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  • 99
    New England Biolabs t4 polynucleotide kinase
    T4 Polynucleotide Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 29387 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t4 rna ligase
    ESCRT-II binds directly to RNA through Vps25 in Xenopus egg extracts. A , Western blotting of ESCRT-II IPs or mock IPs (nonspecific rabbit IgG) performed under native (−) or denaturing (+) conditions. Vps22 was not detectable by Western blotting with our ESCRT-II polyclonal antibody. H.C. , heavy chain. B , autoradiograph of a CLIP experiment from Xenopus egg extract under high RNase conditions (0.1 mg/ml). A radioactive band consistent with the molecular mass of Vps25 (denoted by the red asterisk ) is observed, but no bands at the molecular mass of Vps22 or Vps36 are apparent. The expected migrations of the ESCRT-II subunits are indicated to the left of the gel. <t>T4</t> RNA ligase forms a covalent intermediate with pCp (used to radiolabel the RNA fragments) and appears in every lane. The bands above and below the Vps25 band (denoted by black asterisks ) are nonspecific, as they appeared in the IgG control in some replicates of this experiment. C , autoradiograph of a CLIP experiment from Xenopus egg extract performed as described in B , except under denaturing immunoprecipitation conditions. The same polyclonal ESCRT-II antibody was used for A– C , but under denaturing conditions this antibody only immunoprecipitates Vps25.
    T4 Rna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3116 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t4 rna ligase
    ESCRT-II binds directly to RNA through Vps25 in Xenopus egg extracts. A , Western blotting of ESCRT-II IPs or mock IPs (nonspecific rabbit IgG) performed under native (−) or denaturing (+) conditions. Vps22 was not detectable by Western blotting with our ESCRT-II polyclonal antibody. H.C. , heavy chain. B , autoradiograph of a CLIP experiment from Xenopus egg extract under high RNase conditions (0.1 mg/ml). A radioactive band consistent with the molecular mass of Vps25 (denoted by the red asterisk ) is observed, but no bands at the molecular mass of Vps22 or Vps36 are apparent. The expected migrations of the ESCRT-II subunits are indicated to the left of the gel. <t>T4</t> RNA ligase forms a covalent intermediate with pCp (used to radiolabel the RNA fragments) and appears in every lane. The bands above and below the Vps25 band (denoted by black asterisks ) are nonspecific, as they appeared in the IgG control in some replicates of this experiment. C , autoradiograph of a CLIP experiment from Xenopus egg extract performed as described in B , except under denaturing immunoprecipitation conditions. The same polyclonal ESCRT-II antibody was used for A– C , but under denaturing conditions this antibody only immunoprecipitates Vps25.
    T4 Rna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3749 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t4 rna ligase 1 ssrna ligase
    Endoribonucleolytic cleavage with purified H5 protein results in a 3′-OH. A , schematic of potential cleavage products for the TAP-treated (+TAP) or untreated (−TAP) 430-nt ssRNA substrate. I and II , possible scenarios depending on the nature of 3′ ends after cleavage. Ends denoted in boldface type indicate possible results of cleavage. B , schematic of the potential and actual products after treatment of the cleavage reaction with Terminator exonuclease ( Term ). I and II , possible outcomes depending on the nature of 3′ ends after cleavage. Gray line , <t>RNA</t> degraded by Terminator; black line , RNA not degraded by Terminator. C , schematic of the potential and actual products after [5′- 32 <t>P]pCp</t> treatment of unlabeled, purified RNA substrate and products from a cleavage reaction. Black line , RNA labeled with [5′- 32 P]pCp; gray line , RNA not labeled with [5′- 32 P]pCp. Lane 1 , RNA substrate treated with H5 in cleavage assay, purified, and labeled with [5′- 32 P]pCp; lane 2 , RNA substrate treated with buffer in cleavage assay, purified, and labeled with [5′- 32 P]pCp; lane 3 , RNA substrate labeled with [5′- 32 P]pCp.
    T4 Rna Ligase 1 Ssrna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 463 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    PerkinElmer genescreen plus hybridization transfer membrane
    Northern blot analysis of tRNA Cys , tRNA Tyr , and tRNA Ala . RNA samples were prepared as described under “Experimental Procedures.” Eight micrograms of total RNA were subjected to electrophoresis and then transferred to a <t>GeneScreen</t> Plus membrane. Transfer RNAs were detected using a 32 P-labeled 5′-end-specific probe. M , mature RNA substrate.
    Genescreen Plus Hybridization Transfer Membrane, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 95/100, based on 310 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dpni
    Northern blot analysis of tRNA Cys , tRNA Tyr , and tRNA Ala . RNA samples were prepared as described under “Experimental Procedures.” Eight micrograms of total RNA were subjected to electrophoresis and then transferred to a <t>GeneScreen</t> Plus membrane. Transfer RNAs were detected using a 32 P-labeled 5′-end-specific probe. M , mature RNA substrate.
    Dpni, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2375 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t4 rna ligase buffer
    Northern blot analysis of tRNA Cys , tRNA Tyr , and tRNA Ala . RNA samples were prepared as described under “Experimental Procedures.” Eight micrograms of total RNA were subjected to electrophoresis and then transferred to a <t>GeneScreen</t> Plus membrane. Transfer RNAs were detected using a 32 P-labeled 5′-end-specific probe. M , mature RNA substrate.
    T4 Rna Ligase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 462 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Agilent technologies 2100 bioanalyzer microfluidics based platform
    Northern blot analysis of tRNA Cys , tRNA Tyr , and tRNA Ala . RNA samples were prepared as described under “Experimental Procedures.” Eight micrograms of total RNA were subjected to electrophoresis and then transferred to a <t>GeneScreen</t> Plus membrane. Transfer RNAs were detected using a 32 P-labeled 5′-end-specific probe. M , mature RNA substrate.
    2100 Bioanalyzer Microfluidics Based Platform, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 89/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    thermo fisher 32p pcp
    Northern blot analysis of tRNA Cys , tRNA Tyr , and tRNA Ala . RNA samples were prepared as described under “Experimental Procedures.” Eight micrograms of total RNA were subjected to electrophoresis and then transferred to a <t>GeneScreen</t> Plus membrane. Transfer RNAs were detected using a 32 P-labeled 5′-end-specific probe. M , mature RNA substrate.
    32p Pcp, supplied by thermo fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs alkaline phosphatase
    Northern blot analysis of tRNA Cys , tRNA Tyr , and tRNA Ala . RNA samples were prepared as described under “Experimental Procedures.” Eight micrograms of total RNA were subjected to electrophoresis and then transferred to a <t>GeneScreen</t> Plus membrane. Transfer RNAs were detected using a 32 P-labeled 5′-end-specific probe. M , mature RNA substrate.
    Alkaline Phosphatase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2053 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore monoclonal anti pcpe 1 antibody
