p pcp Perkinelmer Search Results


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  • 95
    New England Biolabs rna 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.
    Rna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 194 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore phencyclidine pcp
    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.
    Phencyclidine Pcp, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    75
    PerkinElmer pcp 5 32p
    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.
    Pcp 5 32p, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    PerkinElmer p pcp
    Effect of RBP16 or RBP16(F14,16A) on in vitro insertion editing of A6 with natural gRNA. RBP16 ( A ) or RBP16(F14,16A) ( B ) was titrated into in vitro insertion reactions (20 μL final volume) containing [ 32 <t>P]pCp</t> 3′-labeled A6 <t>pre-mRNA</t> (∼10
    P Pcp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 90/100, based on 149 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    PerkinElmer pcp 5 32p lead
    Effect of RBP16 or RBP16(F14,16A) on in vitro insertion editing of A6 with natural gRNA. RBP16 ( A ) or RBP16(F14,16A) ( B ) was titrated into in vitro insertion reactions (20 μL final volume) containing [ 32 <t>P]pCp</t> 3′-labeled A6 <t>pre-mRNA</t> (∼10
    Pcp 5 32p Lead, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 81/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    PerkinElmer 3 5 cytidine
    FinO binding to SLII requires a terminal 3′-OH on the 3′-tail of SLII. Native gels (8%) of binding reactions between FinO constructs and SLII RNA derivatives. ( A ) FinO does not bind SLII RNA containing a <t>3′,5′-cytidine</t> diphosphate 3′-terminus. T4 RNA ligase I was used to ligate 3′,5′-cytidine [5′- 32 P] disphosphate (pCp) to the 3′-tail resulting in a 3′-phosphate. NP, no protein. Triangles represent increasing concentrations of FinO or the indicated mutants: 0.25, 0.5, 1, 2.5, 5 and 10 µM. The positions of free 32 P-SLII and the FinO- 32 P-SLII are noted by arrows. ( B ) Treatment of SLII RNA containing a 3′,5′-cytidine diphosphate 3′-terminus to give a 2′,3′ cis -diol (3′-hydroxyl) restores FinO binding. ( C ) Oxidation of SLII with sodium periodate to give a 2′,3′ dialdehyde reduces binding affinity. The protein concentrations were 1 µM in each of the binding reactions in B and C.
    3 5 Cytidine, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 78/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    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: 92/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    PerkinElmer pcp cytidine 3 5 bis phosphate
    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.
    Pcp Cytidine 3 5 Bis Phosphate, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 87/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer ci mmol
    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.
    Ci Mmol, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 1860 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    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: 90/100, based on 278 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    PerkinElmer rna
    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.
    Rna, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 90/100, based on 1156 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    PerkinElmer thymidine phosphoramidites
    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.
    Thymidine Phosphoramidites, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    PerkinElmer myo inositol
    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.
    Myo Inositol, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 90/100, based on 114 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer γ 32 p gtp
    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.
    γ 32 P Gtp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 247 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    PerkinElmer h oh dpat
    (A) Effect of <t>8-OH-DPAT</t> on [ 35 <t>S]-GTPγS</t> binding to Sprague–Dawley rat brainstem membranes in the presence of DMSO or 100 nM THCV ( n = 11). (B) Effect of 8-OH-DPAT ( n = 4) and THCV ( n = 6) on [ 35 S]-GTPγS binding to Sprague–Dawley
    H Oh Dpat, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer volocity software
    (A) Effect of <t>8-OH-DPAT</t> on [ 35 <t>S]-GTPγS</t> binding to Sprague–Dawley rat brainstem membranes in the presence of DMSO or 100 nM THCV ( n = 11). (B) Effect of 8-OH-DPAT ( n = 4) and THCV ( n = 6) on [ 35 S]-GTPγS binding to Sprague–Dawley
    Volocity Software, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 9735 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    PerkinElmer ultima gold
    (A) Effect of <t>8-OH-DPAT</t> on [ 35 <t>S]-GTPγS</t> binding to Sprague–Dawley rat brainstem membranes in the presence of DMSO or 100 nM THCV ( n = 11). (B) Effect of 8-OH-DPAT ( n = 4) and THCV ( n = 6) on [ 35 S]-GTPγS binding to Sprague–Dawley
    Ultima Gold, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 90/100, based on 611 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    PerkinElmer lambda 950 uv vis spectrophotometer
    (A) Effect of <t>8-OH-DPAT</t> on [ 35 <t>S]-GTPγS</t> binding to Sprague–Dawley rat brainstem membranes in the presence of DMSO or 100 nM THCV ( n = 11). (B) Effect of 8-OH-DPAT ( n = 4) and THCV ( n = 6) on [ 35 S]-GTPγS binding to Sprague–Dawley
    Lambda 950 Uv Vis Spectrophotometer, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 93/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    PerkinElmer phencyclidine hydrochloride
    (A) Effect of <t>8-OH-DPAT</t> on [ 35 <t>S]-GTPγS</t> binding to Sprague–Dawley rat brainstem membranes in the presence of DMSO or 100 nM THCV ( n = 11). (B) Effect of 8-OH-DPAT ( n = 4) and THCV ( n = 6) on [ 35 S]-GTPγS binding to Sprague–Dawley
    Phencyclidine Hydrochloride, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    PerkinElmer α 32 p utp
    (A) Effect of <t>8-OH-DPAT</t> on [ 35 <t>S]-GTPγS</t> binding to Sprague–Dawley rat brainstem membranes in the presence of DMSO or 100 nM THCV ( n = 11). (B) Effect of 8-OH-DPAT ( n = 4) and THCV ( n = 6) on [ 35 S]-GTPγS binding to Sprague–Dawley
    α 32 P Utp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 90/100, based on 1695 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    PerkinElmer γ32 atp
    (A) Effect of <t>8-OH-DPAT</t> on [ 35 <t>S]-GTPγS</t> binding to Sprague–Dawley rat brainstem membranes in the presence of DMSO or 100 nM THCV ( n = 11). (B) Effect of 8-OH-DPAT ( n = 4) and THCV ( n = 6) on [ 35 S]-GTPγS binding to Sprague–Dawley
    γ32 Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 90/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    PerkinElmer lambda 50b spectrofluorimeter
    (A) Effect of <t>8-OH-DPAT</t> on [ 35 <t>S]-GTPγS</t> binding to Sprague–Dawley rat brainstem membranes in the presence of DMSO or 100 nM THCV ( n = 11). (B) Effect of 8-OH-DPAT ( n = 4) and THCV ( n = 6) on [ 35 S]-GTPγS binding to Sprague–Dawley
    Lambda 50b Spectrofluorimeter, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    PerkinElmer t4 rna ligase
    (A) Effect of <t>8-OH-DPAT</t> on [ 35 <t>S]-GTPγS</t> binding to Sprague–Dawley rat brainstem membranes in the presence of DMSO or 100 nM THCV ( n = 11). (B) Effect of 8-OH-DPAT ( n = 4) and THCV ( n = 6) on [ 35 S]-GTPγS binding to Sprague–Dawley
    T4 Rna Ligase, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    PerkinElmer na4 ppi
    (A) Effect of <t>8-OH-DPAT</t> on [ 35 <t>S]-GTPγS</t> binding to Sprague–Dawley rat brainstem membranes in the presence of DMSO or 100 nM THCV ( n = 11). (B) Effect of 8-OH-DPAT ( n = 4) and THCV ( n = 6) on [ 35 S]-GTPγS binding to Sprague–Dawley
    Na4 Ppi, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer γ32 p atp
    (A) Effect of <t>8-OH-DPAT</t> on [ 35 <t>S]-GTPγS</t> binding to Sprague–Dawley rat brainstem membranes in the presence of DMSO or 100 nM THCV ( n = 11). (B) Effect of 8-OH-DPAT ( n = 4) and THCV ( n = 6) on [ 35 S]-GTPγS binding to Sprague–Dawley
    γ32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 1116 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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. <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.
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    Image Search Results


