A single-stranded (ss) DNA 20-mer (ssDNA20) was purchased from TriLink Biotechnologies (San Diego, CA) with 6-carboxyfluorescein (FAM) at the 5′ end: 5′-FAM-CTT CTT TGG GAG TGA ATT AG-3′. Two RNAs were designed based on predicted stem-loop elements present in the 5′ region of FIV gRNA (Kenyon et al., 2008 (link); Rizvi et al., 2010 (link)) with the following sequences: micro R: 5′-UUU GAG GAC GUU UGC GUU CUC-3′; micro SL2: 5′-UUU GCU GAC CUA AAU AGG GAA GC-3′. The predicted secondary structures of these two RNAs are shown in Fig. 1D . These RNAs were purchased with 5′-FAM labels from Dharmacon (now Thermo Scientific, Waltham, MA). HIV-1 TAR RNA was prepared as described (Vo et al., 2009b (link)) and TAR DNA was purchased from Integrated DNA Technologies (Coralville, IA). TAR RNA was 5′-radiolabeled using γ-[32P]-GTP (Perkin Elmer) and T4 polynucleotide kinase (New England Biolabs) using standard protocols, followed by purification by denaturing PAGE. Concentrations of all NAs were determined by measuring UV absorption at 260 nm and using the following extinction coefficients: ssDNA20: 192,900 M−1cm−1, 377,800 M−1cm−1, micro R: 222,400 M−1cm−1, micro SL2: 253,700 M−1cm−1, TAR RNA: 533,700 M−1cm−1, TAR DNA: 564,800 M−1cm−1.
γ 32p gtp
Manufactured by PerkinElmer
Sourced in United Kingdom, United States
[γ-32P]GTP is a radioactive nucleotide analog used in various biochemical and molecular biology applications. It is a guanosine triphosphate (GTP) molecule with a radioactive phosphorus-32 (32P) atom incorporated into the gamma phosphate group. This product can be utilized in techniques such as in vitro transcription, RNA labeling, and GTPase activity assays.
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Lab products found in correlation
γ 32p atp, by PerkinElmer (3 mentions)
T4 polynucleotide kinase, by New England Biolabs (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Pfu ultra dna polymerase, by Agilent Technologies (2 mentions)
35s gtpγs, by PerkinElmer (2 mentions)
Phosphorimager, by GE Healthcare (1 mentions)
Fastap alkaline phosphatase, by Thermo Fisher Scientific (1 mentions)
Pluronic f 68, by Thermo Fisher Scientific (1 mentions)
Mutagenesis primers, by Integrated DNA Technologies (1 mentions)
Anti flag antibody f7425, by Merck Group (1 mentions)
Pcmv5 mouse src, by Addgene (1 mentions)
Gsh agarose, by Merck Group (1 mentions)
Protein a g sepharose, by Thermo Fisher Scientific (1 mentions)
Ipl 41, by Thermo Fisher Scientific (1 mentions)
Anti src antibody, by Santa Cruz Biotechnology (1 mentions)
Anti myc antibody, by Merck Group (1 mentions)
Ni nta, by Qiagen (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Glutathione (gsh), by Merck Group (1 mentions)
17 protocols using γ 32p gtp
1
Fluorescent Nucleic Acid Probes for Structural Studies
2
GTPase Activity Assay of OLA1 Protein
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OLA1 protein (1 μM) was incubated with [γ-32P]GTP (6000 Ci/mmol, PerkinElmer) or [γ-32P]ATP (3000 Ci/mmol) in the presence of 100 μM of the corresponding unlabeled nucleotides in a 50 μl reaction buffer (25 mM HEPES at pH 7.5, 80 mM K2OAc, 2.5 mM MgOAc, and 1 mM DTT) at 35 °C. At various time points, the reaction was stopped by the addition of 200 μl of activated charcoal suspension (100 mg/ml charcoal (Sigma Aldrich) in 1 N HCL). After two centrifugations at 100000 × g, release of [32P] phosphate in the supernatant was measured by scintillation counting.
3
Kinetics of SRP-Mediated GTPase Activation
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One micromolar of M. jannaschii SRP RNA (nucleotides G123–C258, which include the helix 5 region required for GTPase activation11 (link)17 (link)), 1.5 μM of SRP19 and 0.5 μM of SRP54 were assembled in binding buffer containing 10 mM Tris-Cl (pH 7.5), 150 mM KCl, 5 mM MgCl2, and 1% (v/v) ß-mercaptoethanol to form 0.5 μM of M. jannaschii SRP. M. jannaschii SR(aa93-408) was added to the SRP complex to final concentrations of 0, 1, 2, 5, 10 and 25 μM. GTP hydrolysis reactions, carried out at 25 °C, were started by adding 100 μM GTP with trace amounts of γ-32P-GTP (PerkinElmer). At different time points, 0.5 μl aliquots of the reactions were spotted on a polyethyleneimine–cellulose thin-layer chromatography (PEI-TLC) plate (Merck). Inorganic phosphate (Pi) and GTP were separated by chromatography in 0.75 M KH2PO4 and quantified with a Phosphorimager (Molecular Dynamics). Rates of GTP hydrolysis were averaged from three independent experiments and fitted to a single exponential model.
