atp New England Biolabs Search Results


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  • 95
    New England Biolabs atp
    <t>RIG-I:RNA</t> conformation as probed by limited trypsin digestion time course. (A to D) RIG-I fragments were detected by Western blotting with a monoclonal antibody to the helicase domain. Cell extracts were incubated with trypsin in the absence (A [–RNA]) or presence of RNA ligands, including nonbinding control yeast tRNA (B [tRNA]), polyI:C (C), or polyU/UC (D), and with 1 mM <t>ATP</t> or AMP-PNP. Aliquots were removed from the reaction at various time points (minutes) post-addition of trypsin. Additional data are provided in Fig. S6 in the supplemental material. (E) Biotinylated RNAs were used in pulldown experiments to test for RNA binding by the 80-kDa and 55-kDa RIG-I fragments. Trypsin digests (+ tryp) and control lysates (− tryp) were incubated without RNA (-RNA), with nonbiotinylated polyU/UC RNA (-btn), with biotinylated polyU/UC RNA (can), or with biotinylated X RNA (X) in the presence of AMP-PNP. After 1.5 h, trypsin digestions were quenched by adding protease inhibitor and incubated with streptavidin paramagnetic beads. RIG-I present in the bead-bound fraction (BND) versus the input fraction (INP) was detected by Western blotting.
    Atp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1267 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore atp
    <t>RIG-I:RNA</t> conformation as probed by limited trypsin digestion time course. (A to D) RIG-I fragments were detected by Western blotting with a monoclonal antibody to the helicase domain. Cell extracts were incubated with trypsin in the absence (A [–RNA]) or presence of RNA ligands, including nonbinding control yeast tRNA (B [tRNA]), polyI:C (C), or polyU/UC (D), and with 1 mM <t>ATP</t> or AMP-PNP. Aliquots were removed from the reaction at various time points (minutes) post-addition of trypsin. Additional data are provided in Fig. S6 in the supplemental material. (E) Biotinylated RNAs were used in pulldown experiments to test for RNA binding by the 80-kDa and 55-kDa RIG-I fragments. Trypsin digests (+ tryp) and control lysates (− tryp) were incubated without RNA (-RNA), with nonbiotinylated polyU/UC RNA (-btn), with biotinylated polyU/UC RNA (can), or with biotinylated X RNA (X) in the presence of AMP-PNP. After 1.5 h, trypsin digestions were quenched by adding protease inhibitor and incubated with streptavidin paramagnetic beads. RIG-I present in the bead-bound fraction (BND) versus the input fraction (INP) was detected by Western blotting.
    Atp, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 7359 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer p γ atp
    <t>RIG-I:RNA</t> conformation as probed by limited trypsin digestion time course. (A to D) RIG-I fragments were detected by Western blotting with a monoclonal antibody to the helicase domain. Cell extracts were incubated with trypsin in the absence (A [–RNA]) or presence of RNA ligands, including nonbinding control yeast tRNA (B [tRNA]), polyI:C (C), or polyU/UC (D), and with 1 mM <t>ATP</t> or AMP-PNP. Aliquots were removed from the reaction at various time points (minutes) post-addition of trypsin. Additional data are provided in Fig. S6 in the supplemental material. (E) Biotinylated RNAs were used in pulldown experiments to test for RNA binding by the 80-kDa and 55-kDa RIG-I fragments. Trypsin digests (+ tryp) and control lysates (− tryp) were incubated without RNA (-RNA), with nonbiotinylated polyU/UC RNA (-btn), with biotinylated polyU/UC RNA (can), or with biotinylated X RNA (X) in the presence of AMP-PNP. After 1.5 h, trypsin digestions were quenched by adding protease inhibitor and incubated with streptavidin paramagnetic beads. RIG-I present in the bead-bound fraction (BND) versus the input fraction (INP) was detected by Western blotting.