    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

    Effect of RBP16 or RBP16(F14,16A) on in vitro insertion editing of A6 with natural gRNA. RBP16 ( A ) or RBP16(F14,16A) ( B ) was titrated into in vitro insertion reactions (20 μL final volume) containing [ 32 P]pCp 3′-labeled A6 pre-mRNA (∼10

    Journal:

    Article Title: RBP16 stimulates trypanosome RNA editing in vitro at an early step in the editing reaction

    doi: 10.1261/rna.2331506

    Figure Lengend Snippet: Effect of RBP16 or RBP16(F14,16A) on in vitro insertion editing of A6 with natural gRNA. RBP16 ( A ) or RBP16(F14,16A) ( B ) was titrated into in vitro insertion reactions (20 μL final volume) containing [ 32 P]pCp 3′-labeled A6 pre-mRNA (∼10

    Article Snippet: Radiolabeling of pre-mRNA at the 3′ end was performed by ligation of [5′-32 P]pCp (Perkin Elmer) by T4 RNA ligase (Promega).

    Techniques: In Vitro, Labeling

    FinO binding to SLII requires a terminal 3′-OH on the 3′-tail of SLII. Native gels (8%) of binding reactions between FinO constructs and SLII RNA derivatives. ( A ) FinO does not bind SLII RNA containing a 3′,5′-cytidine diphosphate 3′-terminus. T4 RNA ligase I was used to ligate 3′,5′-cytidine [5′- 32 P] disphosphate (pCp) to the 3′-tail resulting in a 3′-phosphate. NP, no protein. Triangles represent increasing concentrations of FinO or the indicated mutants: 0.25, 0.5, 1, 2.5, 5 and 10 µM. The positions of free 32 P-SLII and the FinO- 32 P-SLII are noted by arrows. ( B ) Treatment of SLII RNA containing a 3′,5′-cytidine diphosphate 3′-terminus to give a 2′,3′ cis -diol (3′-hydroxyl) restores FinO binding. ( C ) Oxidation of SLII with sodium periodate to give a 2′,3′ dialdehyde reduces binding affinity. The protein concentrations were 1 µM in each of the binding reactions in B and C.

