Structured Review

DuPont de Nemours α 32 p atp
Effect of CoA and organic acids on the synthesis of p 4 A catalyzed by acyl-CoA synthetase. (A) Effect of CoA on the synthesis of p 4 A. The reaction mixture (50 μl) contained 50 mM MES-KOH (pH 6.3), 0.1 mM dithiothreitol, 11 mM MgCl 2 , 1 mM [2,8- 3 <t>H]ATP,</t> 10 mM P 3 , and the indicated concentrations of CoA and acyl-CoA synthetase (8.3 μg of protein). (B) Effect of organic acids on the inhibitory effect of CoA on the synthesis of p 4 A. Reaction mixtures (28 μl) containing 72 mM MES-KOH (pH 6.3), 0.14 mM dithiothreitol, 7.9 mM MgCl 2 , 0.76 mM [α- 32 P]ATP, 7.2 mM P 3 , 0.14 mM CoA, 0.7 μl of desalted inorganic pyrophosphatase, and acyl-CoA synthetase (5.2 μg of protein) were preincubated at 30°C for 20 min. Thereafter, they were supplemented with 12 μl of the following solutions: water (lane b), 1% Triton X-100–5% ethanol (solution C; lane c), 1 mM solutions of palmitic acid in solution C (lane d), octanoic acid in water (lane e), or other possible effectors (acetic acid, lysine, methionine, phenylalanine, tryptophan, and luciferin; lanes f to k, respectively). One hour after the addition of organic acids, aliquots of the reaction were analyzed by TLC. Control without acyl-CoA synthetase is shown in lane a.
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Images

1) Product Images from "Acyl Coenzyme A Synthetase from Pseudomonas fragi Catalyzes the Synthesis of Adenosine 5?-Polyphosphates and Dinucleoside Polyphosphates †"

Article Title: Acyl Coenzyme A Synthetase from Pseudomonas fragi Catalyzes the Synthesis of Adenosine 5?-Polyphosphates and Dinucleoside Polyphosphates †

Journal: Journal of Bacteriology

doi:

Effect of CoA and organic acids on the synthesis of p 4 A catalyzed by acyl-CoA synthetase. (A) Effect of CoA on the synthesis of p 4 A. The reaction mixture (50 μl) contained 50 mM MES-KOH (pH 6.3), 0.1 mM dithiothreitol, 11 mM MgCl 2 , 1 mM [2,8- 3 H]ATP, 10 mM P 3 , and the indicated concentrations of CoA and acyl-CoA synthetase (8.3 μg of protein). (B) Effect of organic acids on the inhibitory effect of CoA on the synthesis of p 4 A. Reaction mixtures (28 μl) containing 72 mM MES-KOH (pH 6.3), 0.14 mM dithiothreitol, 7.9 mM MgCl 2 , 0.76 mM [α- 32 P]ATP, 7.2 mM P 3 , 0.14 mM CoA, 0.7 μl of desalted inorganic pyrophosphatase, and acyl-CoA synthetase (5.2 μg of protein) were preincubated at 30°C for 20 min. Thereafter, they were supplemented with 12 μl of the following solutions: water (lane b), 1% Triton X-100–5% ethanol (solution C; lane c), 1 mM solutions of palmitic acid in solution C (lane d), octanoic acid in water (lane e), or other possible effectors (acetic acid, lysine, methionine, phenylalanine, tryptophan, and luciferin; lanes f to k, respectively). One hour after the addition of organic acids, aliquots of the reaction were analyzed by TLC. Control without acyl-CoA synthetase is shown in lane a.
Figure Legend Snippet: Effect of CoA and organic acids on the synthesis of p 4 A catalyzed by acyl-CoA synthetase. (A) Effect of CoA on the synthesis of p 4 A. The reaction mixture (50 μl) contained 50 mM MES-KOH (pH 6.3), 0.1 mM dithiothreitol, 11 mM MgCl 2 , 1 mM [2,8- 3 H]ATP, 10 mM P 3 , and the indicated concentrations of CoA and acyl-CoA synthetase (8.3 μg of protein). (B) Effect of organic acids on the inhibitory effect of CoA on the synthesis of p 4 A. Reaction mixtures (28 μl) containing 72 mM MES-KOH (pH 6.3), 0.14 mM dithiothreitol, 7.9 mM MgCl 2 , 0.76 mM [α- 32 P]ATP, 7.2 mM P 3 , 0.14 mM CoA, 0.7 μl of desalted inorganic pyrophosphatase, and acyl-CoA synthetase (5.2 μg of protein) were preincubated at 30°C for 20 min. Thereafter, they were supplemented with 12 μl of the following solutions: water (lane b), 1% Triton X-100–5% ethanol (solution C; lane c), 1 mM solutions of palmitic acid in solution C (lane d), octanoic acid in water (lane e), or other possible effectors (acetic acid, lysine, methionine, phenylalanine, tryptophan, and luciferin; lanes f to k, respectively). One hour after the addition of organic acids, aliquots of the reaction were analyzed by TLC. Control without acyl-CoA synthetase is shown in lane a.

Techniques Used: Thin Layer Chromatography

2) Product Images from "Mutagenesis of the Dengue Virus Type 2 NS3 Protein within and outside Helicase Motifs: Effects on Enzyme Activity and Virus Replication"

Article Title: Mutagenesis of the Dengue Virus Type 2 NS3 Protein within and outside Helicase Motifs: Effects on Enzyme Activity and Virus Replication

Journal: Journal of Virology

doi: 10.1128/JVI.75.20.9633-9643.2001

RNA helicase assay of 74%NS3 fusion proteins. (A) Structure of the 3′-tailed dsRNA substrate; the lower strand of RNA was labeled with [α- 32 P]ATP. (B) RNA helicase activity of the parental G2 protein. Lane 1, heated RNA substrate (hS); lane 2, untreated RNA substrate (S); lane 3, RNA helicase activity of 1 pmol of purified G2 protein; lane 4, same as lane 3 but omitting ATP and Mn 2+ . (C) RNA helicase activity of mutant fusion proteins. (D) Percent unwinding of dsRNA to single-stranded RNA (ssRNA). Means (columns) and range of values (error bars) from three independent experiments are shown.
Figure Legend Snippet: RNA helicase assay of 74%NS3 fusion proteins. (A) Structure of the 3′-tailed dsRNA substrate; the lower strand of RNA was labeled with [α- 32 P]ATP. (B) RNA helicase activity of the parental G2 protein. Lane 1, heated RNA substrate (hS); lane 2, untreated RNA substrate (S); lane 3, RNA helicase activity of 1 pmol of purified G2 protein; lane 4, same as lane 3 but omitting ATP and Mn 2+ . (C) RNA helicase activity of mutant fusion proteins. (D) Percent unwinding of dsRNA to single-stranded RNA (ssRNA). Means (columns) and range of values (error bars) from three independent experiments are shown.