    P γ Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 574 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    New England Biolabs cofactor atp
    Detecting backbone damage in a <t>DNA</t> duplex. (a–b) Current histograms showing the measured blockade currents for 14 µM solutions of the (a) nicked and (b) repaired duplexes, respectively. (c) Current histogram of a mixture of the same amount (7 µM) of nicked and repaired duplexes. (d) Sample current-time traces generated during dsDNA residence events, showing the difference in blockade currents, I N and I R , for nicked DNA and repaired DNA, respectively. The trace was post-filtered at 1 kHz for presentation. An expanded view of the same I-t trace is also shown in (d) to present the difference in blockade current of the two types of events. The expanded I-t trace was post-filtered at 0.1 kHz for presentation. Experiments were carried out at 20.0 °C in a 100 mM KCl (7.5% PEG, 5 mM MgCl 2 , 1 mM <t>ATP)</t> solution buffered to pH 7.6 by 66 mM Tris-HCl. Counts indicate the number of individual dsDNA unzipping events.
    Cofactor Atp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer γ 32 p atp
    Detecting backbone damage in a <t>DNA</t> duplex. (a–b) Current histograms showing the measured blockade currents for 14 µM solutions of the (a) nicked and (b) repaired duplexes, respectively. (c) Current histogram of a mixture of the same amount (7 µM) of nicked and repaired duplexes. (d) Sample current-time traces generated during dsDNA residence events, showing the difference in blockade currents, I N and I R , for nicked DNA and repaired DNA, respectively. The trace was post-filtered at 1 kHz for presentation. An expanded view of the same I-t trace is also shown in (d) to present the difference in blockade current of the two types of events. The expanded I-t trace was post-filtered at 0.1 kHz for presentation. Experiments were carried out at 20.0 °C in a 100 mM KCl (7.5% PEG, 5 mM MgCl 2 , 1 mM <t>ATP)</t> solution buffered to pH 7.6 by 66 mM Tris-HCl. Counts indicate the number of individual dsDNA unzipping events.
    γ 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 940 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    New England Biolabs acyclic atp
    Detecting backbone damage in a <t>DNA</t> duplex. (a–b) Current histograms showing the measured blockade currents for 14 µM solutions of the (a) nicked and (b) repaired duplexes, respectively. (c) Current histogram of a mixture of the same amount (7 µM) of nicked and repaired duplexes. (d) Sample current-time traces generated during dsDNA residence events, showing the difference in blockade currents, I N and I R , for nicked DNA and repaired DNA, respectively. The trace was post-filtered at 1 kHz for presentation. An expanded view of the same I-t trace is also shown in (d) to present the difference in blockade current of the two types of events. The expanded I-t trace was post-filtered at 0.1 kHz for presentation. Experiments were carried out at 20.0 °C in a 100 mM KCl (7.5% PEG, 5 mM MgCl 2 , 1 mM <t>ATP)</t> solution buffered to pH 7.6 by 66 mM Tris-HCl. Counts indicate the number of individual dsDNA unzipping events.
    Acyclic Atp, supplied by New England Biolabs, 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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    76
    New England Biolabs atp sulphurylase
    Detecting backbone damage in a <t>DNA</t> duplex. (a–b) Current histograms showing the measured blockade currents for 14 µM solutions of the (a) nicked and (b) repaired duplexes, respectively. (c) Current histogram of a mixture of the same amount (7 µM) of nicked and repaired duplexes. (d) Sample current-time traces generated during dsDNA residence events, showing the difference in blockade currents, I N and I R , for nicked DNA and repaired DNA, respectively. The trace was post-filtered at 1 kHz for presentation. An expanded view of the same I-t trace is also shown in (d) to present the difference in blockade current of the two types of events. The expanded I-t trace was post-filtered at 0.1 kHz for presentation. Experiments were carried out at 20.0 °C in a 100 mM KCl (7.5% PEG, 5 mM MgCl 2 , 1 mM <t>ATP)</t> solution buffered to pH 7.6 by 66 mM Tris-HCl. Counts indicate the number of individual dsDNA unzipping events.
