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Promega α 32 p atp
α 32 P Atp, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 86 stars, based on 4 article reviews
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α 32 p atp - by Bioz Stars, 2020-04
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Related Articles

Amplification:

Article Title: Genetic organization of the psbAD region in phages infecting marine Synechococcus strains
Article Snippet: A hybridization probe was prepared from a 525-bp PCR product from S-PM2, which was amplified by using the following primers: S-PM2F1S, 5′-GCTGCTTCTCTTGATGAGTG-3′; and S-PM2R2S, 5′-AGTGTAGCGAACGAGAGTTG-3′. .. The PCR product was gel-extracted and labeled with [α-32 P]ATP by using DNA polymerase I Klenow fragment in 1× labeling buffer (Promega) at 25°C for 2 h. Unincorporated nucleotides were removed by purification through a Sephadex G-25 column ( ).

Article Title: Direct Response Elements of BMP within the PV.1A Promoter Are Essential for Its Transcriptional Regulation during Early Xenopus Development
Article Snippet: .. Promoter fragments or 3x-repeat oligonucleotides were amplified by annealing two complementary oligos, respectively, and labeled with α-32 P-ATP using T4 polynucleotide kinase (Promega, Madison, WI). .. Labeled DNA probes were incubated with 10 µg animal cap protein extract or 1 µg in vitro translated protein at room temperature for 20 minutes in 10 µL binding buffer (2.5% glycerol, 5 mM MgCl2 , 50 ng/µL Poly [dI·dC], and 0.05% NP-40).

Filtration:

Article Title: Regions of RNase E Important for 5?-End-Dependent RNA Cleavage and Autoregulated Synthesis
Article Snippet: The reaction mixture (20 μl) contained Tris Cl (40 mM, pH 7.9), MgCl2 (6 mM), NaCl (10 mM), dithiothreitol (7.5 mM), spermidine (2 mM), GTP (0.25 mM), CTP (0.25 mM), UTP (0.25 mM), ATP (0.025 mM), [α-32 P]ATP (30 μCi), GMP (0 or 15 mM), RNasin (20 U; Promega), plasmid pT7-R25 linearized by Hin dIII cleavage (0.2 μg), and T7 RNA polymerase (40 U). .. After incubation for 16 h at 37°C, the reaction mixture was diluted with water (30 μl) and the RNA was isolated by gel filtration on Sephadex G-50 and stored at −20°C.

Synthesized:

Article Title: Regions of RNase E Important for 5?-End-Dependent RNA Cleavage and Autoregulated Synthesis
Article Snippet: Internally radiolabeled RNA was synthesized by in vitro transcription with T7 RNA polymerase. .. The reaction mixture (20 μl) contained Tris Cl (40 mM, pH 7.9), MgCl2 (6 mM), NaCl (10 mM), dithiothreitol (7.5 mM), spermidine (2 mM), GTP (0.25 mM), CTP (0.25 mM), UTP (0.25 mM), ATP (0.025 mM), [α-32 P]ATP (30 μCi), GMP (0 or 15 mM), RNasin (20 U; Promega), plasmid pT7-R25 linearized by Hin dIII cleavage (0.2 μg), and T7 RNA polymerase (40 U).

Article Title: Interplay between the Poly(A) Tail, Poly(A)-Binding Protein, and Coronavirus Nucleocapsid Protein Regulates Gene Expression of Coronavirus and the Host Cell
Article Snippet: An in vitro transcription reaction for synthesizing 32 P-labeled RNA for electrophoretic mobility shift assay (EMSA) was carried out using T7 RNA polymerase and [α-32 P]ATP as specified by the manufacturer (Promega). .. To purify 32 P-labeled RNA, the synthesized 32 P-labeled RNA was separated on 6% sequencing gels, and passive elution was performed, followed by phenol-chloroform extraction.

Article Title: MicroRNA–Directed siRNA Biogenesis in Caenorhabditis elegans
Article Snippet: .. For detection of miRNAs, oligonucleotides corresponding to the antisense sequences of the small RNAs were synthesized and radioactively end-labeled with [α-32 P]ATP by using T4 polynucleotide kinase (Promega). ..

Article Title: RNA Stability Regulates Differential Expression Of The Metastasis Protein, Osteopontin, In Hepatocellular Cancer
Article Snippet: .. Briefly, 5’-UTR from the human OPN promoter sequence was synthesized and end-labeled with (α-32 P) ATP (2500 Ci/mmol) using T4 polynucleotide kinase (Promega Co.) followed by G-50 column purification. .. The final products were resolved on 6% poly-acrylamide gel electrophoresis using 0.5× Tris/borate EDTA buffer and visualized by autoradiography.

Article Title: Expression of a novel mRNA transcript for human microsomal epoxide hydrolase (EPHX1) is regulated by short open reading frames within its 5?-untranslated region
Article Snippet: .. The full-length EPHX1 E1-b mRNA transcript was synthesized using RiboMAX Large Scale RNA Production System (Promega) and labeled with [α-32 P]ATP and used to program rabbit reticulocyte lysate (nuclease treated; Promega) as described previously. .. In vitro translation was performed with unlabeled methionine in the presence or absence of 40 µM uORF1 or uORF 2 peptides.

Neutralization:

Article Title: Single-strand DNA intermediates in phage ?'s Red recombination pathway
Article Snippet: Equivalent aliquots of total DNA were digested with restriction endonucleases, fractionated by agarose gel electrophoresis, and transferred through capillary action to nitrocellulose membranes either in the absence of prior denaturation [native conditions ( )] or after denaturation and neutralization ( ). .. One unit of S1 nuclease (Boehringer Mannheim) was added followed by incubation at 30°C for 30 min. Blots were probed either with defined oligonucleotides that had been end-labeled with [γ-32 P]ATP by using T4 polynucleotide kinase (New England Biolabs) or with various defined random primed DNA (either to the left or the right of the Xho I cut site) fragments labeled with [α-32 P]ATP by using a random prime labeling kit as specified by the manufacturer (Promega).

Autoradiography:

Article Title: A cytoplasmic RNA virus generates functional viral small RNAs and regulates viral IRES activity in mammalian cells
Article Snippet: The oligonucleotide probes were generated by 5′-end-labelled with [α-32 P]ATP using T4 polynucleotide kinase (Promega). .. After probe hybridization, the probed membranes were washed twice with 2× SSC, and 0.1% SDS at 42°C for 15 min, and were then processed by autoradiography.

Article Title: Single-strand DNA intermediates in phage ?'s Red recombination pathway
Article Snippet: One unit of S1 nuclease (Boehringer Mannheim) was added followed by incubation at 30°C for 30 min. Blots were probed either with defined oligonucleotides that had been end-labeled with [γ-32 P]ATP by using T4 polynucleotide kinase (New England Biolabs) or with various defined random primed DNA (either to the left or the right of the Xho I cut site) fragments labeled with [α-32 P]ATP by using a random prime labeling kit as specified by the manufacturer (Promega). .. The blots were visualized by autoradiography at −70°C using Kodak X-Omat film.