    Northern blot analysis of tRNA Cys , tRNA Tyr , and tRNA Ala . RNA samples were prepared as described under “Experimental Procedures.” Eight micrograms of total RNA were subjected to electrophoresis and then transferred to a <t>GeneScreen</t> Plus membrane. Transfer RNAs were detected using a 32 P-labeled 5′-end-specific probe. M , mature RNA substrate.
    Monoclonal Anti Pcpe 1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t4 polynucleotide kinase
    Northern blot analysis of tRNA Cys , tRNA Tyr , and tRNA Ala . RNA samples were prepared as described under “Experimental Procedures.” Eight micrograms of total RNA were subjected to electrophoresis and then transferred to a <t>GeneScreen</t> Plus membrane. Transfer RNAs were detected using a 32 P-labeled 5′-end-specific probe. M , mature RNA substrate.
    T4 Polynucleotide Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9937 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega rnasin
    Northern blot analysis of tRNA Cys , tRNA Tyr , and tRNA Ala . RNA samples were prepared as described under “Experimental Procedures.” Eight micrograms of total RNA were subjected to electrophoresis and then transferred to a <t>GeneScreen</t> Plus membrane. Transfer RNAs were detected using a 32 P-labeled 5′-end-specific probe. M , mature RNA substrate.
    Rnasin, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 22066 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore β estradiol
    Effect of PAP, quercetin, and b‐estradiol on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. Cytosols from HK‐2 cells (25 ng/100 μL) were incubated with 50 μM of AL‐I‐NOH in the presence of ssDNA for 30 min at 37°C. Reactions were set in triplicate for each condition, without or with 100 μM one of the following: PAP, quercetin and <t>β‐estradiol.</t> DNA was extracted and five micrograms from each sample was subjected to adduct analysis. Fragment of PAGE and quantification of obtained results are shown. St‐ mixture of standard at 30 fmol each. dG‐ and dA‐AL are upper and lower bands, respectively.
    β Estradiol, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2636 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore bis p nitrophenyl phosphate
    Effect of PAP, quercetin, and b‐estradiol on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. Cytosols from HK‐2 cells (25 ng/100 μL) were incubated with 50 μM of AL‐I‐NOH in the presence of ssDNA for 30 min at 37°C. Reactions were set in triplicate for each condition, without or with 100 μM one of the following: PAP, quercetin and <t>β‐estradiol.</t> DNA was extracted and five micrograms from each sample was subjected to adduct analysis. Fragment of PAGE and quantification of obtained results are shown. St‐ mixture of standard at 30 fmol each. dG‐ and dA‐AL are upper and lower bands, respectively.
    Bis P Nitrophenyl Phosphate, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher polyacrylamide gel
    Effect of PAP, quercetin, and b‐estradiol on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. Cytosols from HK‐2 cells (25 ng/100 μL) were incubated with 50 μM of AL‐I‐NOH in the presence of ssDNA for 30 min at 37°C. Reactions were set in triplicate for each condition, without or with 100 μM one of the following: PAP, quercetin and <t>β‐estradiol.</t> DNA was extracted and five micrograms from each sample was subjected to adduct analysis. Fragment of PAGE and quantification of obtained results are shown. St‐ mixture of standard at 30 fmol each. dG‐ and dA‐AL are upper and lower bands, respectively.
    Polyacrylamide Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6595 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Bio-Rad p 30 size exclusion columns
    Effect of PAP, quercetin, and b‐estradiol on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. Cytosols from HK‐2 cells (25 ng/100 μL) were incubated with 50 μM of AL‐I‐NOH in the presence of ssDNA for 30 min at 37°C. Reactions were set in triplicate for each condition, without or with 100 μM one of the following: PAP, quercetin and <t>β‐estradiol.</t> DNA was extracted and five micrograms from each sample was subjected to adduct analysis. Fragment of PAGE and quantification of obtained results are shown. St‐ mixture of standard at 30 fmol each. dG‐ and dA‐AL are upper and lower bands, respectively.
    P 30 Size Exclusion Columns, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 96/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher calf intestine alkaline phosphatase
    Effect of PAP, quercetin, and b‐estradiol on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. Cytosols from HK‐2 cells (25 ng/100 μL) were incubated with 50 μM of AL‐I‐NOH in the presence of ssDNA for 30 min at 37°C. Reactions were set in triplicate for each condition, without or with 100 μM one of the following: PAP, quercetin and <t>β‐estradiol.</t> DNA was extracted and five micrograms from each sample was subjected to adduct analysis. Fragment of PAGE and quantification of obtained results are shown. St‐ mixture of standard at 30 fmol each. dG‐ and dA‐AL are upper and lower bands, respectively.
    Calf Intestine Alkaline Phosphatase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 555 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher atp
    Effect of PAP, quercetin, and b‐estradiol on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. Cytosols from HK‐2 cells (25 ng/100 μL) were incubated with 50 μM of AL‐I‐NOH in the presence of ssDNA for 30 min at 37°C. Reactions were set in triplicate for each condition, without or with 100 μM one of the following: PAP, quercetin and <t>β‐estradiol.</t> DNA was extracted and five micrograms from each sample was subjected to adduct analysis. Fragment of PAGE and quantification of obtained results are shown. St‐ mixture of standard at 30 fmol each. dG‐ and dA‐AL are upper and lower bands, respectively.
    Atp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2321 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore pentachlorophenol pcp
    Effect of dialysis on 10 kDa MWCO and pentachlorophenol on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. (A) Cytosols from HK‐2 cells (25 ng/100 μL) were incubated with 1 (filled circles), 10 (empty circles), 25 (filled triangles), or 50 μM (empty triangles) AL‐I‐NOH in the presence of ssDNA for 15–60 min at 37°C. DNA was extracted and five micrograms from each sample was subjected to adduct analysis as described in the section Materials and Methods. Results are presented as the dependence of AL‐DNA (dG‐AL and dA‐AL adducts) levels on the reaction time. (B) HK‐2 cytosols were incubated with AL‐I‐NOH and ssDNA in the presence and absence of pentachlorophenol <t>(PCP)</t> for 30 min as indicated. Each reaction with PCP was conducted in duplicate, and reaction without PCP was set in triplicate. A fragment of representative PAGE with DNA adduct analysis for five microgram of DNA is shown. AL‐DNA levels without PCP were 58 ± 4 adducts per 10 7 nucleotides, and 60 ± 4 with PCP, combined across all concentrations of PCP. c1‐ssDNA incubated with AL‐I‐NOH. C. HK‐2 cytosol was dialyzed on 10 MWCO membrane against Tris–HCl buffer (pH 7.5) overnight at 4°C. The protein amount was quantified by Bradford assay, and various amount of protein (25 ng, 50 ng, 200 ng, 1000, 5000, 10,000 and 25,000/100 μL) was incubated with ssDNA and AL‐I‐NOH with or without <t>PAPS</t> for 30 min. c2—DNA; c3—DNA, AL‐I‐NOH; c4—DNA, AL‐I‐NOH, PAPS. St‐ standard mixture of oligonucleotides containing dG‐AL‐II and dA‐AL‐II adducts, 15 and 30 fmol each. AL‐DNA levels were 0.3 (25 ng protein) and 8 (10,000 ng protein) adducts per 10 7 nucleotides, which is ~500‐times less than before dialysis. Reactions in (B) and (C) were conducted in parallel and analyzed in the same digestion assay and resolved on the same gel. The vertical line was introduced manually to indicate the border between two different experiments. Upper band is dG‐AL, lower band is dA‐AL.