    Atp Sulphurylase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 76/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    New England Biolabs nonradiolabeled atp
    Phosphorylation of E6AP at T485 confers its interaction with 14-3-3γ. (Left) PKA and 14-3-3 phosphoconsensus motifs. (Right) Direct interaction assay with purified 14-3-3γ. Purified GST fusion proteins were either untreated or subjected to phosphorylation (indicated as “P”) with PKA in the presence of <t>nonradiolabeled</t> <t>ATP.</t> They were then incubated with purified recombinant 14-3-3γ. (Upper) After extensive washing, the bound protein was detected by Western blotting using anti-14-3-3γ antibody. (Lower) Ponceau staining of the nitrocellulose membrane.
    Nonradiolabeled Atp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 83/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Valiant γ 32 p atp
    Phosphorylation of E6AP at T485 confers its interaction with 14-3-3γ. (Left) PKA and 14-3-3 phosphoconsensus motifs. (Right) Direct interaction assay with purified 14-3-3γ. Purified GST fusion proteins were either untreated or subjected to phosphorylation (indicated as “P”) with PKA in the presence of <t>nonradiolabeled</t> <t>ATP.</t> They were then incubated with purified recombinant 14-3-3γ. (Upper) After extensive washing, the bound protein was detected by Western blotting using anti-14-3-3γ antibody. (Lower) Ponceau staining of the nitrocellulose membrane.
    γ 32 P Atp, supplied by Valiant, used in various techniques. Bioz Stars score: 95/100, based on 199 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    New England Biolabs dideoxy atp
    Phosphorylation of E6AP at T485 confers its interaction with 14-3-3γ. (Left) PKA and 14-3-3 phosphoconsensus motifs. (Right) Direct interaction assay with purified 14-3-3γ. Purified GST fusion proteins were either untreated or subjected to phosphorylation (indicated as “P”) with PKA in the presence of <t>nonradiolabeled</t> <t>ATP.</t> They were then incubated with purified recombinant 14-3-3γ. (Upper) After extensive washing, the bound protein was detected by Western blotting using anti-14-3-3γ antibody. (Lower) Ponceau staining of the nitrocellulose membrane.
    Dideoxy Atp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 78/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    New England Biolabs datp atp
    Amino acid 128–165 is responsible for direct binding with Hsp70 to prevent Apaf-1 binding and to aid in caspase-9 processing induced by cytochrome c <t>/ATP.</t> ( a ) His-tagged M1or M1Δ128–165 (10 μ g) was immobilized on Ni +2 overnight in HBS at 4 °C, and then washed and incubated with recombinant Hsp70 (10 μ g) for 4 h at 4 °C. Associated Hsp70 was detected by immunoblotting. Wild-type Hsp70 associated with native M1 but not with M1Δ128–165. ( b ) Jurkat cell extracts were Hsp70 immunodepleted, before recombinant Hsp70 and M1 or M1Δ128–165 proteins were added to it incubated and Apaf-1 was immunoprecipitated. Hsp70 did not associate with Apaf-1 when native M1 was present, but did associate in the absence of M1 or in the presence of M1Δ128–165 protein. ( c ) Co-addition of recombinant M1 enhances processing of procaspase-9 induced by <t>dATP</t> or ATP and cytochrome c in Jurkat cell extracts in the presence of wild-type Hsp70, whereas in the presence of M1Δ128–165 protein, caspase-9 activation is inhibited
    Datp Atp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 82/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    New England Biolabs atp containing buffer