Article Title: RNA Stability Regulates Differential Expression Of The Metastasis Protein, Osteopontin, In Hepatocellular Cancer
Article Snippet: Briefly, 5’-UTR from the human OPN promoter sequence was synthesized and end-labeled with (α-32 P) ATP (2500 Ci/mmol) using T4 polynucleotide kinase (Promega Co.) followed by G-50 column purification. .. The final products were resolved on 6% poly-acrylamide gel electrophoresis using 0.5× Tris/borate EDTA buffer and visualized by autoradiography.

Electrophoresis:

Article Title: Reprogramming the tRNA-splicing activity of a bacterial RNA repair enzyme
Article Snippet: A reaction mixture (100 µl) containing 80 mM HEPES-KOH (pH 7.5), 24 mM MgCl2 , 40 mM DTT, 2 mM spermidine, 2.5 mM CTP, UTP and GTP, 0.5 mM [α-32 P]ATP, 5 µg template DNA, 120 units RNAsin (Promega), 0.5 units yeast inorganic pyrophosphatase (Sigma) and 300 units T7 RNA polymerase (New England Biolabs) was incubated for 3 h at 37°C. .. The labeled pre-tRNA was purified by electrophoresis through an 8% polyacrylamide-urea gel.

Electro Mobility Shift Assay:

Article Title: Distinct and stage specific nuclear factors regulate the expression of falcipains, Plasmodium falciparum cysteine proteases
Article Snippet: Paragraph title: Electromobility Shift Assay (EMSA) ... The reaction consisted of 10 ng of template, 10 pmoles of the specific primers (Table ), 100 μM of each dNTPS, 5 μl of α-32 P ATP and 1 unit of Taq DNA polymerase (Promega).

Hybridization:

Article Title: Genetic organization of the psbAD region in phages infecting marine Synechococcus strains
Article Snippet: A hybridization probe was prepared from a 525-bp PCR product from S-PM2, which was amplified by using the following primers: S-PM2F1S, 5′-GCTGCTTCTCTTGATGAGTG-3′; and S-PM2R2S, 5′-AGTGTAGCGAACGAGAGTTG-3′. .. The PCR product was gel-extracted and labeled with [α-32 P]ATP by using DNA polymerase I Klenow fragment in 1× labeling buffer (Promega) at 25°C for 2 h. Unincorporated nucleotides were removed by purification through a Sephadex G-25 column ( ).

Article Title: A cytoplasmic RNA virus generates functional viral small RNAs and regulates viral IRES activity in mammalian cells
Article Snippet: The oligonucleotide probes were generated by 5′-end-labelled with [α-32 P]ATP using T4 polynucleotide kinase (Promega). .. Prehybridization and hybridization of the membrane were conducted with an ULTRAhyb Hybridization Buffer (Ambion) containing denatured salmon sperm DNA at 42°C.

Article Title: Nervous System-Specific Expression of a Novel Serine Protease: Regulation in the Adult Rat Spinal Cord by Excitotoxic Injury
Article Snippet: The distribution of MSP and tPA mRNA in human brain and peripheral tissues was explored by Northern hybridization of a multiple tissue RNA dot blot containing human poly(A+ ) RNA from 15 different human adult brain regions and 28 peripheral non-neural tissues (Clontech). .. A 409 bp human tPA cDNA probe corresponding to bp 1134–1543 ( ) was PCR-amplified from clone (40403; American Type Culture Collection, Rockville, MD). cDNA fragments were purified from 1% agarose gels (QIAEX II) and were labeled with 5 μCi [α-32 P]-ATP by random hexamer priming (Promega).

Article Title: MicroRNA–Directed siRNA Biogenesis in Caenorhabditis elegans
Article Snippet: Pre-hybridization/hybridization and washes were done at 32°C for siRNAs and 37°C for microRNAs in ULTRAhyb-oligo hybridization buffer (Ambion). .. For detection of miRNAs, oligonucleotides corresponding to the antisense sequences of the small RNAs were synthesized and radioactively end-labeled with [α-32 P]ATP by using T4 polynucleotide kinase (Promega).

Incubation:

Article Title: Regions of RNase E Important for 5?-End-Dependent RNA Cleavage and Autoregulated Synthesis
Article Snippet: The reaction mixture (20 μl) contained Tris Cl (40 mM, pH 7.9), MgCl2 (6 mM), NaCl (10 mM), dithiothreitol (7.5 mM), spermidine (2 mM), GTP (0.25 mM), CTP (0.25 mM), UTP (0.25 mM), ATP (0.025 mM), [α-32 P]ATP (30 μCi), GMP (0 or 15 mM), RNasin (20 U; Promega), plasmid pT7-R25 linearized by Hin dIII cleavage (0.2 μg), and T7 RNA polymerase (40 U). .. After incubation for 16 h at 37°C, the reaction mixture was diluted with water (30 μl) and the RNA was isolated by gel filtration on Sephadex G-50 and stored at −20°C.

Article Title: Interplay between the Poly(A) Tail, Poly(A)-Binding Protein, and Coronavirus Nucleocapsid Protein Regulates Gene Expression of Coronavirus and the Host Cell
Article Snippet: An in vitro transcription reaction for synthesizing 32 P-labeled RNA for electrophoretic mobility shift assay (EMSA) was carried out using T7 RNA polymerase and [α-32 P]ATP as specified by the manufacturer (Promega). .. The 32 P-labeled RNA and N protein were added to a binding reaction mixture containing 20 mM HEPES (pH 7.5), 6 mM MgCl2 , 1.5 μM EGTA, 22.5 mM NaCl, 330 mM KCl, 36% glycerol, 3.6 mM dithiothreitol (DTT), 82.5 μg/ml of bovine serum albumin (BSA), and 36% glycerol and incubated for 15 min at 37°C with 1 U/ml of RNasin (Promega) (final concentrations for 32 P-labeled RNA and N protein were 1 nM and 5 nM, respectively).

Article Title: Single-strand DNA intermediates in phage ?'s Red recombination pathway
Article Snippet: .. One unit of S1 nuclease (Boehringer Mannheim) was added followed by incubation at 30°C for 30 min. Blots were probed either with defined oligonucleotides that had been end-labeled with [γ-32 P]ATP by using T4 polynucleotide kinase (New England Biolabs) or with various defined random primed DNA (either to the left or the right of the Xho I cut site) fragments labeled with [α-32 P]ATP by using a random prime labeling kit as specified by the manufacturer (Promega). ..

Article Title: Reprogramming the tRNA-splicing activity of a bacterial RNA repair enzyme
Article Snippet: .. A reaction mixture (100 µl) containing 80 mM HEPES-KOH (pH 7.5), 24 mM MgCl2 , 40 mM DTT, 2 mM spermidine, 2.5 mM CTP, UTP and GTP, 0.5 mM [α-32 P]ATP, 5 µg template DNA, 120 units RNAsin (Promega), 0.5 units yeast inorganic pyrophosphatase (Sigma) and 300 units T7 RNA polymerase (New England Biolabs) was incubated for 3 h at 37°C. .. The labeled pre-tRNA was purified by electrophoresis through an 8% polyacrylamide-urea gel.