    Pentachlorophenol Pcp, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore phencyclidine pcp
    Effect of dialysis on 10 kDa MWCO and pentachlorophenol on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. (A) Cytosols from HK‐2 cells (25 ng/100 μL) were incubated with 1 (filled circles), 10 (empty circles), 25 (filled triangles), or 50 μM (empty triangles) AL‐I‐NOH in the presence of ssDNA for 15–60 min at 37°C. DNA was extracted and five micrograms from each sample was subjected to adduct analysis as described in the section Materials and Methods. Results are presented as the dependence of AL‐DNA (dG‐AL and dA‐AL adducts) levels on the reaction time. (B) HK‐2 cytosols were incubated with AL‐I‐NOH and ssDNA in the presence and absence of pentachlorophenol <t>(PCP)</t> for 30 min as indicated. Each reaction with PCP was conducted in duplicate, and reaction without PCP was set in triplicate. A fragment of representative PAGE with DNA adduct analysis for five microgram of DNA is shown. AL‐DNA levels without PCP were 58 ± 4 adducts per 10 7 nucleotides, and 60 ± 4 with PCP, combined across all concentrations of PCP. c1‐ssDNA incubated with AL‐I‐NOH. C. HK‐2 cytosol was dialyzed on 10 MWCO membrane against Tris–HCl buffer (pH 7.5) overnight at 4°C. The protein amount was quantified by Bradford assay, and various amount of protein (25 ng, 50 ng, 200 ng, 1000, 5000, 10,000 and 25,000/100 μL) was incubated with ssDNA and AL‐I‐NOH with or without <t>PAPS</t> for 30 min. c2—DNA; c3—DNA, AL‐I‐NOH; c4—DNA, AL‐I‐NOH, PAPS. St‐ standard mixture of oligonucleotides containing dG‐AL‐II and dA‐AL‐II adducts, 15 and 30 fmol each. AL‐DNA levels were 0.3 (25 ng protein) and 8 (10,000 ng protein) adducts per 10 7 nucleotides, which is ~500‐times less than before dialysis. Reactions in (B) and (C) were conducted in parallel and analyzed in the same digestion assay and resolved on the same gel. The vertical line was introduced manually to indicate the border between two different experiments. Upper band is dG‐AL, lower band is dA‐AL.
    Phencyclidine Pcp, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rna elution buffer
    Effect of dialysis on 10 kDa MWCO and pentachlorophenol on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. (A) Cytosols from HK‐2 cells (25 ng/100 μL) were incubated with 1 (filled circles), 10 (empty circles), 25 (filled triangles), or 50 μM (empty triangles) AL‐I‐NOH in the presence of ssDNA for 15–60 min at 37°C. DNA was extracted and five micrograms from each sample was subjected to adduct analysis as described in the section Materials and Methods. Results are presented as the dependence of AL‐DNA (dG‐AL and dA‐AL adducts) levels on the reaction time. (B) HK‐2 cytosols were incubated with AL‐I‐NOH and ssDNA in the presence and absence of pentachlorophenol <t>(PCP)</t> for 30 min as indicated. Each reaction with PCP was conducted in duplicate, and reaction without PCP was set in triplicate. A fragment of representative PAGE with DNA adduct analysis for five microgram of DNA is shown. AL‐DNA levels without PCP were 58 ± 4 adducts per 10 7 nucleotides, and 60 ± 4 with PCP, combined across all concentrations of PCP. c1‐ssDNA incubated with AL‐I‐NOH. C. HK‐2 cytosol was dialyzed on 10 MWCO membrane against Tris–HCl buffer (pH 7.5) overnight at 4°C. The protein amount was quantified by Bradford assay, and various amount of protein (25 ng, 50 ng, 200 ng, 1000, 5000, 10,000 and 25,000/100 μL) was incubated with ssDNA and AL‐I‐NOH with or without <t>PAPS</t> for 30 min. c2—DNA; c3—DNA, AL‐I‐NOH; c4—DNA, AL‐I‐NOH, PAPS. St‐ standard mixture of oligonucleotides containing dG‐AL‐II and dA‐AL‐II adducts, 15 and 30 fmol each. AL‐DNA levels were 0.3 (25 ng protein) and 8 (10,000 ng protein) adducts per 10 7 nucleotides, which is ~500‐times less than before dialysis. Reactions in (B) and (C) were conducted in parallel and analyzed in the same digestion assay and resolved on the same gel. The vertical line was introduced manually to indicate the border between two different experiments. Upper band is dG‐AL, lower band is dA‐AL.
    Rna Elution Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore salmon sperm dna ssdna
    Effect of dialysis on 10 kDa MWCO and pentachlorophenol on activation of AL‐I‐NOH in <t>AL‐DNA</t> by HK‐2 cytosols. (A) Cytosols from HK‐2 cells (25 ng/100 μL) were incubated with 1 (filled circles), 10 (empty circles), 25 (filled triangles), or 50 μM (empty triangles) AL‐I‐NOH in the presence of <t>ssDNA</t> for 15–60 min at 37°C. DNA was extracted and five micrograms from each sample was subjected to adduct analysis as described in the section Materials and Methods. Results are presented as the dependence of AL‐DNA (dG‐AL and dA‐AL adducts) levels on the reaction time. (B) HK‐2 cytosols were incubated with AL‐I‐NOH and ssDNA in the presence and absence of pentachlorophenol (PCP) for 30 min as indicated. Each reaction with PCP was conducted in duplicate, and reaction without PCP was set in triplicate. A fragment of representative PAGE with DNA adduct analysis for five microgram of DNA is shown. AL‐DNA levels without PCP were 58 ± 4 adducts per 10 7 nucleotides, and 60 ± 4 with PCP, combined across all concentrations of PCP. c1‐ssDNA incubated with AL‐I‐NOH. C. HK‐2 cytosol was dialyzed on 10 MWCO membrane against Tris–HCl buffer (pH 7.5) overnight at 4°C. The protein amount was quantified by Bradford assay, and various amount of protein (25 ng, 50 ng, 200 ng, 1000, 5000, 10,000 and 25,000/100 μL) was incubated with ssDNA and AL‐I‐NOH with or without PAPS for 30 min. c2—DNA; c3—DNA, AL‐I‐NOH; c4—DNA, AL‐I‐NOH, PAPS. St‐ standard mixture of oligonucleotides containing dG‐AL‐II and dA‐AL‐II adducts, 15 and 30 fmol each. AL‐DNA levels were 0.3 (25 ng protein) and 8 (10,000 ng protein) adducts per 10 7 nucleotides, which is ~500‐times less than before dialysis. Reactions in (B) and (C) were conducted in parallel and analyzed in the same digestion assay and resolved on the same gel. The vertical line was introduced manually to indicate the border between two different experiments. Upper band is dG‐AL, lower band is dA‐AL.
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    ESCRT-II binds directly to RNA through Vps25 in Xenopus egg extracts. A , Western blotting of ESCRT-II IPs or mock IPs (nonspecific rabbit IgG) performed under native (−) or denaturing (+) conditions. Vps22 was not detectable by Western blotting with our ESCRT-II polyclonal antibody. H.C. , heavy chain. B , autoradiograph of a CLIP experiment from Xenopus egg extract under high RNase conditions (0.1 mg/ml). A radioactive band consistent with the molecular mass of Vps25 (denoted by the red asterisk ) is observed, but no bands at the molecular mass of Vps22 or Vps36 are apparent. The expected migrations of the ESCRT-II subunits are indicated to the left of the gel. T4 RNA ligase forms a covalent intermediate with pCp (used to radiolabel the RNA fragments) and appears in every lane. The bands above and below the Vps25 band (denoted by black asterisks ) are nonspecific, as they appeared in the IgG control in some replicates of this experiment. C , autoradiograph of a CLIP experiment from Xenopus egg extract performed as described in B , except under denaturing immunoprecipitation conditions. The same polyclonal ESCRT-II antibody was used for A– C , but under denaturing conditions this antibody only immunoprecipitates Vps25.