    Amino acid 128–165 is responsible for direct binding with Hsp70 to prevent Apaf-1 binding and to aid in caspase-9 processing induced by cytochrome c <t>/ATP.</t> ( a ) His-tagged M1or M1Δ128–165 (10 μ g) was immobilized on Ni +2 overnight in HBS at 4 °C, and then washed and incubated with recombinant Hsp70 (10 μ g) for 4 h at 4 °C. Associated Hsp70 was detected by immunoblotting. Wild-type Hsp70 associated with native M1 but not with M1Δ128–165. ( b ) Jurkat cell extracts were Hsp70 immunodepleted, before recombinant Hsp70 and M1 or M1Δ128–165 proteins were added to it incubated and Apaf-1 was immunoprecipitated. Hsp70 did not associate with Apaf-1 when native M1 was present, but did associate in the absence of M1 or in the presence of M1Δ128–165 protein. ( c ) Co-addition of recombinant M1 enhances processing of procaspase-9 induced by <t>dATP</t> or ATP and cytochrome c in Jurkat cell extracts in the presence of wild-type Hsp70, whereas in the presence of M1Δ128–165 protein, caspase-9 activation is inhibited
    Atp Containing Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    New England Biolabs biotin atp
    Amino acid 128–165 is responsible for direct binding with Hsp70 to prevent Apaf-1 binding and to aid in caspase-9 processing induced by cytochrome c <t>/ATP.</t> ( a ) His-tagged M1or M1Δ128–165 (10 μ g) was immobilized on Ni +2 overnight in HBS at 4 °C, and then washed and incubated with recombinant Hsp70 (10 μ g) for 4 h at 4 °C. Associated Hsp70 was detected by immunoblotting. Wild-type Hsp70 associated with native M1 but not with M1Δ128–165. ( b ) Jurkat cell extracts were Hsp70 immunodepleted, before recombinant Hsp70 and M1 or M1Δ128–165 proteins were added to it incubated and Apaf-1 was immunoprecipitated. Hsp70 did not associate with Apaf-1 when native M1 was present, but did associate in the absence of M1 or in the presence of M1Δ128–165 protein. ( c ) Co-addition of recombinant M1 enhances processing of procaspase-9 induced by <t>dATP</t> or ATP and cytochrome c in Jurkat cell extracts in the presence of wild-type Hsp70, whereas in the presence of M1Δ128–165 protein, caspase-9 activation is inhibited
    Biotin Atp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 81/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    New England Biolabs atp sulfurylase
    General outline of the chemiluminescence detection of nitric oxide. Soluble guanylyl cyclase converts GTP to cGMP and pyrophosphate (PP i ) in reaction accelerated by NO. The product of the reaction, PP i , is converted to <t>ATP</t> by <t>ATP-sulfurylase.</t> Finally,
    Atp Sulfurylase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs t4 polynucleotide kinases
    Extending the half-life of siRNA duplexes prolongs the persistence of RNA interference in vivo. ( A ) Comparing the stability of unmodified siRNAs with siRNAs containing 2′-fluoro-uridine and 2′-fluoro-cytidine (2′-FU, 2′-FC) modifications ( a ) and thioate linkage (P–S) modifications ( b ). Unmodified or modified EGFP antisense strand siRNAs (AS) were 5′-labeled with [γ- 32 P]ATP by <t>T4</t> polynucleotide kinases. Duplex siRNAs were formed by annealing equal molar ratios of sense-strand (SS) siRNAs with the 5′- 32 P-labeled antisense strand. To analyze siRNA stability in HeLa cell extract, 50 pmole of siRNA was incubated with 500 μg of HeLa cell extract in 50 μL of reaction mixture containing 20 mM HEPES (pH 7.9), 100 mM KCl, 10 mM NaCl, 2 mM MgCl 2 , and 10% glycerol. At various time points, siRNAs were extracted and analyzed on 20% polyacrylamide gels containing 7 M urea followed by phosphorimage analysis (Fugi). ( B ) Kinetics of RNAi effects of duplex siRNA with 2′-fluoro-uridine and 2′-fluoro-cytidine modification in HeLa cells over a 144-h time course. The fluorescence intensity ratio of target (GFP) to control (RFP) protein was determined in the presence of unmodified dsRNA (blue bars) and duplex siRNA with 2′-fluoro-uridine and -cytidine modifications (DS-2′-FU, 2′-FC, cyan bar) and normalized to the ratio observed in the presence of mock-treated cells (red bars). Normalized ratios at
    T4 Polynucleotide Kinases, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs t4 dna ligase reaction buffer