Article Title: Direct Response Elements of BMP within the PV.1A Promoter Are Essential for Its Transcriptional Regulation during Early Xenopus Development
Article Snippet: Promoter fragments or 3x-repeat oligonucleotides were amplified by annealing two complementary oligos, respectively, and labeled with α-32 P-ATP using T4 polynucleotide kinase (Promega, Madison, WI). .. Labeled DNA probes were incubated with 10 µg animal cap protein extract or 1 µg in vitro translated protein at room temperature for 20 minutes in 10 µL binding buffer (2.5% glycerol, 5 mM MgCl2 , 50 ng/µL Poly [dI·dC], and 0.05% NP-40).

Southern Blot:

Article Title: Genetic organization of the psbAD region in phages infecting marine Synechococcus strains
Article Snippet: Homologs of psbA in other cyanophages were initially detected by Southern blotting. .. The PCR product was gel-extracted and labeled with [α-32 P]ATP by using DNA polymerase I Klenow fragment in 1× labeling buffer (Promega) at 25°C for 2 h. Unincorporated nucleotides were removed by purification through a Sephadex G-25 column ( ).

Transferring:

Article Title: Expression of a novel mRNA transcript for human microsomal epoxide hydrolase (EPHX1) is regulated by short open reading frames within its 5?-untranslated region
Article Snippet: The full-length EPHX1 E1-b mRNA transcript was synthesized using RiboMAX Large Scale RNA Production System (Promega) and labeled with [α-32 P]ATP and used to program rabbit reticulocyte lysate (nuclease treated; Promega) as described previously. .. In vitro translation was performed for 0, 20, 40, or 60 min at 30°C, and the reactions were stopped by transferring the tubes to ice.

Northern Blot:

Article Title: Nervous System-Specific Expression of a Novel Serine Protease: Regulation in the Adult Rat Spinal Cord by Excitotoxic Injury
Article Snippet: The distribution of MSP and tPA mRNA in human brain and peripheral tissues was explored by Northern hybridization of a multiple tissue RNA dot blot containing human poly(A+ ) RNA from 15 different human adult brain regions and 28 peripheral non-neural tissues (Clontech). .. A 409 bp human tPA cDNA probe corresponding to bp 1134–1543 ( ) was PCR-amplified from clone (40403; American Type Culture Collection, Rockville, MD). cDNA fragments were purified from 1% agarose gels (QIAEX II) and were labeled with 5 μCi [α-32 P]-ATP by random hexamer priming (Promega).

Infection:

Article Title: A cytoplasmic RNA virus generates functional viral small RNAs and regulates viral IRES activity in mammalian cells
Article Snippet: vsRNA detection in EV71-infected cells SF268 and RD cells were mock-infected or infected with EV71/4642/MP4 at an MOI of 40. .. The oligonucleotide probes were generated by 5′-end-labelled with [α-32 P]ATP using T4 polynucleotide kinase (Promega).

Generated:

Article Title: A cytoplasmic RNA virus generates functional viral small RNAs and regulates viral IRES activity in mammalian cells
Article Snippet: .. The oligonucleotide probes were generated by 5′-end-labelled with [α-32 P]ATP using T4 polynucleotide kinase (Promega). .. Prehybridization and hybridization of the membrane were conducted with an ULTRAhyb Hybridization Buffer (Ambion) containing denatured salmon sperm DNA at 42°C.

Article Title: Reprogramming the tRNA-splicing activity of a bacterial RNA repair enzyme
Article Snippet: This pre-tRNA was generated by in vitro transcription of BstN1-cut plasmid pNtY9-T7-M1 by T7 RNA polymerase in the presence of [α32 P]ATP. .. A reaction mixture (100 µl) containing 80 mM HEPES-KOH (pH 7.5), 24 mM MgCl2 , 40 mM DTT, 2 mM spermidine, 2.5 mM CTP, UTP and GTP, 0.5 mM [α-32 P]ATP, 5 µg template DNA, 120 units RNAsin (Promega), 0.5 units yeast inorganic pyrophosphatase (Sigma) and 300 units T7 RNA polymerase (New England Biolabs) was incubated for 3 h at 37°C.

DNA Sequencing:

Article Title: Genetic organization of the psbAD region in phages infecting marine Synechococcus strains
Article Snippet: PCRs, Southern Blotting, and DNA Sequencing. .. The PCR product was gel-extracted and labeled with [α-32 P]ATP by using DNA polymerase I Klenow fragment in 1× labeling buffer (Promega) at 25°C for 2 h. Unincorporated nucleotides were removed by purification through a Sephadex G-25 column ( ).

Isolation:

Article Title: A cytoplasmic RNA virus generates functional viral small RNAs and regulates viral IRES activity in mammalian cells
Article Snippet: At 6 h p.i., small RNA fractions with sizes ≤ 200 nt were isolated and enriched using the mirVana miRNA Isolation Kit (Ambion). .. The oligonucleotide probes were generated by 5′-end-labelled with [α-32 P]ATP using T4 polynucleotide kinase (Promega).

Article Title: Regions of RNase E Important for 5?-End-Dependent RNA Cleavage and Autoregulated Synthesis
Article Snippet: The reaction mixture (20 μl) contained Tris Cl (40 mM, pH 7.9), MgCl2 (6 mM), NaCl (10 mM), dithiothreitol (7.5 mM), spermidine (2 mM), GTP (0.25 mM), CTP (0.25 mM), UTP (0.25 mM), ATP (0.025 mM), [α-32 P]ATP (30 μCi), GMP (0 or 15 mM), RNasin (20 U; Promega), plasmid pT7-R25 linearized by Hin dIII cleavage (0.2 μg), and T7 RNA polymerase (40 U). .. After incubation for 16 h at 37°C, the reaction mixture was diluted with water (30 μl) and the RNA was isolated by gel filtration on Sephadex G-50 and stored at −20°C.

Sequencing:

Article Title: Interplay between the Poly(A) Tail, Poly(A)-Binding Protein, and Coronavirus Nucleocapsid Protein Regulates Gene Expression of Coronavirus and the Host Cell
Article Snippet: An in vitro transcription reaction for synthesizing 32 P-labeled RNA for electrophoretic mobility shift assay (EMSA) was carried out using T7 RNA polymerase and [α-32 P]ATP as specified by the manufacturer (Promega). .. To purify 32 P-labeled RNA, the synthesized 32 P-labeled RNA was separated on 6% sequencing gels, and passive elution was performed, followed by phenol-chloroform extraction.

Article Title: Reprogramming the tRNA-splicing activity of a bacterial RNA repair enzyme
Article Snippet: Synthesis of pre-tRNA in vitro The intron-containing pre-tRNA is a chimera consisting of the mature tRNA sequence of plant pre-tRNATyr plus the intron and anticodon of Methanocaldococcus pre-tRNATrp ( ). .. A reaction mixture (100 µl) containing 80 mM HEPES-KOH (pH 7.5), 24 mM MgCl2 , 40 mM DTT, 2 mM spermidine, 2.5 mM CTP, UTP and GTP, 0.5 mM [α-32 P]ATP, 5 µg template DNA, 120 units RNAsin (Promega), 0.5 units yeast inorganic pyrophosphatase (Sigma) and 300 units T7 RNA polymerase (New England Biolabs) was incubated for 3 h at 37°C.

