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 …
Divulgations
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