Protocol for Photoaffinity Labeling of Membrane Associated G-Proteins with Guanosine 5'-[g32P]triphosphate [g] 4-Azidoanilide ([g32P]GTP-AA): A Stepwise Approach
by Ashok Chavan Ph.D. 1. Membranes are prepared and added to 2-ml screw-top microcentrifuge tubes. Total assay volumes should be kept to 30-50 ml, but can go as high as 100 ml. The total amount of membranes will be dependent upon the identification step. If SDS-PAGE and autoradiography are planned, 30 mg of membrane protein/gel lane is probably sufficient. If a western blot is planned, 90 mg are better and if immunoprecipitation of the sample will be done, use 150 mg. For photolabeling 10 mM HEPES pH 7.4, 1 mM MgCl2, 0.1 mM b-mercaptoethanol is a good universal buffer to use. 2. If agonist-dependent labeling is planned, membranes should be incubated with GDP (amount determined experimentally but 1-100 mM is the likely range) for 5-10 minutes at 23°C. Following this, Guanosine 5'-[g32P]triphosphate [g] 4-Azidoanilide ([g32P]GTPgAA, Order No. GP01) is added (remove methanol under a gentle stream of air or nitrogen in a fume hood or in a speed vac). If agonist is not present, 0.1 mM is a good concentration to use. When doing agonist dependent labeling, GTP-Azidoanilide concentrations of 1 nM will give diminished overall labeling, but are likely to show increased agonist specificity. 3. After 3 minutes incubation with [g32P]GTP-AA, agonist is added and incubation continued for 10 minutes (this time will also need to be determined experimentally). 4. Following incubation, tubes are placed in a metal block on ice and subjected to UV irradiation (254 nm; 9W manufacturer’s rating, 1-6 mW/cm2 determined by light meter) with a hand-held lamp held at 4 cm for 3 minutes. 5. Photoincorporation is stopped by adding dithiothreitol to 10 mM. Membranes can then be washed by the addition of 1 ml labeling buffer and centrifugation in a microcentrifuge. Pellets are resuspended in sample buffer and subjected to SDS PAGE. Alternately, concentrated (3X) sample buffer is added to the solution and the sample is applied to SDS PAGE. Gels can be dried and either exposed to film or phosphorimaging plates, or proteins from wet gels can be transferred to nitrocellulose and processed for immunoblot analysis. 6. Immunoprecipitation: Extract proteins from membrane in RIPA buffer for 30-60 min at 4oC with continuous shaking and vortexing every 10 min (use 2x volume of membrane pellet). Centrifuge at 14,000 x g for 10 min. Save supernatant and repeat extraction on pellet. Combine supernatants for immunoprecipitation (volume 50-60 ml).
7. SDS-PAGE: Load and run the samples on 9-11% SDS-PAGE gel. Stain gel for 30 min-2 hrs shaking at room temp. Destain gel for 2-10 hrs shaking at room temp. Dry the gel on gel dryer and expose to X-ray film 12 hrs-2 days or to phosphorimager plates. RIPA Buffer: 50 mM Na2HPO4, pH 7.2-7.4; 1% deoxycholate; 1% Triton X-100; 0.5% SDS; 150 mM NaCl; 2 mM EDTA, pH 7.2-7.4; 2 mg/ml aprotinin, 2 mg/ml leupeptin, 1mM PMSF (added at the time of use). Wash Buffer: 50 mM Na2HPO4, pH 7.2-7.4; 0.5% Triton X-100; 150 mM NaCl; 2 mM EDTA, pH 7.2-7.4
Additional References on Immunoprecipitation of Radiolabeled Proteins From
Upstate
http://www.protocol-online.org/prot/Molecular_Biology/Protein/Immunoprecipitation/
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(Bjorkman
Group, Howard Hughes Medical Institute at California Institute of Technology)