    Journal: Nucleic Acids Research

    Article Title: Mapping interactions between the RNA chaperone FinO and its RNA targets

    doi: 10.1093/nar/gkr025

    Figure Lengend Snippet: FinO binding to SLII requires a terminal 3′-OH on the 3′-tail of SLII. Native gels (8%) of binding reactions between FinO constructs and SLII RNA derivatives. ( A ) FinO does not bind SLII RNA containing a 3′,5′-cytidine diphosphate 3′-terminus. T4 RNA ligase I was used to ligate 3′,5′-cytidine [5′- 32 P] disphosphate (pCp) to the 3′-tail resulting in a 3′-phosphate. NP, no protein. Triangles represent increasing concentrations of FinO or the indicated mutants: 0.25, 0.5, 1, 2.5, 5 and 10 µM. The positions of free 32 P-SLII and the FinO- 32 P-SLII are noted by arrows. ( B ) Treatment of SLII RNA containing a 3′,5′-cytidine diphosphate 3′-terminus to give a 2′,3′ cis -diol (3′-hydroxyl) restores FinO binding. ( C ) Oxidation of SLII with sodium periodate to give a 2′,3′ dialdehyde reduces binding affinity. The protein concentrations were 1 µM in each of the binding reactions in B and C.

    Article Snippet: The 3′-end-labeled RNAs were labeled with 3′,5′-cytidine [5′-32 P] diphosphate (pCp) (Perkin Elmer) and T4 RNA Ligase I (New England Biolabs).

    Techniques: Binding Assay, Construct

    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

    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

    (A) Effect of 8-OH-DPAT on [ 35 S]-GTPγS binding to Sprague–Dawley rat brainstem membranes in the presence of DMSO or 100 nM THCV ( n = 11). (B) Effect of 8-OH-DPAT ( n = 4) and THCV ( n = 6) on [ 35 S]-GTPγS binding to Sprague–Dawley

    Journal: British Journal of Pharmacology

    Article Title: The phytocannabinoid, Δ9-tetrahydrocannabivarin, can act through 5-HT1A receptors to produce antipsychotic effects

    doi: 10.1111/bph.13000

    Figure Lengend Snippet: (A) Effect of 8-OH-DPAT on [ 35 S]-GTPγS binding to Sprague–Dawley rat brainstem membranes in the presence of DMSO or 100 nM THCV ( n = 11). (B) Effect of 8-OH-DPAT ( n = 4) and THCV ( n = 6) on [ 35 S]-GTPγS binding to Sprague–Dawley

    Article Snippet: [35 S]=GTPγS (1250 Ci·mmol−1 ) and 8-[3 H]-OH-DPAT (135.2 Ci·mmol−1 ) were purchased from PerkinElmer Life Sciences, Inc. (Boston, MA, USA), GTPγS and adenosine deaminase from Roche Diagnostic (Indianapolis, IN, USA), and GDP, DMSO and PCP from Sigma-Aldrich UK (Dorset, UK).

    Techniques: Binding Assay

    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

    Schematic representation of radiolabeling of RNA at its 3′ end. T4 RNA ligase catalyzes the ligation reaction where 5′[ 32 P]pCp is covalently attached to the 3′ end of the single-stranded RNA substrate. The radiolabeled RNA molecule

    Journal: Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]

    Article Title: Synthesis and Labeling of RNA In Vitro

    doi: 10.1002/0471142727.mb0415s102

    Figure Lengend Snippet: Schematic representation of radiolabeling of RNA at its 3′ end. T4 RNA ligase catalyzes the ligation reaction where 5′[ 32 P]pCp is covalently attached to the 3′ end of the single-stranded RNA substrate. The radiolabeled RNA molecule

    Article Snippet: 10 × buffer for T4 RNA ligase (see recipe) 10 mM ATP (Thermo Scientific) RNA substrate with 3′ hydroxyl end derived from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 5′ 10 µCi/µl [32 P]pCp (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 RNA ligase (Thermo Scientific) G50 buffer (see recipe) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for T4 RNA ligase 1 µl distilled, deionized H2 O 1 µl 10 mM ATP 5 µl RNA substrate with a 3′-hydroxyl end (30 pmol) 10 µl 10 µCi/µl 5′ [32 P]pCp (3000 Ci/mmol) 1 µl 10 U/µl T4 RNA ligase.

    Techniques: Radioactivity, Ligation