    Journal: The Journal of Biological Chemistry

    Article Title: The RNA-binding complex ESCRT-II in Xenopus laevis eggs recognizes purine-rich sequences through its subunit, Vps25

    doi: 10.1074/jbc.RA118.003718

    Figure Lengend Snippet: ESCRT-II binds directly to RNA through Vps25 in Xenopus egg extracts. A , Western blotting of ESCRT-II IPs or mock IPs (nonspecific rabbit IgG) performed under native (−) or denaturing (+) conditions. Vps22 was not detectable by Western blotting with our ESCRT-II polyclonal antibody. H.C. , heavy chain. B , autoradiograph of a CLIP experiment from Xenopus egg extract under high RNase conditions (0.1 mg/ml). A radioactive band consistent with the molecular mass of Vps25 (denoted by the red asterisk ) is observed, but no bands at the molecular mass of Vps22 or Vps36 are apparent. The expected migrations of the ESCRT-II subunits are indicated to the left of the gel. T4 RNA ligase forms a covalent intermediate with pCp (used to radiolabel the RNA fragments) and appears in every lane. The bands above and below the Vps25 band (denoted by black asterisks ) are nonspecific, as they appeared in the IgG control in some replicates of this experiment. C , autoradiograph of a CLIP experiment from Xenopus egg extract performed as described in B , except under denaturing immunoprecipitation conditions. The same polyclonal ESCRT-II antibody was used for A– C , but under denaturing conditions this antibody only immunoprecipitates Vps25.

