Assay to Study Competing GTPase-Binding Protein Partners. J. Vis. Exp. (Pending Publication), e21454, doi: (2023).

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