Article Title: RNA Stability Regulates Differential Expression Of The Metastasis Protein, Osteopontin, In Hepatocellular Cancer
Article Snippet: .. Briefly, 5’-UTR from the human OPN promoter sequence was synthesized and end-labeled with (α-32 P) ATP (2500 Ci/mmol) using T4 polynucleotide kinase (Promega Co.) followed by G-50 column purification. .. The final products were resolved on 6% poly-acrylamide gel electrophoresis using 0.5× Tris/borate EDTA buffer and visualized by autoradiography.

Binding Assay:

Article Title: Distinct and stage specific nuclear factors regulate the expression of falcipains, Plasmodium falciparum cysteine proteases
Article Snippet: The reaction consisted of 10 ng of template, 10 pmoles of the specific primers (Table ), 100 μM of each dNTPS, 5 μl of α-32 P ATP and 1 unit of Taq DNA polymerase (Promega). .. Binding reactions were analyzed on a 6% native polyacrylamide gel in 0.5% TBE.

Article Title: Interplay between the Poly(A) Tail, Poly(A)-Binding Protein, and Coronavirus Nucleocapsid Protein Regulates Gene Expression of Coronavirus and the Host Cell
Article Snippet: An in vitro transcription reaction for synthesizing 32 P-labeled RNA for electrophoretic mobility shift assay (EMSA) was carried out using T7 RNA polymerase and [α-32 P]ATP as specified by the manufacturer (Promega). .. The 32 P-labeled RNA and N protein were added to a binding reaction mixture containing 20 mM HEPES (pH 7.5), 6 mM MgCl2 , 1.5 μM EGTA, 22.5 mM NaCl, 330 mM KCl, 36% glycerol, 3.6 mM dithiothreitol (DTT), 82.5 μg/ml of bovine serum albumin (BSA), and 36% glycerol and incubated for 15 min at 37°C with 1 U/ml of RNasin (Promega) (final concentrations for 32 P-labeled RNA and N protein were 1 nM and 5 nM, respectively).

Article Title: Direct Response Elements of BMP within the PV.1A Promoter Are Essential for Its Transcriptional Regulation during Early Xenopus Development
Article Snippet: Promoter fragments or 3x-repeat oligonucleotides were amplified by annealing two complementary oligos, respectively, and labeled with α-32 P-ATP using T4 polynucleotide kinase (Promega, Madison, WI). .. Labeled DNA probes were incubated with 10 µg animal cap protein extract or 1 µg in vitro translated protein at room temperature for 20 minutes in 10 µL binding buffer (2.5% glycerol, 5 mM MgCl2 , 50 ng/µL Poly [dI·dC], and 0.05% NP-40).

Random Primed:

Article Title: Single-strand DNA intermediates in phage ?'s Red recombination pathway
Article Snippet: .. One unit of S1 nuclease (Boehringer Mannheim) was added followed by incubation at 30°C for 30 min. Blots were probed either with defined oligonucleotides that had been end-labeled with [γ-32 P]ATP by using T4 polynucleotide kinase (New England Biolabs) or with various defined random primed DNA (either to the left or the right of the Xho I cut site) fragments labeled with [α-32 P]ATP by using a random prime labeling kit as specified by the manufacturer (Promega). ..

Stability Assay:

Article Title: Expression of a novel mRNA transcript for human microsomal epoxide hydrolase (EPHX1) is regulated by short open reading frames within its 5?-untranslated region
Article Snippet: Paragraph title: RNA stability assay ... The full-length EPHX1 E1-b mRNA transcript was synthesized using RiboMAX Large Scale RNA Production System (Promega) and labeled with [α-32 P]ATP and used to program rabbit reticulocyte lysate (nuclease treated; Promega) as described previously.

Labeling:

Article Title: Genetic organization of the psbAD region in phages infecting marine Synechococcus strains
Article Snippet: .. The PCR product was gel-extracted and labeled with [α-32 P]ATP by using DNA polymerase I Klenow fragment in 1× labeling buffer (Promega) at 25°C for 2 h. Unincorporated nucleotides were removed by purification through a Sephadex G-25 column ( ). .. The labeled S-PM2 psbA PCR product was used as a hybridization probe against cyanophage DNA that had been digested with Eco RI and run on a 0.75% agarose gel.

Article Title: Distinct and stage specific nuclear factors regulate the expression of falcipains, Plasmodium falciparum cysteine proteases
Article Snippet: Electromobility Shift Assay (EMSA) Complementary strands of the probes used for EMSA were labeled with α-32 P ATP using PCR in a 50 μl reaction mixture. .. The reaction consisted of 10 ng of template, 10 pmoles of the specific primers (Table ), 100 μM of each dNTPS, 5 μl of α-32 P ATP and 1 unit of Taq DNA polymerase (Promega).

Article Title: Regions of RNase E Important for 5?-End-Dependent RNA Cleavage and Autoregulated Synthesis
Article Snippet: The reaction mixture (20 μl) contained Tris Cl (40 mM, pH 7.9), MgCl2 (6 mM), NaCl (10 mM), dithiothreitol (7.5 mM), spermidine (2 mM), GTP (0.25 mM), CTP (0.25 mM), UTP (0.25 mM), ATP (0.025 mM), [α-32 P]ATP (30 μCi), GMP (0 or 15 mM), RNasin (20 U; Promega), plasmid pT7-R25 linearized by Hin dIII cleavage (0.2 μg), and T7 RNA polymerase (40 U). .. The suitability of these reaction conditions for synthesizing 5′-monophosphorylated or -capped RNA was confirmed in experiments involving the synthesis of RNA that was internally labeled with a fluorescent nucleotide analog and 5′ end labeled with [γ-32 P]GTP.

Article Title: Single-strand DNA intermediates in phage ?'s Red recombination pathway
Article Snippet: .. One unit of S1 nuclease (Boehringer Mannheim) was added followed by incubation at 30°C for 30 min. Blots were probed either with defined oligonucleotides that had been end-labeled with [γ-32 P]ATP by using T4 polynucleotide kinase (New England Biolabs) or with various defined random primed DNA (either to the left or the right of the Xho I cut site) fragments labeled with [α-32 P]ATP by using a random prime labeling kit as specified by the manufacturer (Promega). ..

Article Title: Nervous System-Specific Expression of a Novel Serine Protease: Regulation in the Adult Rat Spinal Cord by Excitotoxic Injury
Article Snippet: .. A 409 bp human tPA cDNA probe corresponding to bp 1134–1543 ( ) was PCR-amplified from clone (40403; American Type Culture Collection, Rockville, MD). cDNA fragments were purified from 1% agarose gels (QIAEX II) and were labeled with 5 μCi [α-32 P]-ATP by random hexamer priming (Promega). .. Northern hybridization of mRNA blotted on Zetta membranes was performed according to Maniatis ( ).