    Article Snippet: The beads were then washed three times in PBS, transferred to a fresh tube, and then resuspended in 1× T4 RNA ligase buffer (50 m m Tris-HCl, 10 m m MgCl2 , and 10 m m DTT, pH 7.5) with 1 m m ATP, 0.02 mg/ml BSA, and 1 unit/μl RNase inhibitor (Roche Applied Science) and 3′-end–labeled by the addition of 1 μl of pCp (cytidine 3′-5′-(bis)phosphate, 5′-32 P, 3000 Ci/mmol, PerkinElmer) and 0.5 μl of T4 RNA ligase (Fermentas) per 20-μl reaction.

    Techniques: Western Blot, Autoradiography, Cross-linking Immunoprecipitation, Immunoprecipitation

    Endoribonucleolytic cleavage with purified H5 protein results in a 3′-OH. A , schematic of potential cleavage products for the TAP-treated (+TAP) or untreated (−TAP) 430-nt ssRNA substrate. I and II , possible scenarios depending on the nature of 3′ ends after cleavage. Ends denoted in boldface type indicate possible results of cleavage. B , schematic of the potential and actual products after treatment of the cleavage reaction with Terminator exonuclease ( Term ). I and II , possible outcomes depending on the nature of 3′ ends after cleavage. Gray line , RNA degraded by Terminator; black line , RNA not degraded by Terminator. C , schematic of the potential and actual products after [5′- 32 P]pCp treatment of unlabeled, purified RNA substrate and products from a cleavage reaction. Black line , RNA labeled with [5′- 32 P]pCp; gray line , RNA not labeled with [5′- 32 P]pCp. Lane 1 , RNA substrate treated with H5 in cleavage assay, purified, and labeled with [5′- 32 P]pCp; lane 2 , RNA substrate treated with buffer in cleavage assay, purified, and labeled with [5′- 32 P]pCp; lane 3 , RNA substrate labeled with [5′- 32 P]pCp.

    Journal: The Journal of Biological Chemistry

    Article Title: Biochemical and Biophysical Properties of a Putative Hub Protein Expressed by Vaccinia Virus *

    doi: 10.1074/jbc.M112.442012

    Figure Lengend Snippet: Endoribonucleolytic cleavage with purified H5 protein results in a 3′-OH. A , schematic of potential cleavage products for the TAP-treated (+TAP) or untreated (−TAP) 430-nt ssRNA substrate. I and II , possible scenarios depending on the nature of 3′ ends after cleavage. Ends denoted in boldface type indicate possible results of cleavage. B , schematic of the potential and actual products after treatment of the cleavage reaction with Terminator exonuclease ( Term ). I and II , possible outcomes depending on the nature of 3′ ends after cleavage. Gray line , RNA degraded by Terminator; black line , RNA not degraded by Terminator. C , schematic of the potential and actual products after [5′- 32 P]pCp treatment of unlabeled, purified RNA substrate and products from a cleavage reaction. Black line , RNA labeled with [5′- 32 P]pCp; gray line , RNA not labeled with [5′- 32 P]pCp. Lane 1 , RNA substrate treated with H5 in cleavage assay, purified, and labeled with [5′- 32 P]pCp; lane 2 , RNA substrate treated with buffer in cleavage assay, purified, and labeled with [5′- 32 P]pCp; lane 3 , RNA substrate labeled with [5′- 32 P]pCp.

    Article Snippet: This RNA (and RNA not subjected to the cleavage reaction) was then labeled in a reaction containing 150 μCi of [5′-32 P]pCp (3000 Ci/mmol; PerkinElmer Life Sciences) and 10 units of RNA ligase (New England Biolabs) at 4 °C overnight.

    Techniques: Purification, Labeling, Cleavage Assay

    Northern blot analysis of tRNA Cys , tRNA Tyr , and tRNA Ala . RNA samples were prepared as described under “Experimental Procedures.” Eight micrograms of total RNA were subjected to electrophoresis and then transferred to a GeneScreen Plus membrane. Transfer RNAs were detected using a 32 P-labeled 5′-end-specific probe. M , mature RNA substrate.

    Journal: The Journal of Biological Chemistry

    Article Title: Exoribonuclease and Endoribonuclease Activities of RNase BN/RNase Z both Function in Vivo *

    doi: 10.1074/jbc.M112.407403

    Figure Lengend Snippet: Northern blot analysis of tRNA Cys , tRNA Tyr , and tRNA Ala . RNA samples were prepared as described under “Experimental Procedures.” Eight micrograms of total RNA were subjected to electrophoresis and then transferred to a GeneScreen Plus membrane. Transfer RNAs were detected using a 32 P-labeled 5′-end-specific probe. M , mature RNA substrate.

    Article Snippet: [γ-32 P]ATP, 5′-[32 P]pCp, and GeneScreen Plus hybridization transfer membrane were obtained from PerkinElmer Life Sciences.

    Techniques: Northern Blot, Electrophoresis, Labeling

    Effect of PAP, quercetin, and b‐estradiol on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. Cytosols from HK‐2 cells (25 ng/100 μL) were incubated with 50 μM of AL‐I‐NOH in the presence of ssDNA for 30 min at 37°C. Reactions were set in triplicate for each condition, without or with 100 μM one of the following: PAP, quercetin and β‐estradiol. DNA was extracted and five micrograms from each sample was subjected to adduct analysis. Fragment of PAGE and quantification of obtained results are shown. St‐ mixture of standard at 30 fmol each. dG‐ and dA‐AL are upper and lower bands, respectively.

    Journal: Environmental and Molecular Mutagenesis

    Article Title: Bioactivation mechanisms of N‐hydroxyaristolactams: Nitroreduction metabolites of aristolochic acids

    doi: 10.1002/em.22321

    Figure Lengend Snippet: Effect of PAP, quercetin, and b‐estradiol on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. Cytosols from HK‐2 cells (25 ng/100 μL) were incubated with 50 μM of AL‐I‐NOH in the presence of ssDNA for 30 min at 37°C. Reactions were set in triplicate for each condition, without or with 100 μM one of the following: PAP, quercetin and β‐estradiol. DNA was extracted and five micrograms from each sample was subjected to adduct analysis. Fragment of PAGE and quantification of obtained results are shown. St‐ mixture of standard at 30 fmol each. dG‐ and dA‐AL are upper and lower bands, respectively.