Techniques Used: Helicase Assay, Labeling, Activity Assay, Purification, Mutagenesis

3) Product Images from "A protein tyrosine phosphatase-like protein from baculovirus has RNA 5?-triphosphatase and diphosphatase activities"

Article Title: A protein tyrosine phosphatase-like protein from baculovirus has RNA 5?-triphosphatase and diphosphatase activities

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

BVP leaves a 5′-monophosphate end. The RNA triphosphatase assay was performed with [α- 32 P]ATP-terminated trinucleotide RNA (label is indicated by an asterisk). After incubation for 10 min at 30°C, the reaction mixture was analyzed by ( A ) PEI–cellulose TLC or ( B ) 25% polyacrylamide gel electrophoresis. The positions of ADP- and AMP-terminated RNA trinucleotides were determined by using ADP- and AMP-terminated trimers as standards (not shown). In A , RNA substrate was incubated with the following: buffer (lane 1), the indicated amount of BVP (BVP, lanes 2–4), an active site mutant of BVP [BVP(C119S), lanes 5–7], 1 unit of CIP (lane 8), the C. elegans capping enzyme triphosphatase (Cel-1[1–236], lanes 9–11), or the corresponding active site mutant [Cel-1[1–236](C124S), lanes 12–14]. Reactions in lanes 1–5 of B were identical to those in lanes 1–4 and 8 of A .
Figure Legend Snippet: BVP leaves a 5′-monophosphate end. The RNA triphosphatase assay was performed with [α- 32 P]ATP-terminated trinucleotide RNA (label is indicated by an asterisk). After incubation for 10 min at 30°C, the reaction mixture was analyzed by ( A ) PEI–cellulose TLC or ( B ) 25% polyacrylamide gel electrophoresis. The positions of ADP- and AMP-terminated RNA trinucleotides were determined by using ADP- and AMP-terminated trimers as standards (not shown). In A , RNA substrate was incubated with the following: buffer (lane 1), the indicated amount of BVP (BVP, lanes 2–4), an active site mutant of BVP [BVP(C119S), lanes 5–7], 1 unit of CIP (lane 8), the C. elegans capping enzyme triphosphatase (Cel-1[1–236], lanes 9–11), or the corresponding active site mutant [Cel-1[1–236](C124S), lanes 12–14]. Reactions in lanes 1–5 of B were identical to those in lanes 1–4 and 8 of A .

Techniques Used: Incubation, Thin Layer Chromatography, Polyacrylamide Gel Electrophoresis, Mutagenesis

4) Product Images from "Sensing Infection by Adenovirus: Toll-Like Receptor-Independent Viral DNA Recognition Signals Activation of the Interferon Regulatory Factor 3 Master Regulator ▿"

Article Title: Sensing Infection by Adenovirus: Toll-Like Receptor-Independent Viral DNA Recognition Signals Activation of the Interferon Regulatory Factor 3 Master Regulator ▿

Journal: Journal of Virology

doi: 10.1128/JVI.02685-06

Biochemical analysis of signaling responses to AdV, MyD88, and MyD88/TRIF agonists. (A) Western blot analysis of signal transduction using the indicated antibodies. At each indicated time point, whole-cell lysates from mock-treated macrophages were compared to lysates from macrophages stimulated with either 25,000 AdV particles/cell, 10 ng/ml LPS, or 2 μM CpG DNA. Data are representative of three independent experiments. (B) NF-κB gel shift assay. Nuclear extracts from macrophages stimulated for the indicated times were used in gel shift assay with an [α- 32 P]ATP-labeled probe containing a consensus NF-κB-binding sequence.
Figure Legend Snippet: Biochemical analysis of signaling responses to AdV, MyD88, and MyD88/TRIF agonists. (A) Western blot analysis of signal transduction using the indicated antibodies. At each indicated time point, whole-cell lysates from mock-treated macrophages were compared to lysates from macrophages stimulated with either 25,000 AdV particles/cell, 10 ng/ml LPS, or 2 μM CpG DNA. Data are representative of three independent experiments. (B) NF-κB gel shift assay. Nuclear extracts from macrophages stimulated for the indicated times were used in gel shift assay with an [α- 32 P]ATP-labeled probe containing a consensus NF-κB-binding sequence.

Techniques Used: Western Blot, Transduction, Electrophoretic Mobility Shift Assay, Labeling, Binding Assay, Sequencing

Related Articles

Transduction:

Article Title: A dileucine motif in the C terminus of the ?2-adrenergic receptor is involved in receptor internalization
Article Snippet: Adenylyl cyclase activity was determined in freshly prepared membranes (70 μg of protein) in 100 μl of 50 mM Tris⋅HCl, pH 7.4/1 mM EDTA/100 μM cAMP/50 μM GTP/5 mM creatine phosphate/creatine kinase (0.4 mg/ml)/BSA (1 mg/ml)/100 μM [α-32 P]ATP (0.2 μCi per tube; 1 Ci = 37 GBq; NEN/DuPont)/10−9 –10−4 M (−)-isoproterenol. .. Signaling efficiencies of different receptor mutants were determined by simultaneous curve fitting and calculation of the “transducer ratio” τ ( ) by using the algorithm: E = E 0 + E max (τ A )/[( K A + A ) + τ A ], with E max denoting the maximum possible effect (which was shared between all curves) and K A denoting the agonist dissociation constant. τ describes the signal transduction efficacy of the system and is estimated individually for each curve ( ).

Centrifugation:

Article Title: The Lumenal Domain of Sec63p Stimulates the ATPase Activity of BiP and Mediates BiP Recruitment to the Translocon in Saccharomyces cerevisiae
Article Snippet: Unbound proteins were collected by centrifugation at 3000 g (2 min) at 4°C followed by two 50-μl washes with binding buffer. .. To determine the nucleotide-bound state of BiP associated with 63Jp, we performed standard binding assays adding BiP to the 63Jp or GST glutathione agarose affinity matrices described above with 50 μM ATP and 5 μCi [α-32 P]ATP ( DuPont /NEN).

Amplification:

Article Title: Archaeal-type lysyl-tRNA synthetase in the Lyme disease spirochete Borrelia burgdorferi
Article Snippet: .. Flagellin ( ) and lysS (BBKRS1-BBKRS2) probe fragments were generated by PCR amplification and radiolabeled with [α-32 P]ATP (DuPont) by random priming (Life Technologies). ..