Article Title: Reprogramming the tRNA-splicing activity of a bacterial RNA repair enzyme
Article Snippet: A reaction mixture (100 µl) containing 80 mM HEPES-KOH (pH 7.5), 24 mM MgCl2 , 40 mM DTT, 2 mM spermidine, 2.5 mM CTP, UTP and GTP, 0.5 mM [α-32 P]ATP, 5 µg template DNA, 120 units RNAsin (Promega), 0.5 units yeast inorganic pyrophosphatase (Sigma) and 300 units T7 RNA polymerase (New England Biolabs) was incubated for 3 h at 37°C. .. The labeled pre-tRNA was purified by electrophoresis through an 8% polyacrylamide-urea gel.

Article Title: Direct Response Elements of BMP within the PV.1A Promoter Are Essential for Its Transcriptional Regulation during Early Xenopus Development
Article Snippet: .. Promoter fragments or 3x-repeat oligonucleotides were amplified by annealing two complementary oligos, respectively, and labeled with α-32 P-ATP using T4 polynucleotide kinase (Promega, Madison, WI). .. Labeled DNA probes were incubated with 10 µg animal cap protein extract or 1 µg in vitro translated protein at room temperature for 20 minutes in 10 µL binding buffer (2.5% glycerol, 5 mM MgCl2 , 50 ng/µL Poly [dI·dC], and 0.05% NP-40).

Article Title: Expression of a novel mRNA transcript for human microsomal epoxide hydrolase (EPHX1) is regulated by short open reading frames within its 5?-untranslated region
Article Snippet: .. The full-length EPHX1 E1-b mRNA transcript was synthesized using RiboMAX Large Scale RNA Production System (Promega) and labeled with [α-32 P]ATP and used to program rabbit reticulocyte lysate (nuclease treated; Promega) as described previously. .. In vitro translation was performed with unlabeled methionine in the presence or absence of 40 µM uORF1 or uORF 2 peptides.

Purification:

Article Title: Genetic organization of the psbAD region in phages infecting marine Synechococcus strains
Article Snippet: .. The PCR product was gel-extracted and labeled with [α-32 P]ATP by using DNA polymerase I Klenow fragment in 1× labeling buffer (Promega) at 25°C for 2 h. Unincorporated nucleotides were removed by purification through a Sephadex G-25 column ( ). .. The labeled S-PM2 psbA PCR product was used as a hybridization probe against cyanophage DNA that had been digested with Eco RI and run on a 0.75% agarose gel.

Article Title: Distinct and stage specific nuclear factors regulate the expression of falcipains, Plasmodium falciparum cysteine proteases
Article Snippet: The reaction consisted of 10 ng of template, 10 pmoles of the specific primers (Table ), 100 μM of each dNTPS, 5 μl of α-32 P ATP and 1 unit of Taq DNA polymerase (Promega). .. Labeled probes were purified using QIAquick nucleotide removal kit (Qiagen) and the counts were checked by using scintillation counter.

Article Title: Nervous System-Specific Expression of a Novel Serine Protease: Regulation in the Adult Rat Spinal Cord by Excitotoxic Injury
Article Snippet: .. A 409 bp human tPA cDNA probe corresponding to bp 1134–1543 ( ) was PCR-amplified from clone (40403; American Type Culture Collection, Rockville, MD). cDNA fragments were purified from 1% agarose gels (QIAEX II) and were labeled with 5 μCi [α-32 P]-ATP by random hexamer priming (Promega). .. Northern hybridization of mRNA blotted on Zetta membranes was performed according to Maniatis ( ).

Article Title: Reprogramming the tRNA-splicing activity of a bacterial RNA repair enzyme
Article Snippet: A reaction mixture (100 µl) containing 80 mM HEPES-KOH (pH 7.5), 24 mM MgCl2 , 40 mM DTT, 2 mM spermidine, 2.5 mM CTP, UTP and GTP, 0.5 mM [α-32 P]ATP, 5 µg template DNA, 120 units RNAsin (Promega), 0.5 units yeast inorganic pyrophosphatase (Sigma) and 300 units T7 RNA polymerase (New England Biolabs) was incubated for 3 h at 37°C. .. The labeled pre-tRNA was purified by electrophoresis through an 8% polyacrylamide-urea gel.

Article Title: RNA Stability Regulates Differential Expression Of The Metastasis Protein, Osteopontin, In Hepatocellular Cancer
Article Snippet: .. Briefly, 5’-UTR from the human OPN promoter sequence was synthesized and end-labeled with (α-32 P) ATP (2500 Ci/mmol) using T4 polynucleotide kinase (Promega Co.) followed by G-50 column purification. .. The final products were resolved on 6% poly-acrylamide gel electrophoresis using 0.5× Tris/borate EDTA buffer and visualized by autoradiography.

Dot Blot:

Article Title: Nervous System-Specific Expression of a Novel Serine Protease: Regulation in the Adult Rat Spinal Cord by Excitotoxic Injury
Article Snippet: The distribution of MSP and tPA mRNA in human brain and peripheral tissues was explored by Northern hybridization of a multiple tissue RNA dot blot containing human poly(A+ ) RNA from 15 different human adult brain regions and 28 peripheral non-neural tissues (Clontech). .. A 409 bp human tPA cDNA probe corresponding to bp 1134–1543 ( ) was PCR-amplified from clone (40403; American Type Culture Collection, Rockville, MD). cDNA fragments were purified from 1% agarose gels (QIAEX II) and were labeled with 5 μCi [α-32 P]-ATP by random hexamer priming (Promega).

Polymerase Chain Reaction:

Article Title: Genetic organization of the psbAD region in phages infecting marine Synechococcus strains
Article Snippet: .. The PCR product was gel-extracted and labeled with [α-32 P]ATP by using DNA polymerase I Klenow fragment in 1× labeling buffer (Promega) at 25°C for 2 h. Unincorporated nucleotides were removed by purification through a Sephadex G-25 column ( ). .. The labeled S-PM2 psbA PCR product was used as a hybridization probe against cyanophage DNA that had been digested with Eco RI and run on a 0.75% agarose gel.

Article Title: Distinct and stage specific nuclear factors regulate the expression of falcipains, Plasmodium falciparum cysteine proteases
Article Snippet: Electromobility Shift Assay (EMSA) Complementary strands of the probes used for EMSA were labeled with α-32 P ATP using PCR in a 50 μl reaction mixture. .. The reaction consisted of 10 ng of template, 10 pmoles of the specific primers (Table ), 100 μM of each dNTPS, 5 μl of α-32 P ATP and 1 unit of Taq DNA polymerase (Promega).