    Extending the half-life of siRNA duplexes prolongs the persistence of RNA interference in vivo. ( A ) Comparing the stability of unmodified siRNAs with siRNAs containing 2′-fluoro-uridine and 2′-fluoro-cytidine (2′-FU, 2′-FC) modifications ( a ) and thioate linkage (P–S) modifications ( b ). Unmodified or modified EGFP antisense strand siRNAs (AS) were 5′-labeled with [γ- 32 P]ATP by <t>T4</t> polynucleotide kinases. Duplex siRNAs were formed by annealing equal molar ratios of sense-strand (SS) siRNAs with the 5′- 32 P-labeled antisense strand. To analyze siRNA stability in HeLa cell extract, 50 pmole of siRNA was incubated with 500 μg of HeLa cell extract in 50 μL of reaction mixture containing 20 mM HEPES (pH 7.9), 100 mM KCl, 10 mM NaCl, 2 mM MgCl 2 , and 10% glycerol. At various time points, siRNAs were extracted and analyzed on 20% polyacrylamide gels containing 7 M urea followed by phosphorimage analysis (Fugi). ( B ) Kinetics of RNAi effects of duplex siRNA with 2′-fluoro-uridine and 2′-fluoro-cytidine modification in HeLa cells over a 144-h time course. The fluorescence intensity ratio of target (GFP) to control (RFP) protein was determined in the presence of unmodified dsRNA (blue bars) and duplex siRNA with 2′-fluoro-uridine and -cytidine modifications (DS-2′-FU, 2′-FC, cyan bar) and normalized to the ratio observed in the presence of mock-treated cells (red bars). Normalized ratios at
    T4 Dna Ligase Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 887 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    New England Biolabs γ 32 p atp labeled 50 bp dna ladder
    BACE1 mRNA in human brain and pancreas contains the full-length 5′ UTR. ( A ) Schematic representation of the riboprobe. The riboprobe was designed to protect most of the transcript leader and 166 nt of the ORF. A full-length BACE1 5′ UTR was expected to protect a fragment of 536 nt (from 85 to 621), while the putative alternatively spliced variant was expected to protect a fragment of 403 nt (from 218 to 621). Arrows represent uAUGs, while black boxes represent uORFs. ( B ) Total RNAs from human brain (Br) and pancreas (Pan) have similar quality. Equal amounts of total RNAs were separate on agarose gel. ( C ) RNase protection assay. 10 μg of total RNA either from human brain or pancreas were incubated with 32 P-labeled riboprobe for BACE1 alone, or BACE1 and GAPDH together. Protected signal for BACE1 (∼500 bp) and GAPDH (∼240 bp) are indicated with arrows. Cont − RNA = control reaction without RNA, Br = human brain, Pan = human pancreas, cont + RNA = control reaction with yeast RNA, ladder is <t>γ-ATP</t> <t>labeled</t> 50 bp <t>DNA</t> marker. ( D ) qPCR analysis. The values were normalized on L19 expression (L19 ratio brain/pancreas = 0.6).
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    Millipore adenosine 5 o 3 thiotriphosphate
    BACE1 mRNA in human brain and pancreas contains the full-length 5′ UTR. ( A ) Schematic representation of the riboprobe. The riboprobe was designed to protect most of the transcript leader and 166 nt of the ORF. A full-length BACE1 5′ UTR was expected to protect a fragment of 536 nt (from 85 to 621), while the putative alternatively spliced variant was expected to protect a fragment of 403 nt (from 218 to 621). Arrows represent uAUGs, while black boxes represent uORFs. ( B ) Total RNAs from human brain (Br) and pancreas (Pan) have similar quality. Equal amounts of total RNAs were separate on agarose gel. ( C ) RNase protection assay. 10 μg of total RNA either from human brain or pancreas were incubated with 32 P-labeled riboprobe for BACE1 alone, or BACE1 and GAPDH together. Protected signal for BACE1 (∼500 bp) and GAPDH (∼240 bp) are indicated with arrows. Cont − RNA = control reaction without RNA, Br = human brain, Pan = human pancreas, cont + RNA = control reaction with yeast RNA, ladder is <t>γ-ATP</t> <t>labeled</t> 50 bp <t>DNA</t> marker. ( D ) qPCR analysis. The values were normalized on L19 expression (L19 ratio brain/pancreas = 0.6).