Synthesized:

Article Title: Molecular Characterization and Chromosomal Localization of a Third α-Class Hypoxia Inducible Factor Subunit, HIF3α
Article Snippet: The core HRE element was generated by end-labeling 50 ng of oligonucleotide OF414 with [α-32 P]ATP (3,000 Ci/mmol, DuPont NEN) and then annealing with 10-fold excess of unlabeled complementary oligonucleotide, OL415 ( ). .. For expression of the bHLH-PAS proteins, mHIF3α (PL1025) and hARNT (PL87) were synthesized in a reticulocyte lysate in the presence of [35 S]methionine ( ).

Incubation:

Article Title: The Lumenal Domain of Sec63p Stimulates the ATPase Activity of BiP and Mediates BiP Recruitment to the Translocon in Saccharomyces cerevisiae
Article Snippet: To determine the nucleotide-bound state of BiP associated with 63Jp, we performed standard binding assays adding BiP to the 63Jp or GST glutathione agarose affinity matrices described above with 50 μM ATP and 5 μCi [α-32 P]ATP ( DuPont /NEN). .. After a 75-min incubation at 4°C, the radioactivity was observed in the unbound and wash fractions by spotting 1 μl of the bead supernatant to polyethyleneimine cellulose thin layer plates.

Article Title: Sensing Infection by Adenovirus: Toll-Like Receptor-Independent Viral DNA Recognition Signals Activation of the Interferon Regulatory Factor 3 Master Regulator ▿
Article Snippet: Nuclei were spun down, resuspended in 50 μl of hypertonic buffer (10 mM HEPES [pH 7.9], 400 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA), and incubated on ice for 30 min. .. Probe labeling was carried out according to the manufacturer's instructions, using [α-32 P]ATP (3,000 Ci/mmol, 10 mCi/ml; DuPont NEN Research Products).

Article Title: Mutagenesis of the Dengue Virus Type 2 NS3 Protein within and outside Helicase Motifs: Effects on Enzyme Activity and Virus Replication
Article Snippet: Briefly, the final volume of the standard assay used to test mutated proteins was 10 μl, containing 50 mM Tris-HCl (pH 8.0), 10 mM NaCl, 2.5 mM MgCl2 , 1 μCi of [α-32 P]ATP (800 Ci/mmol; DuPont) and 0.4 pmol of protein sample. .. Reaction mixes were incubated for 1 h at 24°C, and the reactions were terminated by the addition of EDTA to a final concentration of 20 mM.

Article Title: A protein tyrosine phosphatase-like protein from baculovirus has RNA 5?-triphosphatase and diphosphatase activities
Article Snippet: The standard reaction (100 μl) contained 40 mM Tris⋅HCl (pH 7.5), 10 mM MgCl2 , 10 mM DTT, 50 μg/ml BSA, 50 mM potassium glutamate, 2 mM dTTP, 2 mM CTP, 0.3 mM [γ-32 P]- or [α-32 P]ATP (500–1,000 cpm/pmol) (NEN/DuPont), 1 nM DNA template [5′-CCCCCGGTC(T)25 -3′], and 1 μM (hexamer) T7 primase. .. The reaction was incubated at 37°C for 4 h, and RNA was extracted with phenol-chloroform (1:1).

Article Title: Intragenic suppressors of Hsp70 mutants: Interplay between the ATPase- and peptide-binding domains
Article Snippet: .. For complex formation, DnaK (16 μg) was incubated with 100 μCi (1 Ci = 37 GBq) of [α-32 P]ATP (DuPont NEG-003H, 3,000 Ci/mmol) in buffer A [25 mM Hepes⋅KOH, pH 7.5/100 mM KCl/11 mM Mg(OAc)2 ] containing 100 nM ATP for 15 minutes at 30°C. .. Complex was separated from free ATP on a Nick column (Amersham Pharmacia), adjusted to 10% glycerol, and frozen at −70°C.

Activity Assay:

Article Title: A dileucine motif in the C terminus of the ?2-adrenergic receptor is involved in receptor internalization
Article Snippet: .. Adenylyl cyclase activity was determined in freshly prepared membranes (70 μg of protein) in 100 μl of 50 mM Tris⋅HCl, pH 7.4/1 mM EDTA/100 μM cAMP/50 μM GTP/5 mM creatine phosphate/creatine kinase (0.4 mg/ml)/BSA (1 mg/ml)/100 μM [α-32 P]ATP (0.2 μCi per tube; 1 Ci = 37 GBq; NEN/DuPont)/10−9 –10−4 M (−)-isoproterenol. ..

Expressing:

Article Title: Molecular Characterization and Chromosomal Localization of a Third α-Class Hypoxia Inducible Factor Subunit, HIF3α
Article Snippet: The core HRE element was generated by end-labeling 50 ng of oligonucleotide OF414 with [α-32 P]ATP (3,000 Ci/mmol, DuPont NEN) and then annealing with 10-fold excess of unlabeled complementary oligonucleotide, OL415 ( ). .. For expression of the bHLH-PAS proteins, mHIF3α (PL1025) and hARNT (PL87) were synthesized in a reticulocyte lysate in the presence of [35 S]methionine ( ).

Western Blot:

Article Title: Sensing Infection by Adenovirus: Toll-Like Receptor-Independent Viral DNA Recognition Signals Activation of the Interferon Regulatory Factor 3 Master Regulator ▿
Article Snippet: Both buffers contained the same cocktail of protease and phosphatase inhibitors as described for Western blots. .. Probe labeling was carried out according to the manufacturer's instructions, using [α-32 P]ATP (3,000 Ci/mmol, 10 mCi/ml; DuPont NEN Research Products).

Hybridization:

Article Title: Archaeal-type lysyl-tRNA synthetase in the Lyme disease spirochete Borrelia burgdorferi
Article Snippet: Prehybridization and hybridization were at 55°C in 1% (wt/vol) BSA/7% (wt/vol) SDS/0.5 M sodium phosphate, pH 7.0/1 mM EDTA in rotating bottles in a hybridization oven (Bellco). .. Flagellin ( ) and lysS (BBKRS1-BBKRS2) probe fragments were generated by PCR amplification and radiolabeled with [α-32 P]ATP (DuPont) by random priming (Life Technologies).

Countercurrent Chromatography:

Article Title: Sensing Infection by Adenovirus: Toll-Like Receptor-Independent Viral DNA Recognition Signals Activation of the Interferon Regulatory Factor 3 Master Regulator ▿
Article Snippet: The sequence of the double-stranded consensus NF-κB-binding oligonucleotide was 5′-AGT TGA GGG GAC TTT CCC AGG C-3′. .. Probe labeling was carried out according to the manufacturer's instructions, using [α-32 P]ATP (3,000 Ci/mmol, 10 mCi/ml; DuPont NEN Research Products).