Article Title: Nervous System-Specific Expression of a Novel Serine Protease: Regulation in the Adult Rat Spinal Cord by Excitotoxic Injury
Article Snippet: .. A 409 bp human tPA cDNA probe corresponding to bp 1134–1543 ( ) was PCR-amplified from clone (40403; American Type Culture Collection, Rockville, MD). cDNA fragments were purified from 1% agarose gels (QIAEX II) and were labeled with 5 μCi [α-32 P]-ATP by random hexamer priming (Promega). .. Northern hybridization of mRNA blotted on Zetta membranes was performed according to Maniatis ( ).

Article Title: Direct Response Elements of BMP within the PV.1A Promoter Are Essential for Its Transcriptional Regulation during Early Xenopus Development
Article Snippet: In vitro translation and electrophoretic mobility shift assays cDNAs encoding Smads 1, 3, and 4 were PCR-amplified and used as templates for in vitro translation. .. Promoter fragments or 3x-repeat oligonucleotides were amplified by annealing two complementary oligos, respectively, and labeled with α-32 P-ATP using T4 polynucleotide kinase (Promega, Madison, WI).

Electrophoretic Mobility Shift Assay:

Article Title: Interplay between the Poly(A) Tail, Poly(A)-Binding Protein, and Coronavirus Nucleocapsid Protein Regulates Gene Expression of Coronavirus and the Host Cell
Article Snippet: .. An in vitro transcription reaction for synthesizing 32 P-labeled RNA for electrophoretic mobility shift assay (EMSA) was carried out using T7 RNA polymerase and [α-32 P]ATP as specified by the manufacturer (Promega). .. To purify 32 P-labeled RNA, the synthesized 32 P-labeled RNA was separated on 6% sequencing gels, and passive elution was performed, followed by phenol-chloroform extraction.

Article Title: Direct Response Elements of BMP within the PV.1A Promoter Are Essential for Its Transcriptional Regulation during Early Xenopus Development
Article Snippet: Paragraph title: In vitro translation and electrophoretic mobility shift assays ... Promoter fragments or 3x-repeat oligonucleotides were amplified by annealing two complementary oligos, respectively, and labeled with α-32 P-ATP using T4 polynucleotide kinase (Promega, Madison, WI).

Article Title: RNA Stability Regulates Differential Expression Of The Metastasis Protein, Osteopontin, In Hepatocellular Cancer
Article Snippet: Cytoplasmic protein extracts obtained from HepG2 and Hep3B cells using lysis buffer containing 1× PBS, 0.1% Triton X-100, 0.5% sodium deoxycholic acid and protein cocktail inhibitors were subjected to gel-shift assay analysis as described previously [ ]. .. Briefly, 5’-UTR from the human OPN promoter sequence was synthesized and end-labeled with (α-32 P) ATP (2500 Ci/mmol) using T4 polynucleotide kinase (Promega Co.) followed by G-50 column purification.

Polyacrylamide Gel Electrophoresis:

Article Title: RNA Stability Regulates Differential Expression Of The Metastasis Protein, Osteopontin, In Hepatocellular Cancer
Article Snippet: Briefly, 5’-UTR from the human OPN promoter sequence was synthesized and end-labeled with (α-32 P) ATP (2500 Ci/mmol) using T4 polynucleotide kinase (Promega Co.) followed by G-50 column purification. .. The final products were resolved on 6% poly-acrylamide gel electrophoresis using 0.5× Tris/borate EDTA buffer and visualized by autoradiography.

Plasmid Preparation:

Article Title: Regions of RNase E Important for 5?-End-Dependent RNA Cleavage and Autoregulated Synthesis
Article Snippet: .. The reaction mixture (20 μl) contained Tris Cl (40 mM, pH 7.9), MgCl2 (6 mM), NaCl (10 mM), dithiothreitol (7.5 mM), spermidine (2 mM), GTP (0.25 mM), CTP (0.25 mM), UTP (0.25 mM), ATP (0.025 mM), [α-32 P]ATP (30 μCi), GMP (0 or 15 mM), RNasin (20 U; Promega), plasmid pT7-R25 linearized by Hin dIII cleavage (0.2 μg), and T7 RNA polymerase (40 U). .. After incubation for 16 h at 37°C, the reaction mixture was diluted with water (30 μl) and the RNA was isolated by gel filtration on Sephadex G-50 and stored at −20°C.

Article Title: Nervous System-Specific Expression of a Novel Serine Protease: Regulation in the Adult Rat Spinal Cord by Excitotoxic Injury
Article Snippet: Rat MSP (pM444-4) and human MSP (pCD-2) cDNA inserts were cut from plasmid DNA with a combination of Sph I and Nco I. .. A 409 bp human tPA cDNA probe corresponding to bp 1134–1543 ( ) was PCR-amplified from clone (40403; American Type Culture Collection, Rockville, MD). cDNA fragments were purified from 1% agarose gels (QIAEX II) and were labeled with 5 μCi [α-32 P]-ATP by random hexamer priming (Promega).

Article Title: Reprogramming the tRNA-splicing activity of a bacterial RNA repair enzyme
Article Snippet: This pre-tRNA was generated by in vitro transcription of BstN1-cut plasmid pNtY9-T7-M1 by T7 RNA polymerase in the presence of [α32 P]ATP. .. A reaction mixture (100 µl) containing 80 mM HEPES-KOH (pH 7.5), 24 mM MgCl2 , 40 mM DTT, 2 mM spermidine, 2.5 mM CTP, UTP and GTP, 0.5 mM [α-32 P]ATP, 5 µg template DNA, 120 units RNAsin (Promega), 0.5 units yeast inorganic pyrophosphatase (Sigma) and 300 units T7 RNA polymerase (New England Biolabs) was incubated for 3 h at 37°C.

RNA Expression:

Article Title: Nervous System-Specific Expression of a Novel Serine Protease: Regulation in the Adult Rat Spinal Cord by Excitotoxic Injury
Article Snippet: Paragraph title: MSP RNA expression in brain and peripheral tissues ... A 409 bp human tPA cDNA probe corresponding to bp 1134–1543 ( ) was PCR-amplified from clone (40403; American Type Culture Collection, Rockville, MD). cDNA fragments were purified from 1% agarose gels (QIAEX II) and were labeled with 5 μCi [α-32 P]-ATP by random hexamer priming (Promega).

Agarose Gel Electrophoresis:

Article Title: Genetic organization of the psbAD region in phages infecting marine Synechococcus strains
Article Snippet: The PCR product was gel-extracted and labeled with [α-32 P]ATP by using DNA polymerase I Klenow fragment in 1× labeling buffer (Promega) at 25°C for 2 h. Unincorporated nucleotides were removed by purification through a Sephadex G-25 column ( ). .. The labeled S-PM2 psbA PCR product was used as a hybridization probe against cyanophage DNA that had been digested with Eco RI and run on a 0.75% agarose gel.

Article Title: Single-strand DNA intermediates in phage ?'s Red recombination pathway
Article Snippet: Equivalent aliquots of total DNA were digested with restriction endonucleases, fractionated by agarose gel electrophoresis, and transferred through capillary action to nitrocellulose membranes either in the absence of prior denaturation [native conditions ( )] or after denaturation and neutralization ( ). .. One unit of S1 nuclease (Boehringer Mannheim) was added followed by incubation at 30°C for 30 min. Blots were probed either with defined oligonucleotides that had been end-labeled with [γ-32 P]ATP by using T4 polynucleotide kinase (New England Biolabs) or with various defined random primed DNA (either to the left or the right of the Xho I cut site) fragments labeled with [α-32 P]ATP by using a random prime labeling kit as specified by the manufacturer (Promega).

