Recombinant RGS10 proteins were incubated with recombinant purified Gαi3 (200 ng, EMD Biosciences) in HKB buffer (76 (link)) containing 10 μM GDP, or GDP plus 30 μM AlCl3, and 10 mM NaF, together with Ni2+ beads (20 μl of a 50% slurry) for 1 hour at 4°C. Beads were pelleted by centrifugation and washed four times with HKB. Bound proteins were eluted with SDS sample buffer and boiling, followed by resolution on SDS NuPAGE gels and transfer to nitrocellulose. Blots were probed with Gαi3 and RGS10. GAP assays were carried out essentially as described (76 (link)). His6-Gαo1, His6-Gαi1, His6-Gαi2 or His6-Gαi3 (1μM) was mixed with RGS10L, its mutants or equivalent amounts of control protein (mannose binding protein, MBP) (1 or 10 μM) in HKB buffer plus 5 mM MgCl2. BODIPY-FL-GTP (ThermoFisher) was added to the mixture after 10 min preincubation to a final concentration of 1 μM. The kinetics of in vitro G-protein binding to GTP were measured using the VICTOR3 multiwell reader (PerkinElmer).
Bodipy fl gtp
Manufactured by Thermo Fisher Scientific
Sourced in Austria, United States
BODIPY-FL-GTP is a fluorescent guanine nucleotide analog that can be used for various applications in cell biology and biochemistry research. It serves as a probe for studying guanine nucleotide-binding proteins and their interactions.
Automatically generated - may contain errors
Lab products found in correlation
Bodipy fl gtpγs, by Thermo Fisher Scientific (2 mentions)
Victor3 multiwell reader, by PerkinElmer (1 mentions)
Fl winlab software package, by PerkinElmer (1 mentions)
Bodipy fl gdp, by Thermo Fisher Scientific (1 mentions)
Fluoromax 4 spectrophotometer, by Horiba (1 mentions)
Black 384 well plate, by Greiner (1 mentions)
Kanamycin, by GoldBio (1 mentions)
Protease inhibitor cocktail, by Abcam (1 mentions)
Imidazole, by Merck Group (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by GoldBio (1 mentions)
Deuterium oxide, by Cambridge Isotopes (1 mentions)
Lysozyme, by GoldBio (1 mentions)
Hepes, by GoldBio (1 mentions)
Leupeptin, by GoldBio (1 mentions)
Mgcl2, by Merck Group (1 mentions)
Acetonitrile, by Avantor (1 mentions)
Formic acid, by Merck Group (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Phosphoric acid, by Merck Group (1 mentions)
Benzamidine, by Merck Group (1 mentions)
5 protocols using bodipy fl gtp
1
Gαi3 GTPase Regulation by RGS10
2
BODIPY-GTP Hydrolysis Assay
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BODIPYFL GTP (ThermoFisher Scientific, Invitrogen; G12411) (50 nM) was equilibrated in hydrolysis buffer (20 mM Tris pH 8, 10 mM MgCl2) in a 1 mL cuvette. Purified Gα–GDP protein (3 μM) was added to initiate BODIPY-GTP binding and subsequent hydrolysis. A PerkinElmer LS55 luminescence spectrometer (498 nm excitation, 508 nm emission) was used to measure fluorescence using the FLWinLab software package (PerkinElmer).
3
Nucleotide Affinity Assay with BODIPY-FL
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To assay nucleotide affinity, fluorescence intensity of 100 nM of BODIPY-FL-GDP or BODIPY-FL-GTP (Thermo Fisher Scientific) in 180 μl of assay buffer (30 mM HEPES, 100 mM KCl, 10 mM MgCl2, pH 7.4) was measured using a Fluoromax-4 spectrophotometer (Horiba) (490 nm excitation, 509 nm emission). Yeast Met–tRNAi was assayed in a similar manner using 20 nM BOP-N-Met–tRNAi (tRNA probes) but with the addition of 1 mM GTP. Change in fluorescence intensity was measured upon addition of increasing amounts of apo–eIF2, incubating for 5 min at room temperature each time. Each measurement was blanked against a control without nucleotide to account for any affect of eIF2 and data were corrected for dilution effects caused by volume addition and normalized to starting values before being fitted to a single site binding model: y = 1 + [(ΔFmax - 1)*(x/(x + Kd))].
4
Kinetic Analysis of G Protein Binding
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Recombinant human Gαo and Gαi1, wild-type or [Q52P] and [Q52R] mutants were brought into the 20 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl by a 10,000-fold buffer exchange on Amicon 10K ultracentrifugation concentrators (Merck, Kenilworth, NJ USA). For measurement, 2 µM of indicated protein was incubated for 30 min in the black 384-well plate (Greiner, Kremsmünster, Austria) and then mixed with an identical volume of 2 µM BODIPY-FL-GTP or 2 µM BODIPY-FL-GTPγS (both from Thermo Fisher Scientific) in the buffer containing 20 mM Tris-HCl buffer (pH 7.5), 150 mM NaCl, 0.2% BSA and 10mM MgCl2 using the plate reader injector directly before measurement. The kinetics of in vitro G protein binding and/or hydrolysis was measured in the Infinite M200 Pro multiwell reader (Tecan, Männedorf, Switzerland) [28 (link),29 (link)].
5
Purification of Recombinant Proteins
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The protease inhibitor cocktail was purchased from BioVision (Mountain View, CA, USA). Deuterium oxide was purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Chelating sepharose resin for Ni-IDA purification was purchased from Cytiva (Uppsala, Sweden). EDTA, Tris, and NaH2PO4 were purchased from Duchefa Biochemie BV (Haarlem, The Netherlands). HEPES, IPTG, leupeptin, lysozyme, kanamycin, and TCEP were purchased from Goldbio (St. Louis, MO, USA). Acetonitrile, H2O, and MeOH were purchased from JT Baker (Phillipsburg, NJ, USA). DNase I was purchased from Roche (Basel, Switzerland). BODIPY-FL-GTP and BODIPY-FL-GTPγS were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Acetic acid, benzamidine, formic acid, GDP, phosphoric acid, imidazole, and MgCl2 were purchased from Sigma–Aldrich (St. Louis, MO, USA).
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