Chromatography:

Article Title: The Lumenal Domain of Sec63p Stimulates the ATPase Activity of BiP and Mediates BiP Recruitment to the Translocon in Saccharomyces cerevisiae
Article Snippet: To determine the nucleotide-bound state of BiP associated with 63Jp, we performed standard binding assays adding BiP to the 63Jp or GST glutathione agarose affinity matrices described above with 50 μM ATP and 5 μCi [α-32 P]ATP ( DuPont /NEN). .. After chromatography, we determined the nucleotide present in the supernatants in relation to the mobility of nonradioactive ADP and ATP.

Article Title: Mutagenesis of the Dengue Virus Type 2 NS3 Protein within and outside Helicase Motifs: Effects on Enzyme Activity and Virus Replication
Article Snippet: Briefly, the final volume of the standard assay used to test mutated proteins was 10 μl, containing 50 mM Tris-HCl (pH 8.0), 10 mM NaCl, 2.5 mM MgCl2 , 1 μCi of [α-32 P]ATP (800 Ci/mmol; DuPont) and 0.4 pmol of protein sample. .. A 0.5-μl sample of the reaction mixture was spotted onto plastic-backed polyethyleneimine-cellulose sheets, and 32 P-labeled ATP and ADP were separated by ascending chromatography in 0.375 M potassium phosphate (pH 3.5).

Ligation:

Article Title: Evidence for a linear search in bimolecular 3? splice site AG selection
Article Snippet: Tandem repeats containing full-length substrates (RNAs F–K) initially were produced by splinted ligation ( ) of RNA 5A to the corresponding 3′ substrate (RNAs 3F–3K). .. All labeled RNAs were transcribed with T7 RNA polymerase (Stratagene) by using either [α-32 P]UTP or [α-32 P]ATP (DuPont/NEN) under standard conditions ( ).

Northern Blot:

Article Title: Archaeal-type lysyl-tRNA synthetase in the Lyme disease spirochete Borrelia burgdorferi
Article Snippet: Paragraph title: Northern Blot Analysis. ... Flagellin ( ) and lysS (BBKRS1-BBKRS2) probe fragments were generated by PCR amplification and radiolabeled with [α-32 P]ATP (DuPont) by random priming (Life Technologies).

Generated:

Article Title: Evidence for a linear search in bimolecular 3? splice site AG selection
Article Snippet: All 3′ substrates contained the AdMLas exon, and templates for their transcription were generated by PCR from pAdMLpar ( ). .. All labeled RNAs were transcribed with T7 RNA polymerase (Stratagene) by using either [α-32 P]UTP or [α-32 P]ATP (DuPont/NEN) under standard conditions ( ).

Article Title: Molecular Characterization and Chromosomal Localization of a Third α-Class Hypoxia Inducible Factor Subunit, HIF3α
Article Snippet: .. The core HRE element was generated by end-labeling 50 ng of oligonucleotide OF414 with [α-32 P]ATP (3,000 Ci/mmol, DuPont NEN) and then annealing with 10-fold excess of unlabeled complementary oligonucleotide, OL415 ( ). .. Unlabeled oligonucleotides containing either wild-type HRE sequence (TACGTG) or mutant HRE sequences, AACGTG (OF396/397) or GACGTG (OL398/399), were used in competition experiments to demonstrate specificity.

Article Title: Archaeal-type lysyl-tRNA synthetase in the Lyme disease spirochete Borrelia burgdorferi
Article Snippet: .. Flagellin ( ) and lysS (BBKRS1-BBKRS2) probe fragments were generated by PCR amplification and radiolabeled with [α-32 P]ATP (DuPont) by random priming (Life Technologies). ..

other:

Article Title: ATPase-Defective Derivatives of Escherichia coli DnaK That Behave Differently with Respect to ATP-Induced Conformational Change and Peptide Release
Article Snippet: [α-32 P]ATP and [γ-32 P]ATP were purchased from DuPont NEN Life Science Products, Inc. (Boston, Mass.).

Sequencing:

Article Title: Sensing Infection by Adenovirus: Toll-Like Receptor-Independent Viral DNA Recognition Signals Activation of the Interferon Regulatory Factor 3 Master Regulator ▿
Article Snippet: The sequence of the double-stranded consensus NF-κB-binding oligonucleotide was 5′-AGT TGA GGG GAC TTT CCC AGG C-3′. .. Probe labeling was carried out according to the manufacturer's instructions, using [α-32 P]ATP (3,000 Ci/mmol, 10 mCi/ml; DuPont NEN Research Products).

Article Title: A protein tyrosine phosphatase-like protein from baculovirus has RNA 5?-triphosphatase and diphosphatase activities
Article Snippet: This enzyme uses a DNA template to make short RNA primers (2–10 nt) that begin with the sequence pppApC ( ). .. The standard reaction (100 μl) contained 40 mM Tris⋅HCl (pH 7.5), 10 mM MgCl2 , 10 mM DTT, 50 μg/ml BSA, 50 mM potassium glutamate, 2 mM dTTP, 2 mM CTP, 0.3 mM [γ-32 P]- or [α-32 P]ATP (500–1,000 cpm/pmol) (NEN/DuPont), 1 nM DNA template [5′-CCCCCGGTC(T)25 -3′], and 1 μM (hexamer) T7 primase.

Article Title: Molecular Characterization and Chromosomal Localization of a Third α-Class Hypoxia Inducible Factor Subunit, HIF3α
Article Snippet: The core HRE element was generated by end-labeling 50 ng of oligonucleotide OF414 with [α-32 P]ATP (3,000 Ci/mmol, DuPont NEN) and then annealing with 10-fold excess of unlabeled complementary oligonucleotide, OL415 ( ). .. Unlabeled oligonucleotides containing either wild-type HRE sequence (TACGTG) or mutant HRE sequences, AACGTG (OF396/397) or GACGTG (OL398/399), were used in competition experiments to demonstrate specificity.

Binding Assay:

Article Title: The Lumenal Domain of Sec63p Stimulates the ATPase Activity of BiP and Mediates BiP Recruitment to the Translocon in Saccharomyces cerevisiae
Article Snippet: .. To determine the nucleotide-bound state of BiP associated with 63Jp, we performed standard binding assays adding BiP to the 63Jp or GST glutathione agarose affinity matrices described above with 50 μM ATP and 5 μCi [α-32 P]ATP ( DuPont /NEN). .. After a 75-min incubation at 4°C, the radioactivity was observed in the unbound and wash fractions by spotting 1 μl of the bead supernatant to polyethyleneimine cellulose thin layer plates.