In Vitro:

Article Title: Regions of RNase E Important for 5?-End-Dependent RNA Cleavage and Autoregulated Synthesis
Article Snippet: Internally radiolabeled RNA was synthesized by in vitro transcription with T7 RNA polymerase. .. The reaction mixture (20 μl) contained Tris Cl (40 mM, pH 7.9), MgCl2 (6 mM), NaCl (10 mM), dithiothreitol (7.5 mM), spermidine (2 mM), GTP (0.25 mM), CTP (0.25 mM), UTP (0.25 mM), ATP (0.025 mM), [α-32 P]ATP (30 μCi), GMP (0 or 15 mM), RNasin (20 U; Promega), plasmid pT7-R25 linearized by Hin dIII cleavage (0.2 μg), and T7 RNA polymerase (40 U).

Article Title: Interplay between the Poly(A) Tail, Poly(A)-Binding Protein, and Coronavirus Nucleocapsid Protein Regulates Gene Expression of Coronavirus and the Host Cell
Article Snippet: .. An in vitro transcription reaction for synthesizing 32 P-labeled RNA for electrophoretic mobility shift assay (EMSA) was carried out using T7 RNA polymerase and [α-32 P]ATP as specified by the manufacturer (Promega). .. To purify 32 P-labeled RNA, the synthesized 32 P-labeled RNA was separated on 6% sequencing gels, and passive elution was performed, followed by phenol-chloroform extraction.

Article Title: Reprogramming the tRNA-splicing activity of a bacterial RNA repair enzyme
Article Snippet: Paragraph title: Synthesis of pre-tRNA in vitro ... A reaction mixture (100 µl) containing 80 mM HEPES-KOH (pH 7.5), 24 mM MgCl2 , 40 mM DTT, 2 mM spermidine, 2.5 mM CTP, UTP and GTP, 0.5 mM [α-32 P]ATP, 5 µg template DNA, 120 units RNAsin (Promega), 0.5 units yeast inorganic pyrophosphatase (Sigma) and 300 units T7 RNA polymerase (New England Biolabs) was incubated for 3 h at 37°C.

Article Title: Direct Response Elements of BMP within the PV.1A Promoter Are Essential for Its Transcriptional Regulation during Early Xenopus Development
Article Snippet: Paragraph title: In vitro translation and electrophoretic mobility shift assays ... Promoter fragments or 3x-repeat oligonucleotides were amplified by annealing two complementary oligos, respectively, and labeled with α-32 P-ATP using T4 polynucleotide kinase (Promega, Madison, WI).

Article Title: Expression of a novel mRNA transcript for human microsomal epoxide hydrolase (EPHX1) is regulated by short open reading frames within its 5?-untranslated region
Article Snippet: The full-length EPHX1 E1-b mRNA transcript was synthesized using RiboMAX Large Scale RNA Production System (Promega) and labeled with [α-32 P]ATP and used to program rabbit reticulocyte lysate (nuclease treated; Promega) as described previously. .. In vitro translation was performed with unlabeled methionine in the presence or absence of 40 µM uORF1 or uORF 2 peptides.

Random Hexamer Labeling:

Article Title: Nervous System-Specific Expression of a Novel Serine Protease: Regulation in the Adult Rat Spinal Cord by Excitotoxic Injury
Article Snippet: .. A 409 bp human tPA cDNA probe corresponding to bp 1134–1543 ( ) was PCR-amplified from clone (40403; American Type Culture Collection, Rockville, MD). cDNA fragments were purified from 1% agarose gels (QIAEX II) and were labeled with 5 μCi [α-32 P]-ATP by random hexamer priming (Promega). .. Northern hybridization of mRNA blotted on Zetta membranes was performed according to Maniatis ( ).

Produced:

Article Title: Nervous System-Specific Expression of a Novel Serine Protease: Regulation in the Adult Rat Spinal Cord by Excitotoxic Injury
Article Snippet: The signal produced from a nontissue-specific constitutively expressed gene, ubiquitin, has been shown to be consistent for each dot containing RNA samples from different tissues. .. A 409 bp human tPA cDNA probe corresponding to bp 1134–1543 ( ) was PCR-amplified from clone (40403; American Type Culture Collection, Rockville, MD). cDNA fragments were purified from 1% agarose gels (QIAEX II) and were labeled with 5 μCi [α-32 P]-ATP by random hexamer priming (Promega).

Mobility Shift:

Article Title: RNA Stability Regulates Differential Expression Of The Metastasis Protein, Osteopontin, In Hepatocellular Cancer
Article Snippet: Paragraph title: Cytoplasmic extracts and Electrophoric mobility shift assays ... Briefly, 5’-UTR from the human OPN promoter sequence was synthesized and end-labeled with (α-32 P) ATP (2500 Ci/mmol) using T4 polynucleotide kinase (Promega Co.) followed by G-50 column purification.

Lysis:

Article Title: RNA Stability Regulates Differential Expression Of The Metastasis Protein, Osteopontin, In Hepatocellular Cancer
Article Snippet: Cytoplasmic protein extracts obtained from HepG2 and Hep3B cells using lysis buffer containing 1× PBS, 0.1% Triton X-100, 0.5% sodium deoxycholic acid and protein cocktail inhibitors were subjected to gel-shift assay analysis as described previously [ ]. .. Briefly, 5’-UTR from the human OPN promoter sequence was synthesized and end-labeled with (α-32 P) ATP (2500 Ci/mmol) using T4 polynucleotide kinase (Promega Co.) followed by G-50 column purification.