    Adenosine 5 O 3 Thiotriphosphate, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs atp•mgcl2
    BACE1 mRNA in human brain and pancreas contains the full-length 5′ UTR. ( A ) Schematic representation of the riboprobe. The riboprobe was designed to protect most of the transcript leader and 166 nt of the ORF. A full-length BACE1 5′ UTR was expected to protect a fragment of 536 nt (from 85 to 621), while the putative alternatively spliced variant was expected to protect a fragment of 403 nt (from 218 to 621). Arrows represent uAUGs, while black boxes represent uORFs. ( B ) Total RNAs from human brain (Br) and pancreas (Pan) have similar quality. Equal amounts of total RNAs were separate on agarose gel. ( C ) RNase protection assay. 10 μg of total RNA either from human brain or pancreas were incubated with 32 P-labeled riboprobe for BACE1 alone, or BACE1 and GAPDH together. Protected signal for BACE1 (∼500 bp) and GAPDH (∼240 bp) are indicated with arrows. Cont − RNA = control reaction without RNA, Br = human brain, Pan = human pancreas, cont + RNA = control reaction with yeast RNA, ladder is <t>γ-ATP</t> <t>labeled</t> 50 bp <t>DNA</t> marker. ( D ) qPCR analysis. The values were normalized on L19 expression (L19 ratio brain/pancreas = 0.6).
    Atp•Mgcl2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t4 kinase
    BACE1 mRNA in human brain and pancreas contains the full-length 5′ UTR. ( A ) Schematic representation of the riboprobe. The riboprobe was designed to protect most of the transcript leader and 166 nt of the ORF. A full-length BACE1 5′ UTR was expected to protect a fragment of 536 nt (from 85 to 621), while the putative alternatively spliced variant was expected to protect a fragment of 403 nt (from 218 to 621). Arrows represent uAUGs, while black boxes represent uORFs. ( B ) Total RNAs from human brain (Br) and pancreas (Pan) have similar quality. Equal amounts of total RNAs were separate on agarose gel. ( C ) RNase protection assay. 10 μg of total RNA either from human brain or pancreas were incubated with 32 P-labeled riboprobe for BACE1 alone, or BACE1 and GAPDH together. Protected signal for BACE1 (∼500 bp) and GAPDH (∼240 bp) are indicated with arrows. Cont − RNA = control reaction without RNA, Br = human brain, Pan = human pancreas, cont + RNA = control reaction with yeast RNA, ladder is <t>γ-ATP</t> <t>labeled</t> 50 bp <t>DNA</t> marker. ( D ) qPCR analysis. The values were normalized on L19 expression (L19 ratio brain/pancreas = 0.6).
    T4 Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 521 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    New England Biolabs pka kinase reactions
    BACE1 mRNA in human brain and pancreas contains the full-length 5′ UTR. ( A ) Schematic representation of the riboprobe. The riboprobe was designed to protect most of the transcript leader and 166 nt of the ORF. A full-length BACE1 5′ UTR was expected to protect a fragment of 536 nt (from 85 to 621), while the putative alternatively spliced variant was expected to protect a fragment of 403 nt (from 218 to 621). Arrows represent uAUGs, while black boxes represent uORFs. ( B ) Total RNAs from human brain (Br) and pancreas (Pan) have similar quality. Equal amounts of total RNAs were separate on agarose gel. ( C ) RNase protection assay. 10 μg of total RNA either from human brain or pancreas were incubated with 32 P-labeled riboprobe for BACE1 alone, or BACE1 and GAPDH together. Protected signal for BACE1 (∼500 bp) and GAPDH (∼240 bp) are indicated with arrows. Cont − RNA = control reaction without RNA, Br = human brain, Pan = human pancreas, cont + RNA = control reaction with yeast RNA, ladder is <t>γ-ATP</t> <t>labeled</t> 50 bp <t>DNA</t> marker. ( D ) qPCR analysis. The values were normalized on L19 expression (L19 ratio brain/pancreas = 0.6).