DC Protein Assay:

Article Title: Sensing Infection by Adenovirus: Toll-Like Receptor-Independent Viral DNA Recognition Signals Activation of the Interferon Regulatory Factor 3 Master Regulator ▿
Article Snippet: The protein content in the extract was measured with the DC protein assay kit (Bio-Rad Laboratories). .. Probe labeling was carried out according to the manufacturer's instructions, using [α-32 P]ATP (3,000 Ci/mmol, 10 mCi/ml; DuPont NEN Research Products).

Radioactivity:

Article Title: The Lumenal Domain of Sec63p Stimulates the ATPase Activity of BiP and Mediates BiP Recruitment to the Translocon in Saccharomyces cerevisiae
Article Snippet: To determine the nucleotide-bound state of BiP associated with 63Jp, we performed standard binding assays adding BiP to the 63Jp or GST glutathione agarose affinity matrices described above with 50 μM ATP and 5 μCi [α-32 P]ATP ( DuPont /NEN). .. After a 75-min incubation at 4°C, the radioactivity was observed in the unbound and wash fractions by spotting 1 μl of the bead supernatant to polyethyleneimine cellulose thin layer plates.

Article Title: A protein tyrosine phosphatase-like protein from baculovirus has RNA 5?-triphosphatase and diphosphatase activities
Article Snippet: By using either [α-32 P]- or [γ-32 P]ATP, we could produce very short 5′-end labeled RNAs with high specific radioactivity. .. The standard reaction (100 μl) contained 40 mM Tris⋅HCl (pH 7.5), 10 mM MgCl2 , 10 mM DTT, 50 μg/ml BSA, 50 mM potassium glutamate, 2 mM dTTP, 2 mM CTP, 0.3 mM [γ-32 P]- or [α-32 P]ATP (500–1,000 cpm/pmol) (NEN/DuPont), 1 nM DNA template [5′-CCCCCGGTC(T)25 -3′], and 1 μM (hexamer) T7 primase.

Article Title: Molecular Characterization and Chromosomal Localization of a Third α-Class Hypoxia Inducible Factor Subunit, HIF3α
Article Snippet: The core HRE element was generated by end-labeling 50 ng of oligonucleotide OF414 with [α-32 P]ATP (3,000 Ci/mmol, DuPont NEN) and then annealing with 10-fold excess of unlabeled complementary oligonucleotide, OL415 ( ). .. The amount of each protein synthesized was calculated by measuring [35 S]methionine radioactivity and estimated to be approximately 1 fmol in each 10 μl gel-shift reaction.

Mutagenesis:

Article Title: Molecular Characterization and Chromosomal Localization of a Third α-Class Hypoxia Inducible Factor Subunit, HIF3α
Article Snippet: The core HRE element was generated by end-labeling 50 ng of oligonucleotide OF414 with [α-32 P]ATP (3,000 Ci/mmol, DuPont NEN) and then annealing with 10-fold excess of unlabeled complementary oligonucleotide, OL415 ( ). .. Unlabeled oligonucleotides containing either wild-type HRE sequence (TACGTG) or mutant HRE sequences, AACGTG (OF396/397) or GACGTG (OL398/399), were used in competition experiments to demonstrate specificity.

Isolation:

Article Title: Organization of G Proteins and Adenylyl Cyclase at the Plasma Membrane
Article Snippet: [α-32 P]ATP was purchased from DuPont NEN (Boston, MA). .. Five micrograms of S49 cell plasma membranes isolated from the sucrose gradients were assayed as a control for several experiments.

Article Title: Archaeal-type lysyl-tRNA synthetase in the Lyme disease spirochete Borrelia burgdorferi
Article Snippet: Total borrelial RNA was extracted from logarithmic-phase cultures by using the Ultraspec RNA isolation system (Biotecx, Houston, TX) according to the manufacturer’s instructions. .. Flagellin ( ) and lysS (BBKRS1-BBKRS2) probe fragments were generated by PCR amplification and radiolabeled with [α-32 P]ATP (DuPont) by random priming (Life Technologies).

Labeling:

Article Title: Sensing Infection by Adenovirus: Toll-Like Receptor-Independent Viral DNA Recognition Signals Activation of the Interferon Regulatory Factor 3 Master Regulator ▿
Article Snippet: .. Probe labeling was carried out according to the manufacturer's instructions, using [α-32 P]ATP (3,000 Ci/mmol, 10 mCi/ml; DuPont NEN Research Products). .. Reaction mixtures were separated on 6% DNA retardation gels (Invitrogen) at 150 V for 30 min at 4°C.

Article Title: Evidence for a linear search in bimolecular 3? splice site AG selection
Article Snippet: .. All labeled RNAs were transcribed with T7 RNA polymerase (Stratagene) by using either [α-32 P]UTP or [α-32 P]ATP (DuPont/NEN) under standard conditions ( ). .. All 5′ and full-length substrates were capped with G(5′)ppp(5′)G (New England Biolabs); 3′ substrates were initiated with GMP.

Article Title: A protein tyrosine phosphatase-like protein from baculovirus has RNA 5?-triphosphatase and diphosphatase activities
Article Snippet: By using either [α-32 P]- or [γ-32 P]ATP, we could produce very short 5′-end labeled RNAs with high specific radioactivity. .. The standard reaction (100 μl) contained 40 mM Tris⋅HCl (pH 7.5), 10 mM MgCl2 , 10 mM DTT, 50 μg/ml BSA, 50 mM potassium glutamate, 2 mM dTTP, 2 mM CTP, 0.3 mM [γ-32 P]- or [α-32 P]ATP (500–1,000 cpm/pmol) (NEN/DuPont), 1 nM DNA template [5′-CCCCCGGTC(T)25 -3′], and 1 μM (hexamer) T7 primase.

Purification:

Article Title: The Lumenal Domain of Sec63p Stimulates the ATPase Activity of BiP and Mediates BiP Recruitment to the Translocon in Saccharomyces cerevisiae
Article Snippet: The amount of BiP associated with 63Jp, 63-1Jp, or GST on the glutathione beads was determined by a comparison with a standard curve of purified BiP using the software Imagequant v1.1 (Molecular Dynamics). .. To determine the nucleotide-bound state of BiP associated with 63Jp, we performed standard binding assays adding BiP to the 63Jp or GST glutathione agarose affinity matrices described above with 50 μM ATP and 5 μCi [α-32 P]ATP ( DuPont /NEN).

Article Title: Acyl Coenzyme A Synthetase from Pseudomonas fragi Catalyzes the Synthesis of Adenosine 5?-Polyphosphates and Dinucleoside Polyphosphates †
Article Snippet: [2,8-3 H]ATP (45 Ci/mmol) was from Amersham Life Sciences, and [α-32 P]ATP was from DuPont NEN. .. Asymmetrical dinucleoside tetraphosphatase was purified from rat liver as described by Sillero et al. ( ).