    Article Snippet: DMSO, 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS) (70% purity), salmon sperm DNA (ssDNA), pentachlorophenol (PCP), quercetin, β‐estradiol, 4‐nitrophenol were from Sigma‐Aldrich.

    Techniques: Activation Assay, Incubation, Polyacrylamide Gel Electrophoresis

    Effect of dialysis on 10 kDa MWCO and pentachlorophenol on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. (A) Cytosols from HK‐2 cells (25 ng/100 μL) were incubated with 1 (filled circles), 10 (empty circles), 25 (filled triangles), or 50 μM (empty triangles) AL‐I‐NOH in the presence of ssDNA for 15–60 min at 37°C. DNA was extracted and five micrograms from each sample was subjected to adduct analysis as described in the section Materials and Methods. Results are presented as the dependence of AL‐DNA (dG‐AL and dA‐AL adducts) levels on the reaction time. (B) HK‐2 cytosols were incubated with AL‐I‐NOH and ssDNA in the presence and absence of pentachlorophenol (PCP) for 30 min as indicated. Each reaction with PCP was conducted in duplicate, and reaction without PCP was set in triplicate. A fragment of representative PAGE with DNA adduct analysis for five microgram of DNA is shown. AL‐DNA levels without PCP were 58 ± 4 adducts per 10 7 nucleotides, and 60 ± 4 with PCP, combined across all concentrations of PCP. c1‐ssDNA incubated with AL‐I‐NOH. C. HK‐2 cytosol was dialyzed on 10 MWCO membrane against Tris–HCl buffer (pH 7.5) overnight at 4°C. The protein amount was quantified by Bradford assay, and various amount of protein (25 ng, 50 ng, 200 ng, 1000, 5000, 10,000 and 25,000/100 μL) was incubated with ssDNA and AL‐I‐NOH with or without PAPS for 30 min. c2—DNA; c3—DNA, AL‐I‐NOH; c4—DNA, AL‐I‐NOH, PAPS. St‐ standard mixture of oligonucleotides containing dG‐AL‐II and dA‐AL‐II adducts, 15 and 30 fmol each. AL‐DNA levels were 0.3 (25 ng protein) and 8 (10,000 ng protein) adducts per 10 7 nucleotides, which is ~500‐times less than before dialysis. Reactions in (B) and (C) were conducted in parallel and analyzed in the same digestion assay and resolved on the same gel. The vertical line was introduced manually to indicate the border between two different experiments. Upper band is dG‐AL, lower band is dA‐AL.

    Journal: Environmental and Molecular Mutagenesis

    Article Title: Bioactivation mechanisms of N‐hydroxyaristolactams: Nitroreduction metabolites of aristolochic acids

    doi: 10.1002/em.22321

    Figure Lengend Snippet: Effect of dialysis on 10 kDa MWCO and pentachlorophenol on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. (A) Cytosols from HK‐2 cells (25 ng/100 μL) were incubated with 1 (filled circles), 10 (empty circles), 25 (filled triangles), or 50 μM (empty triangles) AL‐I‐NOH in the presence of ssDNA for 15–60 min at 37°C. DNA was extracted and five micrograms from each sample was subjected to adduct analysis as described in the section Materials and Methods. Results are presented as the dependence of AL‐DNA (dG‐AL and dA‐AL adducts) levels on the reaction time. (B) HK‐2 cytosols were incubated with AL‐I‐NOH and ssDNA in the presence and absence of pentachlorophenol (PCP) for 30 min as indicated. Each reaction with PCP was conducted in duplicate, and reaction without PCP was set in triplicate. A fragment of representative PAGE with DNA adduct analysis for five microgram of DNA is shown. AL‐DNA levels without PCP were 58 ± 4 adducts per 10 7 nucleotides, and 60 ± 4 with PCP, combined across all concentrations of PCP. c1‐ssDNA incubated with AL‐I‐NOH. C. HK‐2 cytosol was dialyzed on 10 MWCO membrane against Tris–HCl buffer (pH 7.5) overnight at 4°C. The protein amount was quantified by Bradford assay, and various amount of protein (25 ng, 50 ng, 200 ng, 1000, 5000, 10,000 and 25,000/100 μL) was incubated with ssDNA and AL‐I‐NOH with or without PAPS for 30 min. c2—DNA; c3—DNA, AL‐I‐NOH; c4—DNA, AL‐I‐NOH, PAPS. St‐ standard mixture of oligonucleotides containing dG‐AL‐II and dA‐AL‐II adducts, 15 and 30 fmol each. AL‐DNA levels were 0.3 (25 ng protein) and 8 (10,000 ng protein) adducts per 10 7 nucleotides, which is ~500‐times less than before dialysis. Reactions in (B) and (C) were conducted in parallel and analyzed in the same digestion assay and resolved on the same gel. The vertical line was introduced manually to indicate the border between two different experiments. Upper band is dG‐AL, lower band is dA‐AL.

    Article Snippet: DMSO, 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS) (70% purity), salmon sperm DNA (ssDNA), pentachlorophenol (PCP), quercetin, β‐estradiol, 4‐nitrophenol were from Sigma‐Aldrich.