4
Src Kinase Activation Assay Protocol
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Ni-NTA was from Qiagen (Valencia, CA). IPL-41 and pluronic F-68 for growing Sf9 cells were from Invitrogen (Carlsbad, CA). Mutagenesis primers were from Integrated DNA Technologies (Coralville, IA). Cloning and mutagenesis reagents, Protein A/G-Sepharose, and FastAP alkaline phosphatase were from Thermo Scientific. Anti-FLAG affinity resin (M8823), 3X FLAG peptide (F4799), and anti-FLAG antibody (F7425) were from Sigma. Anti-Src antibody was from Santa Cruz (sc-18). Anti-phosphotyrosine 4G10 Platinum antibody was from Millipore. Phosphatidylinositol 4,5-bisphosphate (PIP2) and brominated phosphatidylcholine (PC), were from Avanti Polar Lipids. [γ-32P]GTP and [γ-32P]ATP were from PerkinElmer Life Sciences. Reagents for electrophoresis and immunoblotting were from Bio-Rad. Fluorescently labeled secondary antibodies for Infrared Imaging System were from LICOR. Other reagents, including anti-myc antibodies, buffers, charcoal-activated Norit A, GSH-Agarose, glutathione, ATP, GTP, protease inhibitors and PMSF were from Sigma (St. Louis MO). pCMV5 mouse Src was from Addgene (plasmid #13663, provided by Joan Brugge and Peter Howley).
5
Preparation of Labeled RNA Substrates
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RNA oligonucleotides 3′-labeled with FAM (6-carboxyfluorescein) were purchased from Bio-Synthesis Inc or Metabion (see sequences of RNA substrates in Supplementary Table S2 ). Radioactively 32P-labeled substrates were obtained by in vitro transcription with T7 RNA polymerase using 300 ng of PCR-generated DNA templates. Templates for guanosine-rich and guanosine-deficient RNAs were prepared from pGEM-T Easy Vector and fragment of the Saccharomyces cerevisiae oriIV-1 DNA, respectively. NAD+-capped substrates were prepared as described (4 (link)) with DNA template that contained T7 φ2.5 promoter and was deprived of adenosines except at the transcription start site (see the list of oligonucleotides in Supplementary Table S3 ). Uniformly labeled RNAs were transcribed in the presence of [α-32P]GTP (Hartmann Analytics) and GMP, AMP or NAD (Sigma) to generate 5′ pG-RNA, pA-RNA or NppA-RNA, respectively. 5′-end labeled pppG-RNA was transcribed in the presence of [γ-32P]GTP and NppA-RNA labeled at adenylate phosphate was transcribed in the presence of [32P]NAD (Perkin Elmer). 5′-end-cap 32P-labeled RNA was generated using 20 pmol of non-radioactive uncapped RNA and ScriptCap™ m7G Capping System (Epicentre) and [α-32P]GTP with or without SAM according to manufacturer's protocol.
6
Radioactive Nucleotide Ligands for CFTR Study
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8-N3-[33P]AMP was from Hartmann Analytic GmbH (Braunschweig, Germany). 2-N3-AMP was from Affinity Photoprobes, LLC (Lexington, KY). The azido-nucleotides were dissolved as triethylammonium salt in absolute methanol. Immediately before use, the methanol was evaporated under a stream of argon, and azido-nucleotides were dissolved in a buffer of 20 mm Hepes (pH 7.5), 50 mm NaCl, 3 mm MgCl2. [γ-32P]GTP, dissolved in 10 mm Tricine, pH 7.6, was from PerkinElmer Inc. Non-radioactive ATP, ADP, AMP, and Ap5A were from Sigma-Aldrich. ATP was used as magnesium salt. ADP, AMP, and Ap5A were sodium salts. Protein kinase A (PKA) catalytic subunit, purified from bovine heart, was from EMD Millipore Corp. (Billerica, MA). The monoclonal CFTR antibodies used were from R&D Systems, Inc. (Minneapolis, MN) (13-1 (31 (link))) and EMD Millipore (Billerica, MA) (M3A7 (32 (link)) and 13-4 (33 (link))). The 769 antibody (34 (link)) was from the University of North Carolina (Chapel Hill, NC) in conjunction with the Cystic Fibrosis Foundation. Anti-ZO-1 was from Zymed Laboratories Inc. (San Francisco, CA).
7
GTP-Loading and GAP Assay for Rab GTPases
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For GTP-loading reactions, 10 µl assay buffer (20 mM HEPES, pH 6.8, 1 mg/ml BSA), 73 µl H2O, 10 µl of 10 mM EDTA, pH 8.0, 5 µl of 1 mM GTP, 2 µl γ-[32P]-GTP (6000 Ci/mmol 10 mCi/ml Perkin Elmer, UK), and 100 pmol Rab-GTPase were mixed on ice. After 30 min of incubation at 30°C, loaded GTPases were stored on ice. GAP reactions were started by the addition of GAP to 1 µM Rab in 50 µl final volume as specified in the figures. A 2.5 μl aliquot of the assay mix was scintillation counted to measure the specific activity in counts per minute per picomole of GTP. Reactions were then incubated at 30°C for 0 to 60 min and then split into two equal aliquots. 5 µl of each aliquot was immediately added to 795 µl of ice-cold 5% (wt/vol) activated charcoal slurry in 50 mM NaH2PO4, left for 1 hr on ice, and centrifuged at 20,000×g to pellet the charcoal. A 400 µl aliquot of the supernatant was scintillation counted, and the amount of GTP hydrolyzed was calculated from the specific activity of the reaction mixture.
8
Purification and Characterization of Arc
9
In vitro Transcription of Labeled tRNA
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T7 RNA polymerase was used for runoff in vitro transcription using digested plasmids encoding tRNAHis variant constructs downstream from the T7 RNA polymerase promoter, as previously described (Rao et al. 2011 (link)). The tRNAHis variant constructs were transcribed in the presence of [γ-32P] GTP (6000 Ci/mmol, Perkin-Elmer) to obtained [γ-32P]pp-labeled tRNAHis (5′-*ppp-tRNA), according to published methods (Jackman and Phizicky 2006b (link)).
10
Molecular Reagents and Antibodies for Cellular Studies
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