Polymerase Chain Reaction:

Article Title: Evidence for a linear search in bimolecular 3? splice site AG selection
Article Snippet: All 3′ substrates contained the AdMLas exon, and templates for their transcription were generated by PCR from pAdMLpar ( ). .. All labeled RNAs were transcribed with T7 RNA polymerase (Stratagene) by using either [α-32 P]UTP or [α-32 P]ATP (DuPont/NEN) under standard conditions ( ).

Article Title: Archaeal-type lysyl-tRNA synthetase in the Lyme disease spirochete Borrelia burgdorferi
Article Snippet: .. Flagellin ( ) and lysS (BBKRS1-BBKRS2) probe fragments were generated by PCR amplification and radiolabeled with [α-32 P]ATP (DuPont) by random priming (Life Technologies). ..

Electrophoretic Mobility Shift Assay:

Article Title: Sensing Infection by Adenovirus: Toll-Like Receptor-Independent Viral DNA Recognition Signals Activation of the Interferon Regulatory Factor 3 Master Regulator ▿
Article Snippet: Paragraph title: Electrophoretic mobility shift assay (EMSA). ... Probe labeling was carried out according to the manufacturer's instructions, using [α-32 P]ATP (3,000 Ci/mmol, 10 mCi/ml; DuPont NEN Research Products).

Article Title: Molecular Characterization and Chromosomal Localization of a Third α-Class Hypoxia Inducible Factor Subunit, HIF3α
Article Snippet: Paragraph title: Gel-Shift Assay ... The core HRE element was generated by end-labeling 50 ng of oligonucleotide OF414 with [α-32 P]ATP (3,000 Ci/mmol, DuPont NEN) and then annealing with 10-fold excess of unlabeled complementary oligonucleotide, OL415 ( ).

ATPase Assay:

Article Title: Mutagenesis of the Dengue Virus Type 2 NS3 Protein within and outside Helicase Motifs: Effects on Enzyme Activity and Virus Replication
Article Snippet: Paragraph title: ATPase assay. ... Briefly, the final volume of the standard assay used to test mutated proteins was 10 μl, containing 50 mM Tris-HCl (pH 8.0), 10 mM NaCl, 2.5 mM MgCl2 , 1 μCi of [α-32 P]ATP (800 Ci/mmol; DuPont) and 0.4 pmol of protein sample.

SDS Page:

Article Title: The Lumenal Domain of Sec63p Stimulates the ATPase Activity of BiP and Mediates BiP Recruitment to the Translocon in Saccharomyces cerevisiae
Article Snippet: Proteins remaining on the glutathione beads were solubilized in Laemmli sample buffer, separated by SDS-PAGE, and visualized by staining with Coomassie brilliant blue R-250 (BioRad Labs, Hercules, CA). .. To determine the nucleotide-bound state of BiP associated with 63Jp, we performed standard binding assays adding BiP to the 63Jp or GST glutathione agarose affinity matrices described above with 50 μM ATP and 5 μCi [α-32 P]ATP ( DuPont /NEN).

Plasmid Preparation:

Article Title: Evidence for a linear search in bimolecular 3? splice site AG selection
Article Snippet: All full-length RNAs subsequently were transcribed from the appropriate plasmid. .. All labeled RNAs were transcribed with T7 RNA polymerase (Stratagene) by using either [α-32 P]UTP or [α-32 P]ATP (DuPont/NEN) under standard conditions ( ).

Software:

Article Title: The Lumenal Domain of Sec63p Stimulates the ATPase Activity of BiP and Mediates BiP Recruitment to the Translocon in Saccharomyces cerevisiae
Article Snippet: The amount of BiP associated with 63Jp, 63-1Jp, or GST on the glutathione beads was determined by a comparison with a standard curve of purified BiP using the software Imagequant v1.1 (Molecular Dynamics). .. To determine the nucleotide-bound state of BiP associated with 63Jp, we performed standard binding assays adding BiP to the 63Jp or GST glutathione agarose affinity matrices described above with 50 μM ATP and 5 μCi [α-32 P]ATP ( DuPont /NEN).

Agarose Gel Electrophoresis:

Article Title: Archaeal-type lysyl-tRNA synthetase in the Lyme disease spirochete Borrelia burgdorferi
Article Snippet: RNA was denatured with glyoxal and dimethyl sulfoxide and electrophoresed in a 1% (wt/vol) agarose gel in 10 mM sodium phosphate buffer, pH 7.0. .. Flagellin ( ) and lysS (BBKRS1-BBKRS2) probe fragments were generated by PCR amplification and radiolabeled with [α-32 P]ATP (DuPont) by random priming (Life Technologies).

Produced:

Article Title: Evidence for a linear search in bimolecular 3? splice site AG selection
Article Snippet: Tandem repeats containing full-length substrates (RNAs F–K) initially were produced by splinted ligation ( ) of RNA 5A to the corresponding 3′ substrate (RNAs 3F–3K). .. All labeled RNAs were transcribed with T7 RNA polymerase (Stratagene) by using either [α-32 P]UTP or [α-32 P]ATP (DuPont/NEN) under standard conditions ( ).

Concentration Assay:

Article Title: Mutagenesis of the Dengue Virus Type 2 NS3 Protein within and outside Helicase Motifs: Effects on Enzyme Activity and Virus Replication
Article Snippet: Briefly, the final volume of the standard assay used to test mutated proteins was 10 μl, containing 50 mM Tris-HCl (pH 8.0), 10 mM NaCl, 2.5 mM MgCl2 , 1 μCi of [α-32 P]ATP (800 Ci/mmol; DuPont) and 0.4 pmol of protein sample. .. Reaction mixes were incubated for 1 h at 24°C, and the reactions were terminated by the addition of EDTA to a final concentration of 20 mM.

Article Title: A dileucine motif in the C terminus of the ?2-adrenergic receptor is involved in receptor internalization
Article Snippet: Adenylyl cyclase activity was determined in freshly prepared membranes (70 μg of protein) in 100 μl of 50 mM Tris⋅HCl, pH 7.4/1 mM EDTA/100 μM cAMP/50 μM GTP/5 mM creatine phosphate/creatine kinase (0.4 mg/ml)/BSA (1 mg/ml)/100 μM [α-32 P]ATP (0.2 μCi per tube; 1 Ci = 37 GBq; NEN/DuPont)/10−9 –10−4 M (−)-isoproterenol. .. Adenylyl cyclase activity was normalized to the protein content and concentration–response curves were analyzed ( ) by curve fitting to the equation: E = E 0 + E max A /(EC50 + A ) with E denoting effect, E 0 denoting basal activity, E max denoting maximum effect, and A denoting agonist concentration.