    Techniques: Activation Assay, Incubation, Polyacrylamide Gel Electrophoresis, Bradford Assay, Papanicolaou Stain

    Effect of dialysis on 10 kDa MWCO and pentachlorophenol on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. (A) Cytosols from HK‐2 cells (25 ng/100 μL) were incubated with 1 (filled circles), 10 (empty circles), 25 (filled triangles), or 50 μM (empty triangles) AL‐I‐NOH in the presence of ssDNA for 15–60 min at 37°C. DNA was extracted and five micrograms from each sample was subjected to adduct analysis as described in the section Materials and Methods. Results are presented as the dependence of AL‐DNA (dG‐AL and dA‐AL adducts) levels on the reaction time. (B) HK‐2 cytosols were incubated with AL‐I‐NOH and ssDNA in the presence and absence of pentachlorophenol (PCP) for 30 min as indicated. Each reaction with PCP was conducted in duplicate, and reaction without PCP was set in triplicate. A fragment of representative PAGE with DNA adduct analysis for five microgram of DNA is shown. AL‐DNA levels without PCP were 58 ± 4 adducts per 10 7 nucleotides, and 60 ± 4 with PCP, combined across all concentrations of PCP. c1‐ssDNA incubated with AL‐I‐NOH. C. HK‐2 cytosol was dialyzed on 10 MWCO membrane against Tris–HCl buffer (pH 7.5) overnight at 4°C. The protein amount was quantified by Bradford assay, and various amount of protein (25 ng, 50 ng, 200 ng, 1000, 5000, 10,000 and 25,000/100 μL) was incubated with ssDNA and AL‐I‐NOH with or without PAPS for 30 min. c2—DNA; c3—DNA, AL‐I‐NOH; c4—DNA, AL‐I‐NOH, PAPS. St‐ standard mixture of oligonucleotides containing dG‐AL‐II and dA‐AL‐II adducts, 15 and 30 fmol each. AL‐DNA levels were 0.3 (25 ng protein) and 8 (10,000 ng protein) adducts per 10 7 nucleotides, which is ~500‐times less than before dialysis. Reactions in (B) and (C) were conducted in parallel and analyzed in the same digestion assay and resolved on the same gel. The vertical line was introduced manually to indicate the border between two different experiments. Upper band is dG‐AL, lower band is dA‐AL.

    Journal: Environmental and Molecular Mutagenesis

    Article Title: Bioactivation mechanisms of N‐hydroxyaristolactams: Nitroreduction metabolites of aristolochic acids

    doi: 10.1002/em.22321

    Figure Lengend Snippet: Effect of dialysis on 10 kDa MWCO and pentachlorophenol on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. (A) Cytosols from HK‐2 cells (25 ng/100 μL) were incubated with 1 (filled circles), 10 (empty circles), 25 (filled triangles), or 50 μM (empty triangles) AL‐I‐NOH in the presence of ssDNA for 15–60 min at 37°C. DNA was extracted and five micrograms from each sample was subjected to adduct analysis as described in the section Materials and Methods. Results are presented as the dependence of AL‐DNA (dG‐AL and dA‐AL adducts) levels on the reaction time. (B) HK‐2 cytosols were incubated with AL‐I‐NOH and ssDNA in the presence and absence of pentachlorophenol (PCP) for 30 min as indicated. Each reaction with PCP was conducted in duplicate, and reaction without PCP was set in triplicate. A fragment of representative PAGE with DNA adduct analysis for five microgram of DNA is shown. AL‐DNA levels without PCP were 58 ± 4 adducts per 10 7 nucleotides, and 60 ± 4 with PCP, combined across all concentrations of PCP. c1‐ssDNA incubated with AL‐I‐NOH. C. HK‐2 cytosol was dialyzed on 10 MWCO membrane against Tris–HCl buffer (pH 7.5) overnight at 4°C. The protein amount was quantified by Bradford assay, and various amount of protein (25 ng, 50 ng, 200 ng, 1000, 5000, 10,000 and 25,000/100 μL) was incubated with ssDNA and AL‐I‐NOH with or without PAPS for 30 min. c2—DNA; c3—DNA, AL‐I‐NOH; c4—DNA, AL‐I‐NOH, PAPS. St‐ standard mixture of oligonucleotides containing dG‐AL‐II and dA‐AL‐II adducts, 15 and 30 fmol each. AL‐DNA levels were 0.3 (25 ng protein) and 8 (10,000 ng protein) adducts per 10 7 nucleotides, which is ~500‐times less than before dialysis. Reactions in (B) and (C) were conducted in parallel and analyzed in the same digestion assay and resolved on the same gel. The vertical line was introduced manually to indicate the border between two different experiments. Upper band is dG‐AL, lower band is dA‐AL.

    Article Snippet: DMSO, 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS) (70% purity), salmon sperm DNA (ssDNA), pentachlorophenol (PCP), quercetin, β‐estradiol, 4‐nitrophenol were from Sigma‐Aldrich.

    Techniques: Activation Assay, Incubation, Polyacrylamide Gel Electrophoresis, Bradford Assay, Papanicolaou Stain

    Effect of PAP, quercetin, and b‐estradiol on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. Cytosols from HK‐2 cells (25 ng/100 μL) were incubated with 50 μM of AL‐I‐NOH in the presence of ssDNA for 30 min at 37°C. Reactions were set in triplicate for each condition, without or with 100 μM one of the following: PAP, quercetin and β‐estradiol. DNA was extracted and five micrograms from each sample was subjected to adduct analysis. Fragment of PAGE and quantification of obtained results are shown. St‐ mixture of standard at 30 fmol each. dG‐ and dA‐AL are upper and lower bands, respectively.

    Journal: Environmental and Molecular Mutagenesis

    Article Title: Bioactivation mechanisms of N‐hydroxyaristolactams: Nitroreduction metabolites of aristolochic acids

    doi: 10.1002/em.22321

    Figure Lengend Snippet: Effect of PAP, quercetin, and b‐estradiol on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. Cytosols from HK‐2 cells (25 ng/100 μL) were incubated with 50 μM of AL‐I‐NOH in the presence of ssDNA for 30 min at 37°C. Reactions were set in triplicate for each condition, without or with 100 μM one of the following: PAP, quercetin and β‐estradiol. DNA was extracted and five micrograms from each sample was subjected to adduct analysis. Fragment of PAGE and quantification of obtained results are shown. St‐ mixture of standard at 30 fmol each. dG‐ and dA‐AL are upper and lower bands, respectively.

    Article Snippet: DMSO, 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS) (70% purity), salmon sperm DNA (ssDNA), pentachlorophenol (PCP), quercetin, β‐estradiol, 4‐nitrophenol were from Sigma‐Aldrich.

