α 32p atp

Manufactured by MP Biomedicals

α-32P-ATP is a radioactive nucleotide that contains the phosphorus-32 isotope. It is commonly used as a tracer or label in various biochemical and molecular biology applications to study DNA, RNA, and protein synthesis, as well as signal transduction pathways.

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Lab products found in correlation

Depc treated, by Thermo Fisher Scientific (2 mentions) Nuclease free sterilized water, by Thermo Fisher Scientific (2 mentions) T7 polymerase, by MP Biomedicals (2 mentions) Phosphor imaging system, by GE Healthcare (2 mentions) E coli rna polymerase core enzyme, by Illumina (2 mentions) Agarose, by Merck Group (1 mentions) Restriction endonuclease ecori, by New England Biolabs (1 mentions) Cisplatin, by Merck Group (1 mentions) Rnase a, by Qiagen (1 mentions) Nitrocellulose membrane, by GE Healthcare (1 mentions) Storm 860 phosphorimager, by GE Healthcare (1 mentions)

6 protocols using α 32p atp

Transcription of Unnatural DNA Bases

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Example 5

To characterize the transcription of the unnatural base pairs formed by dTPT3 and dNaM, or analogs or derivatives thereof (wherein derivatives include linker moieties), ribonucleotides and deoxynucleotides are synthesized and converted to the corresponding triphosphates or deoxyphosphoramidites, and the deoxyphosphoramidites are incorporated into DNA templates using automated DNA synthesis. Transcription experiments are conducted with 100 nM DNA substrate, 1× Takara buffer (40 mM Tris-HCl, pH 8.0, 8 mM MgCl₂, 2 mM spermidine), DEPC-treated and nuclease-free sterilized water (Fisher), T7 polymerase (50 units), 20 μM each natural NTP, α-³²P-ATP (2.5 μCi, MP Biomedicals), and either 5 μM TPT3TP or 5 μM NamTP. After incubation for 2 hr at 37° C., the reaction is quenched by the addition of 10 μL of gel loading solution (10 M urea, 0.05% bromophenol blue), and the reaction mixture is loaded onto a 20% polyacrylamide-7 M urea gel, subjected to electrophoresis, and analyzed by phosphorimaging. Transcription efficiency is examined by measuring (at low percent conversion) the amount of full-length product formed as a function of time.

US10626138B2. Method for the site-specific enzymatic labelling of nucleic acids in vitro by incorporation of unnatural nucleotides (2020-04-21). The Scripps Research Institute [US]. Inventors: Floyd E. Romesberg [US], Denis A. Malyshev [US], Lingjun Li [US], Thomas Lavergne [FR], Zhengtao Li [CN].

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In vitro transcription of PG1660 promoter

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In vitro transcription assay was performed using the E. coli RNA polymerase core enzyme (Epicentre, Madison, WI), and the purified rPG1660 protein. The DNA fragment carrying the PG1660 promoter was used as template. The primers used are listed in Table 2. The reactions were incubated at 37°C for 2 h in buffer containing 40 mM Tris–HCl (pH 7.5), 150 mM KCl, 10 mM MgCl₂, 0.1 mM dithiothreitol, 0.01% Triton X-100, 2.5 mM of NTP mix, ~1 mCi [α-³²P]-ATP (MP Biomedicals, Solon, OH). The samples were analyzed on an 8% polyacrylamide denaturing gel and quantified using a phosphor-imaging system (Molecular Dynamics, Sunnyvale, CA).

Dou Y., Rutanhira H., Chen X., Mishra A., Wang C, & Fletcher H.M. (2017). Role of extracytoplasmic function sigma factor PG1660 (RpoE) in the oxidative stress resistance regulatory network of Porphyromonas gingivalis. Molecular oral microbiology, 33(1), 89-104.

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Transcription of Unnatural Base Pairs

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Example 5

To characterize the transcription of the unnatural base pairs formed by dTPT3 and dNaM, or analogs or derivatives thereof (wherein derivatives include linker moieties), ribonucleotides and deoxynucleotides are synthesized and converted to the corresponding triphosphates or deoxyphosphoramidites, and the deoxyphosphoramidites are incorporated into DNA templates using automated DNA synthesis. Transcription experiments are conducted with 100 nM DNA substrate, lx Takara buffer (40 mM Tris-HCl, pH 8.0, 8 mM MgCl2, 2 mM spermidine), DEPC-treated and nuclease-free sterilized water (Fisher), T7 polymerase (50 units), 20 μM each natural NTP, α-³²P-ATP (2.5 μCi, MP Biomedicals), and either 5 M TPT3TP or 5 μM NamTP. After incubation for 2 hr at 37° C., the reaction is quenched by the addition of 10 μL of gel loading solution (10 M urea, 0.05% bromophenol blue), and the reaction mixture is loaded onto a 20% polyacrylamide-7 M urea gel, subjected to electrophoresis, and analyzed by phosphorimaging. Transcription efficiency is examined by measuring (at low percent conversion) the amount of full-length product formed as a function of time.