End Labeling:

Article Title: Molecular Characterization and Chromosomal Localization of a Third α-Class Hypoxia Inducible Factor Subunit, HIF3α
Article Snippet: .. The core HRE element was generated by end-labeling 50 ng of oligonucleotide OF414 with [α-32 P]ATP (3,000 Ci/mmol, DuPont NEN) and then annealing with 10-fold excess of unlabeled complementary oligonucleotide, OL415 ( ). .. Unlabeled oligonucleotides containing either wild-type HRE sequence (TACGTG) or mutant HRE sequences, AACGTG (OF396/397) or GACGTG (OL398/399), were used in competition experiments to demonstrate specificity.

Staining:

Article Title: The Lumenal Domain of Sec63p Stimulates the ATPase Activity of BiP and Mediates BiP Recruitment to the Translocon in Saccharomyces cerevisiae
Article Snippet: Proteins remaining on the glutathione beads were solubilized in Laemmli sample buffer, separated by SDS-PAGE, and visualized by staining with Coomassie brilliant blue R-250 (BioRad Labs, Hercules, CA). .. To determine the nucleotide-bound state of BiP associated with 63Jp, we performed standard binding assays adding BiP to the 63Jp or GST glutathione agarose affinity matrices described above with 50 μM ATP and 5 μCi [α-32 P]ATP ( DuPont /NEN).

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    DuPont de Nemours α 32 p atp
    Effect of CoA and organic acids on the synthesis of p 4 A catalyzed by acyl-CoA synthetase. (A) Effect of CoA on the synthesis of p 4 A. The reaction mixture (50 μl) contained 50 mM MES-KOH (pH 6.3), 0.1 mM dithiothreitol, 11 mM MgCl 2 , 1 mM [2,8- 3 <t>H]ATP,</t> 10 mM P 3 , and the indicated concentrations of CoA and acyl-CoA synthetase (8.3 μg of protein). (B) Effect of organic acids on the inhibitory effect of CoA on the synthesis of p 4 A. Reaction mixtures (28 μl) containing 72 mM MES-KOH (pH 6.3), 0.14 mM dithiothreitol, 7.9 mM MgCl 2 , 0.76 mM [α- 32 P]ATP, 7.2 mM P 3 , 0.14 mM CoA, 0.7 μl of desalted inorganic pyrophosphatase, and acyl-CoA synthetase (5.2 μg of protein) were preincubated at 30°C for 20 min. Thereafter, they were supplemented with 12 μl of the following solutions: water (lane b), 1% Triton X-100–5% ethanol (solution C; lane c), 1 mM solutions of palmitic acid in solution C (lane d), octanoic acid in water (lane e), or other possible effectors (acetic acid, lysine, methionine, phenylalanine, tryptophan, and luciferin; lanes f to k, respectively). One hour after the addition of organic acids, aliquots of the reaction were analyzed by TLC. Control without acyl-CoA synthetase is shown in lane a.
    α 32 P Atp, supplied by DuPont de Nemours, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of CoA and organic acids on the synthesis of p 4 A catalyzed by acyl-CoA synthetase. (A) Effect of CoA on the synthesis of p 4 A. The reaction mixture (50 μl) contained 50 mM MES-KOH (pH 6.3), 0.1 mM dithiothreitol, 11 mM MgCl 2 , 1 mM [2,8- 3 H]ATP, 10 mM P 3 , and the indicated concentrations of CoA and acyl-CoA synthetase (8.3 μg of protein). (B) Effect of organic acids on the inhibitory effect of CoA on the synthesis of p 4 A. Reaction mixtures (28 μl) containing 72 mM MES-KOH (pH 6.3), 0.14 mM dithiothreitol, 7.9 mM MgCl 2 , 0.76 mM [α- 32 P]ATP, 7.2 mM P 3 , 0.14 mM CoA, 0.7 μl of desalted inorganic pyrophosphatase, and acyl-CoA synthetase (5.2 μg of protein) were preincubated at 30°C for 20 min. Thereafter, they were supplemented with 12 μl of the following solutions: water (lane b), 1% Triton X-100–5% ethanol (solution C; lane c), 1 mM solutions of palmitic acid in solution C (lane d), octanoic acid in water (lane e), or other possible effectors (acetic acid, lysine, methionine, phenylalanine, tryptophan, and luciferin; lanes f to k, respectively). One hour after the addition of organic acids, aliquots of the reaction were analyzed by TLC. Control without acyl-CoA synthetase is shown in lane a.

    Journal: Journal of Bacteriology

    Article Title: Acyl Coenzyme A Synthetase from Pseudomonas fragi Catalyzes the Synthesis of Adenosine 5?-Polyphosphates and Dinucleoside Polyphosphates †

    doi:

    Figure Lengend Snippet: Effect of CoA and organic acids on the synthesis of p 4 A catalyzed by acyl-CoA synthetase. (A) Effect of CoA on the synthesis of p 4 A. The reaction mixture (50 μl) contained 50 mM MES-KOH (pH 6.3), 0.1 mM dithiothreitol, 11 mM MgCl 2 , 1 mM [2,8- 3 H]ATP, 10 mM P 3 , and the indicated concentrations of CoA and acyl-CoA synthetase (8.3 μg of protein). (B) Effect of organic acids on the inhibitory effect of CoA on the synthesis of p 4 A. Reaction mixtures (28 μl) containing 72 mM MES-KOH (pH 6.3), 0.14 mM dithiothreitol, 7.9 mM MgCl 2 , 0.76 mM [α- 32 P]ATP, 7.2 mM P 3 , 0.14 mM CoA, 0.7 μl of desalted inorganic pyrophosphatase, and acyl-CoA synthetase (5.2 μg of protein) were preincubated at 30°C for 20 min. Thereafter, they were supplemented with 12 μl of the following solutions: water (lane b), 1% Triton X-100–5% ethanol (solution C; lane c), 1 mM solutions of palmitic acid in solution C (lane d), octanoic acid in water (lane e), or other possible effectors (acetic acid, lysine, methionine, phenylalanine, tryptophan, and luciferin; lanes f to k, respectively). One hour after the addition of organic acids, aliquots of the reaction were analyzed by TLC. Control without acyl-CoA synthetase is shown in lane a.

    Article Snippet: [2,8-3 H]ATP (45 Ci/mmol) was from Amersham Life Sciences, and [α-32 P]ATP was from DuPont NEN.