    Techniques: Activation Assay, Incubation, Polyacrylamide Gel Electrophoresis

    Activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols, fortified, and not fortified by PAPS. Cytosols from HK‐2 cells (6; 25; 100; 400; 1500; 6250 and 25,000 ng/100 μL) or recombinant SULT1B1 protein (1 ng/100 μL) were incubated with 100 μM AL‐I‐NOH in the presence of ssDNA with or without 0.2 mM PAPS for 2 h at 37°C. DNA was extracted and two micrograms from each sample was subjected to adduct analysis as described in the section Materials and Methods. (A), (B), and (D) are representative fragments of polyacrylamide gels following electrophoresis and exposure for 2 (A and B) or 10 min (D) on a phosphoscreen. (A) and (B) are unaltered fragments of the same gel shown at the same contrast, and analysis in (D) was done in a separate experiment and shown at the similar contrast level but longer exposure time. Control 1 in (A) is DNA/cytosol; Control 2—DNA/AA‐I; Controls 3–5—DNA/AL‐I‐NOH; Control 6—DNA/AL‐I‐NOH/PAPS. AL‐DNA levels in control incubations with AL‐I‐NOH were at 0.2 ± 0.02 adducts/10 6 nucleotides. (C) Quantitative representation of results shown in (B). Filled circles—reactions with PAPS; empty circles—without PAPS. (D) s1 (8 AL‐DNA/10 5 nucleotides) and s2 (6 AL‐DNA/10 5 nucleotides) represent adducted DNA from reactions conducted with PAPS from R D and Sigma‐Aldrich, respectively. St—standard mixture of oligonucleotides containing dG‐AL‐II and dA‐AL‐II adducts, 30 fmol each. AL‐DNA is a combined term for dG‐AL and dA‐AL adducts.

    Journal: Environmental and Molecular Mutagenesis

    Article Title: Bioactivation mechanisms of N‐hydroxyaristolactams: Nitroreduction metabolites of aristolochic acids

    doi: 10.1002/em.22321

    Figure Lengend Snippet: Activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols, fortified, and not fortified by PAPS. Cytosols from HK‐2 cells (6; 25; 100; 400; 1500; 6250 and 25,000 ng/100 μL) or recombinant SULT1B1 protein (1 ng/100 μL) were incubated with 100 μM AL‐I‐NOH in the presence of ssDNA with or without 0.2 mM PAPS for 2 h at 37°C. DNA was extracted and two micrograms from each sample was subjected to adduct analysis as described in the section Materials and Methods. (A), (B), and (D) are representative fragments of polyacrylamide gels following electrophoresis and exposure for 2 (A and B) or 10 min (D) on a phosphoscreen. (A) and (B) are unaltered fragments of the same gel shown at the same contrast, and analysis in (D) was done in a separate experiment and shown at the similar contrast level but longer exposure time. Control 1 in (A) is DNA/cytosol; Control 2—DNA/AA‐I; Controls 3–5—DNA/AL‐I‐NOH; Control 6—DNA/AL‐I‐NOH/PAPS. AL‐DNA levels in control incubations with AL‐I‐NOH were at 0.2 ± 0.02 adducts/10 6 nucleotides. (C) Quantitative representation of results shown in (B). Filled circles—reactions with PAPS; empty circles—without PAPS. (D) s1 (8 AL‐DNA/10 5 nucleotides) and s2 (6 AL‐DNA/10 5 nucleotides) represent adducted DNA from reactions conducted with PAPS from R D and Sigma‐Aldrich, respectively. St—standard mixture of oligonucleotides containing dG‐AL‐II and dA‐AL‐II adducts, 30 fmol each. AL‐DNA is a combined term for dG‐AL and dA‐AL adducts.

    Article Snippet: DMSO, 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS) (70% purity), salmon sperm DNA (ssDNA), pentachlorophenol (PCP), quercetin, β‐estradiol, 4‐nitrophenol were from Sigma‐Aldrich.

    Techniques: Activation Assay, Papanicolaou Stain, Recombinant, Incubation, Electrophoresis

    Effect of dialysis on 3.5 kDa MWCO and prolonged incubation at 4°C on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. Cytosols from HK‐2 cells were dialyzed against Tris–HCl buffer (pH 7.5) using 3.5 MWCO membrane overnight at 4°C. In parallel, a portion of cytosol was incubated above the membrane without the dialysis overnight at 4°C. 100–5000 ng of the protein was incubated with ssDNA and 100 μM AL‐I‐NOH with or without PAPS, as indicated. Reactions were allowed to run for 45 min at 37°C. DNA (5 μg) was used for adduct analysis. Fragments of PAGE (left panel) and quantification of obtained results (right panel) are shown. St‐ mixture of standard at 60 fmol each. Upper band is dG‐AL, and lower band is dA‐AL.

    Journal: Environmental and Molecular Mutagenesis

    Article Title: Bioactivation mechanisms of N‐hydroxyaristolactams: Nitroreduction metabolites of aristolochic acids

    doi: 10.1002/em.22321

    Figure Lengend Snippet: Effect of dialysis on 3.5 kDa MWCO and prolonged incubation at 4°C on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. Cytosols from HK‐2 cells were dialyzed against Tris–HCl buffer (pH 7.5) using 3.5 MWCO membrane overnight at 4°C. In parallel, a portion of cytosol was incubated above the membrane without the dialysis overnight at 4°C. 100–5000 ng of the protein was incubated with ssDNA and 100 μM AL‐I‐NOH with or without PAPS, as indicated. Reactions were allowed to run for 45 min at 37°C. DNA (5 μg) was used for adduct analysis. Fragments of PAGE (left panel) and quantification of obtained results (right panel) are shown. St‐ mixture of standard at 60 fmol each. Upper band is dG‐AL, and lower band is dA‐AL.

    Article Snippet: DMSO, 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS) (70% purity), salmon sperm DNA (ssDNA), pentachlorophenol (PCP), quercetin, β‐estradiol, 4‐nitrophenol were from Sigma‐Aldrich.

    Techniques: Incubation, Activation Assay, Papanicolaou Stain, Polyacrylamide Gel Electrophoresis