Competition Binding Assay to Study Competing GTPase-Binding Protein Partners
Published: June 29, 2023
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 …
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).