The complementary DNA (cDNA) fragments encoding AtGPA1, AtGPA1(S52C), and AtGPA1(Q222L) were subcloned into the pDEST17 vector. The proteins were expressed in ArcticExpress cells upon induction with 80 or 250 μM isopropyl-β-d-thiogalactopyranoside (IPTG) at 14°C. The cells were harvested by centrifugation and lysed in extraction buffer [50 mM tris-HCl (pH 8.0), 100 mM NaCl, 2 mM MgCl2, 5 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF), leupeptin (2 μg/ml), 10 μM GDP, lysozyme (250 μg/ml), and 0.2% C12E10]. The bacterial lysate was centrifuged at 100,000g for 1 hour and then incubated with nickel-NTA agarose. The proteins were eluted from the resin with elution buffer [20 mM tris-HCl (pH 8.0), 250 mM NaCl, 5 mM 2-mercaptoethanol, 1 mM PMSF, and 10% glycerol] with 100 or 250 mM imidazole. The eluted proteins were dialyzed in buffer [25 mM tris-HCl (pH 7.6), 50 mM NaCl, 2 mM MgCl2, 1 mM EDTA, 5 mM 2-mercaptoethanol, 1 mM PMSF, and 10 μM GDP] and frozen at −80°C. The amount of GTPγS bound to the recombinant proteins was assessed using radiolabeled [35S]GTPγS (PerkinElmer Inc.). Recombinant AtGPA1 proteins at 0.5 μM were mixed with a series of concentrations of [35S]GTPγS (1.25, 2.5, 5, 10, 20, 40, and 80 μM) in 60 μl of reaction buffer [50 mM tris-HCl (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol (DTT), and 5 mM MgCl2]. The lowest concentration of [35S]GTPγS used (1.25 μM) was still 2.5-fold greater than the concentration of GPA1 proteins (500 nM = 0.5 μM). After incubation at room temperature for 2.5 hours, 50 μl of the reaction mixture was transferred into 450 μl of ice-cold wash buffer [20 mM tris-HCl (pH 7.4), 100 mM NaCl, and 25 mM MgCl2] with 0.1 mM GTP and then applied to a nitrocellulose membrane filter. The filter was washed three times with ice-cold wash buffer. The amount of [35S]GTPγS on the membrane was measured by scintillation counting. For BODIPY-FL-GDP– and BODIPY-FL-GTP–binding assays, the proteins were purified as described earlier, with the modifications that protein expression was induced with 500 μM IPTG, that GDP was excluded from the extraction buffer, that the lysates were centrifuged at 30,000g for 15 min, and that buffer exchange was performed into 25 mM tris-HCl (pH 7.7), 50 mM NaCl, and 5% glycerol using a centrifugal filter with a 10,000 cutoff. Assays were run at 25°C with a Berthold TriStar2 LB 942 plate reader with 485-nm excitation and 535-nm emission filters, a 15-s cycle time, a 0.1-s counting time per measurement, and 60% lamp intensity. Proteins were diluted to 340 nM in 100 μl of dialysis buffer [25 mM tris-HCl (pH 7.7), 50 mM NaCl, and 5% glycerol] supplemented with 10 mM MgCl2. Binding was initiated with the addition of 100 μl of 100 nM BODIPY-FL-GDP or BODIPY-FL-GTP in 20 mM tris-HCl (pH 8.0) at cycle 2 of 61. Raw data were normalized by fluorescence values obtained for bovine serum albumin, which was used as a negative control for nucleotide binding.

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