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  • 91
    Promega β actin probes
    Nocodazole treatment affects viral mRNA synthesis and stability. (A) HeLa cells were treated with nocodazole 1 h before infection in the presence of the drug. At 90 min postinfection the cells were labeled for 30 min with [ 3 H]uridine (left) or [ 35 S]methionine (right). The incorporation of uridine or methionine into RNAs and proteins, respectively, in total cell extracts (including cytoplasm and nuclei) was measured by liquid scintillation counting after TCA precipitation. The values represent the average and SDs of three experiments performed in duplicate. (B) The stability of H5R and <t>β-actin</t> (indicated) mRNAs was assessed in nocodazole-treated infected HeLa cells. Cells were nocodazole-treated 1 h before infection. At 1 h postinfection act D was added and mRNAs isolated from the infected cells at the indicated times after act D addition. The specific mRNAs were detected by Northern blotting, and the amount in each lane was determined by phospho-imager analysis (indicated at the bottom of each panel). The data show that the H5R mRNA is rapidly degraded in the absence of MTs.
    β Actin Probes, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Promega α 32 p atp
    Nocodazole treatment affects viral mRNA synthesis and stability. (A) HeLa cells were treated with nocodazole 1 h before infection in the presence of the drug. At 90 min postinfection the cells were labeled for 30 min with [ 3 H]uridine (left) or [ 35 S]methionine (right). The incorporation of uridine or methionine into RNAs and proteins, respectively, in total cell extracts (including cytoplasm and nuclei) was measured by liquid scintillation counting after TCA precipitation. The values represent the average and SDs of three experiments performed in duplicate. (B) The stability of H5R and <t>β-actin</t> (indicated) mRNAs was assessed in nocodazole-treated infected HeLa cells. Cells were nocodazole-treated 1 h before infection. At 1 h postinfection act D was added and mRNAs isolated from the infected cells at the indicated times after act D addition. The specific mRNAs were detected by Northern blotting, and the amount in each lane was determined by phospho-imager analysis (indicated at the bottom of each panel). The data show that the H5R mRNA is rapidly degraded in the absence of MTs.
    α 32 P Atp, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α 32 p atp/product/Promega
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    Promega α 32 p atp labelled
    Nocodazole treatment affects viral mRNA synthesis and stability. (A) HeLa cells were treated with nocodazole 1 h before infection in the presence of the drug. At 90 min postinfection the cells were labeled for 30 min with [ 3 H]uridine (left) or [ 35 S]methionine (right). The incorporation of uridine or methionine into RNAs and proteins, respectively, in total cell extracts (including cytoplasm and nuclei) was measured by liquid scintillation counting after TCA precipitation. The values represent the average and SDs of three experiments performed in duplicate. (B) The stability of H5R and <t>β-actin</t> (indicated) mRNAs was assessed in nocodazole-treated infected HeLa cells. Cells were nocodazole-treated 1 h before infection. At 1 h postinfection act D was added and mRNAs isolated from the infected cells at the indicated times after act D addition. The specific mRNAs were detected by Northern blotting, and the amount in each lane was determined by phospho-imager analysis (indicated at the bottom of each panel). The data show that the H5R mRNA is rapidly degraded in the absence of MTs.
    α 32 P Atp Labelled, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Nocodazole treatment affects viral mRNA synthesis and stability. (A) HeLa cells were treated with nocodazole 1 h before infection in the presence of the drug. At 90 min postinfection the cells were labeled for 30 min with [ 3 H]uridine (left) or [ 35 S]methionine (right). The incorporation of uridine or methionine into RNAs and proteins, respectively, in total cell extracts (including cytoplasm and nuclei) was measured by liquid scintillation counting after TCA precipitation. The values represent the average and SDs of three experiments performed in duplicate. (B) The stability of H5R and β-actin (indicated) mRNAs was assessed in nocodazole-treated infected HeLa cells. Cells were nocodazole-treated 1 h before infection. At 1 h postinfection act D was added and mRNAs isolated from the infected cells at the indicated times after act D addition. The specific mRNAs were detected by Northern blotting, and the amount in each lane was determined by phospho-imager analysis (indicated at the bottom of each panel). The data show that the H5R mRNA is rapidly degraded in the absence of MTs.

    Journal: Molecular Biology of the Cell

    Article Title: Microtubule-dependent Organization of Vaccinia Virus Core-derivd Early mRNAs into Distinct Cytoplasmic Structures

    doi:

    Figure Lengend Snippet: Nocodazole treatment affects viral mRNA synthesis and stability. (A) HeLa cells were treated with nocodazole 1 h before infection in the presence of the drug. At 90 min postinfection the cells were labeled for 30 min with [ 3 H]uridine (left) or [ 35 S]methionine (right). The incorporation of uridine or methionine into RNAs and proteins, respectively, in total cell extracts (including cytoplasm and nuclei) was measured by liquid scintillation counting after TCA precipitation. The values represent the average and SDs of three experiments performed in duplicate. (B) The stability of H5R and β-actin (indicated) mRNAs was assessed in nocodazole-treated infected HeLa cells. Cells were nocodazole-treated 1 h before infection. At 1 h postinfection act D was added and mRNAs isolated from the infected cells at the indicated times after act D addition. The specific mRNAs were detected by Northern blotting, and the amount in each lane was determined by phospho-imager analysis (indicated at the bottom of each panel). The data show that the H5R mRNA is rapidly degraded in the absence of MTs.

    Article Snippet: The RNA blots were hybridized with H5R and β-actin probes [α-32 P]ATP that were labeled by random priming (Promega) according to the manufacturer's instructions.

    Techniques: Infection, Labeling, TCA Precipitation, Activated Clotting Time Assay, Isolation, Northern Blot

    Biochemical evidence for MT association of viral mRNAs. (A–C) Cells were infected for 2 h. They were either left untreated or were treated with either 20 μM of nocodazole from 1 h before infection onward or with 1 μM Lat A together with 2.5 μM taxol 20 min before cell lysis. (A and B) Total RNA was extracted from infected cells from either the detergent-soluble (S) or -insoluble cytoskeletal fraction (CK), and the fractions were analyzed by an RNase protection assay with the use of probes to H5R and β-actin mRNA. The position of the H5R (A) and β-actin (B) protected mRNA is indicated on the right. The percentage of protected mRNAs in the cytoskeleton (CK) versus soluble (S) fraction of eachsample as assessed by phosphoimager analysis is indicated at the bottom of each panel. (C) Western blot analysis of actin and tubulin (the position of the respective protein is indicated) after nocodazole or Lat A treatment in the soluble (S) and cytoskeleton (CK) fraction. The proteins were detected with the use of antibodies to β-actin or β-tubulin followed by enhanced chemiluminescence.

    Journal: Molecular Biology of the Cell

    Article Title: Microtubule-dependent Organization of Vaccinia Virus Core-derivd Early mRNAs into Distinct Cytoplasmic Structures

    doi:

    Figure Lengend Snippet: Biochemical evidence for MT association of viral mRNAs. (A–C) Cells were infected for 2 h. They were either left untreated or were treated with either 20 μM of nocodazole from 1 h before infection onward or with 1 μM Lat A together with 2.5 μM taxol 20 min before cell lysis. (A and B) Total RNA was extracted from infected cells from either the detergent-soluble (S) or -insoluble cytoskeletal fraction (CK), and the fractions were analyzed by an RNase protection assay with the use of probes to H5R and β-actin mRNA. The position of the H5R (A) and β-actin (B) protected mRNA is indicated on the right. The percentage of protected mRNAs in the cytoskeleton (CK) versus soluble (S) fraction of eachsample as assessed by phosphoimager analysis is indicated at the bottom of each panel. (C) Western blot analysis of actin and tubulin (the position of the respective protein is indicated) after nocodazole or Lat A treatment in the soluble (S) and cytoskeleton (CK) fraction. The proteins were detected with the use of antibodies to β-actin or β-tubulin followed by enhanced chemiluminescence.

    Article Snippet: The RNA blots were hybridized with H5R and β-actin probes [α-32 P]ATP that were labeled by random priming (Promega) according to the manufacturer's instructions.

    Techniques: Infection, Lysis, Rnase Protection Assay, Western Blot