US11634451B2. Method for the site-specific enzymatic labelling of nucleic acids in vitro by incorporation of unnatural nucleotides (2023-04-25). The Scripps Research Institute [US]. Inventors: Floyd E. Romesberg [US], Denis A. Malyshev [US], Lingjun Li [US], Thomas Lavergne [FR], Zhengtao Li [CN].

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Preparation and Characterization of DNA Complexes

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Commercial reagent grade chemicals, solvents and reagents for the preparation of solutions for biological work were used as received from commercial suppliers without further purification. Calf thymus (CT) DNA (42% G + C, mean molecular mass ca. 2 × 10 7 ) was prepared and characterized as previously described [14, 15] . Plasmid pPUC19 [2686 base pairs (bp)] was isolated according to standard procedures. Restriction endonuclease EcoRI was purchased from New England Biolabs. Agarose was obtained from Merck KgaA (Darmstadt, Germany). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was from Calbiochem (Darmstadt, Germany). [α-32 P]ATP was obtained from MP Biomedicals, LLC (Irvine, CA). RNase A was from Qiagen (USA). DNazol (genomic DNA isolation reagent) was obtained from MRC (Cincinnati, OH). Cisplatin was from Sigma-Aldrich (Prague, Czech Republic). Complexes 1 and 2 were synthesized and characterized as described in the previously published article [11] .

Kasparkova J., Kostrhunova H., Novohradsky V., Pracharova J., Curci A., Margiotta N., Natile G, & Brabec V. (2017). Anticancer kiteplatin pyrophosphate derivatives show unexpected target selectivity for DNA. Dalton transactions (Cambridge, England : 2003), 46(41).

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In vitro Transcription of PG0162

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In vitro transcription assays were performed using the E. coli RNA Polymerase Core enzyme (Epicentre, Madison, WI) and the purified rPG0162 protein. The DNA fragment containing the promoter region of the PG0162 gene was used as a template. The reaction mix which contained 40 mM Tris-HCl (pH 7.5), 150 mM KCl, 10 mM MgCl₂, 0.1 mM DTT, 0.01% Triton X-100, 2.5 mM each of NTP mix, ∼1 mCi [α-32P]-ATP (MP Biomedicals, Solon, OH) was incubated at 37 °C for 2 hr. Samples were analyzed on a denaturing urea polyacrylamide gel and quantified using the phosphor imaging system (Molecular Dynamics, Sunnyvale, CA).

Dou Y., Aruni W., Muthiah A., Roy F., Wang C, & Fletcher H.M. (2015). Studies of the extracytoplasmic function sigma factor PG0162 in Porphyromonas gingivalis. Molecular oral microbiology, 31(3), 270-283.

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Dry Nitrocellulose-Based Ligand-Protein Assay

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DRaCALA is a method based on the ability of dry nitrocellulose to separate the free ligand from bound protein–ligand complexes, which allows detection of specific interactions between nucleotides and their cognate binding proteins (Roelofs et al., 2011 (link), Corrigan et al., 2013 (link)). Briefly, [α-³²P] ATP (MP Biomedicals) was converted to ³²P-labeled c-di-AMP by using recombinant M. tuberculosis DisA protein and purified using a thin layer chromatography (TLC) (Bai et al., 2014 (link)). The reaction (10 µl) for DRaCALA assay was prepared by mixing 1 µL ³²P-labeled c-di-AMP and appropriate concentrations of recombinant CabPA or CabPB in binding buffer (40 mM Tris, 100 mM NaCl, 20 mM MgCl₂ [pH 7.5]). Reaction was incubated at room temperature for 10 min, followed by spotting 5 µL onto a nitrocellulose membrane (GE healthcare). After air dry for 10 min, the membrane was exposed on a phosphor screen for 3 h. The radioactivity was detected using a Storm 860 PhosphorImager (Molecular Dynamics). Dissociation constant was analyzed as previous reported (Roelofs et al., 2011 (link)).

Peng X., Zhang Y., Bai G., Zhou X, & Wu H. (2015). Cyclic di-AMP mediates biofilm formation. Molecular microbiology, 99(5), 945-959.

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