    Techniques: Thin Layer Chromatography

    RNA helicase assay of 74%NS3 fusion proteins. (A) Structure of the 3′-tailed dsRNA substrate; the lower strand of RNA was labeled with [α- 32 P]ATP. (B) RNA helicase activity of the parental G2 protein. Lane 1, heated RNA substrate (hS); lane 2, untreated RNA substrate (S); lane 3, RNA helicase activity of 1 pmol of purified G2 protein; lane 4, same as lane 3 but omitting ATP and Mn 2+ . (C) RNA helicase activity of mutant fusion proteins. (D) Percent unwinding of dsRNA to single-stranded RNA (ssRNA). Means (columns) and range of values (error bars) from three independent experiments are shown.

    Journal: Journal of Virology

    Article Title: Mutagenesis of the Dengue Virus Type 2 NS3 Protein within and outside Helicase Motifs: Effects on Enzyme Activity and Virus Replication

    doi: 10.1128/JVI.75.20.9633-9643.2001

    Figure Lengend Snippet: RNA helicase assay of 74%NS3 fusion proteins. (A) Structure of the 3′-tailed dsRNA substrate; the lower strand of RNA was labeled with [α- 32 P]ATP. (B) RNA helicase activity of the parental G2 protein. Lane 1, heated RNA substrate (hS); lane 2, untreated RNA substrate (S); lane 3, RNA helicase activity of 1 pmol of purified G2 protein; lane 4, same as lane 3 but omitting ATP and Mn 2+ . (C) RNA helicase activity of mutant fusion proteins. (D) Percent unwinding of dsRNA to single-stranded RNA (ssRNA). Means (columns) and range of values (error bars) from three independent experiments are shown.

    Article Snippet: Briefly, the final volume of the standard assay used to test mutated proteins was 10 μl, containing 50 mM Tris-HCl (pH 8.0), 10 mM NaCl, 2.5 mM MgCl2 , 1 μCi of [α-32 P]ATP (800 Ci/mmol; DuPont) and 0.4 pmol of protein sample.

    Techniques: Helicase Assay, Labeling, Activity Assay, Purification, Mutagenesis

    BVP leaves a 5′-monophosphate end. The RNA triphosphatase assay was performed with [α- 32 P]ATP-terminated trinucleotide RNA (label is indicated by an asterisk). After incubation for 10 min at 30°C, the reaction mixture was analyzed by ( A ) PEI–cellulose TLC or ( B ) 25% polyacrylamide gel electrophoresis. The positions of ADP- and AMP-terminated RNA trinucleotides were determined by using ADP- and AMP-terminated trimers as standards (not shown). In A , RNA substrate was incubated with the following: buffer (lane 1), the indicated amount of BVP (BVP, lanes 2–4), an active site mutant of BVP [BVP(C119S), lanes 5–7], 1 unit of CIP (lane 8), the C. elegans capping enzyme triphosphatase (Cel-1[1–236], lanes 9–11), or the corresponding active site mutant [Cel-1[1–236](C124S), lanes 12–14]. Reactions in lanes 1–5 of B were identical to those in lanes 1–4 and 8 of A .

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: A protein tyrosine phosphatase-like protein from baculovirus has RNA 5?-triphosphatase and diphosphatase activities

    doi:

    Figure Lengend Snippet: BVP leaves a 5′-monophosphate end. The RNA triphosphatase assay was performed with [α- 32 P]ATP-terminated trinucleotide RNA (label is indicated by an asterisk). After incubation for 10 min at 30°C, the reaction mixture was analyzed by ( A ) PEI–cellulose TLC or ( B ) 25% polyacrylamide gel electrophoresis. The positions of ADP- and AMP-terminated RNA trinucleotides were determined by using ADP- and AMP-terminated trimers as standards (not shown). In A , RNA substrate was incubated with the following: buffer (lane 1), the indicated amount of BVP (BVP, lanes 2–4), an active site mutant of BVP [BVP(C119S), lanes 5–7], 1 unit of CIP (lane 8), the C. elegans capping enzyme triphosphatase (Cel-1[1–236], lanes 9–11), or the corresponding active site mutant [Cel-1[1–236](C124S), lanes 12–14]. Reactions in lanes 1–5 of B were identical to those in lanes 1–4 and 8 of A .

    Article Snippet: The standard reaction (100 μl) contained 40 mM Tris⋅HCl (pH 7.5), 10 mM MgCl2 , 10 mM DTT, 50 μg/ml BSA, 50 mM potassium glutamate, 2 mM dTTP, 2 mM CTP, 0.3 mM [γ-32 P]- or [α-32 P]ATP (500–1,000 cpm/pmol) (NEN/DuPont), 1 nM DNA template [5′-CCCCCGGTC(T)25 -3′], and 1 μM (hexamer) T7 primase.

    Techniques: Incubation, Thin Layer Chromatography, Polyacrylamide Gel Electrophoresis, Mutagenesis

    Biochemical analysis of signaling responses to AdV, MyD88, and MyD88/TRIF agonists. (A) Western blot analysis of signal transduction using the indicated antibodies. At each indicated time point, whole-cell lysates from mock-treated macrophages were compared to lysates from macrophages stimulated with either 25,000 AdV particles/cell, 10 ng/ml LPS, or 2 μM CpG DNA. Data are representative of three independent experiments. (B) NF-κB gel shift assay. Nuclear extracts from macrophages stimulated for the indicated times were used in gel shift assay with an [α- 32 P]ATP-labeled probe containing a consensus NF-κB-binding sequence.

    Journal: Journal of Virology

    Article Title: Sensing Infection by Adenovirus: Toll-Like Receptor-Independent Viral DNA Recognition Signals Activation of the Interferon Regulatory Factor 3 Master Regulator ▿

    doi: 10.1128/JVI.02685-06

    Figure Lengend Snippet: Biochemical analysis of signaling responses to AdV, MyD88, and MyD88/TRIF agonists. (A) Western blot analysis of signal transduction using the indicated antibodies. At each indicated time point, whole-cell lysates from mock-treated macrophages were compared to lysates from macrophages stimulated with either 25,000 AdV particles/cell, 10 ng/ml LPS, or 2 μM CpG DNA. Data are representative of three independent experiments. (B) NF-κB gel shift assay. Nuclear extracts from macrophages stimulated for the indicated times were used in gel shift assay with an [α- 32 P]ATP-labeled probe containing a consensus NF-κB-binding sequence.

    Article Snippet: Probe labeling was carried out according to the manufacturer's instructions, using [α-32 P]ATP (3,000 Ci/mmol, 10 mCi/ml; DuPont NEN Research Products).

    Techniques: Western Blot, Transduction, Electrophoretic Mobility Shift Assay, Labeling, Binding Assay, Sequencing