    Pka Kinase Reactions, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    New England Biolabs ck2 kinase
    <t>CK2-phosphorylated</t> NS5A-D2D3: phosphorylation sites, kinetics, and conformational changes
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    New England Biolabs datp solution
    <t>CK2-phosphorylated</t> NS5A-D2D3: phosphorylation sites, kinetics, and conformational changes
    Datp Solution, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    New England Biolabs cdc2 kinase
    Mitotic phosphorylation of RCC1 is essential in vivo. ( A ) RCC1 and RCC1S2,11A have the same GEF activity in vitro. ( B ) Phosphorylation of RCC1 does not affect its GEF activity in vitro. Purified 6His-RCC1 or 6His-RCC1S2,11A was phosphorylated by <t>Cdc2</t>
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    New England Biolabs t7 kinase
    Mitotic phosphorylation of RCC1 is essential in vivo. ( A ) RCC1 and RCC1S2,11A have the same GEF activity in vitro. ( B ) Phosphorylation of RCC1 does not affect its GEF activity in vitro. Purified 6His-RCC1 or 6His-RCC1S2,11A was phosphorylated by <t>Cdc2</t>
    T7 Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 80/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs polynuclotide kinase
    Mitotic phosphorylation of RCC1 is essential in vivo. ( A ) RCC1 and RCC1S2,11A have the same GEF activity in vitro. ( B ) Phosphorylation of RCC1 does not affect its GEF activity in vitro. Purified 6His-RCC1 or 6His-RCC1S2,11A was phosphorylated by <t>Cdc2</t>
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    New England Biolabs t4 nucleotide kinase
    Mitotic phosphorylation of RCC1 is essential in vivo. ( A ) RCC1 and RCC1S2,11A have the same GEF activity in vitro. ( B ) Phosphorylation of RCC1 does not affect its GEF activity in vitro. Purified 6His-RCC1 or 6His-RCC1S2,11A was phosphorylated by <t>Cdc2</t>
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    New England Biolabs gsk3 kinase
    Mitotic phosphorylation of RCC1 is essential in vivo. ( A ) RCC1 and RCC1S2,11A have the same GEF activity in vitro. ( B ) Phosphorylation of RCC1 does not affect its GEF activity in vitro. Purified 6His-RCC1 or 6His-RCC1S2,11A was phosphorylated by <t>Cdc2</t>
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    95
    New England Biolabs t4 dna ligase
    <t>T4</t> DNA relaxation within nanoslits after electrokinetic loading. Plots show the relaxation kinetics (0.4 mM ionic strength, New England Biolabs buffer 4) (see Materials and Methods ) of two DNA molecules [the first (black) enters nanoslits in a folded
    T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 38631 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    RIG-I:RNA conformation as probed by limited trypsin digestion time course. (A to D) RIG-I fragments were detected by Western blotting with a monoclonal antibody to the helicase domain. Cell extracts were incubated with trypsin in the absence (A [–RNA]) or presence of RNA ligands, including nonbinding control yeast tRNA (B [tRNA]), polyI:C (C), or polyU/UC (D), and with 1 mM ATP or AMP-PNP. Aliquots were removed from the reaction at various time points (minutes) post-addition of trypsin. Additional data are provided in Fig. S6 in the supplemental material. (E) Biotinylated RNAs were used in pulldown experiments to test for RNA binding by the 80-kDa and 55-kDa RIG-I fragments. Trypsin digests (+ tryp) and control lysates (− tryp) were incubated without RNA (-RNA), with nonbiotinylated polyU/UC RNA (-btn), with biotinylated polyU/UC RNA (can), or with biotinylated X RNA (X) in the presence of AMP-PNP. After 1.5 h, trypsin digestions were quenched by adding protease inhibitor and incubated with streptavidin paramagnetic beads. RIG-I present in the bead-bound fraction (BND) versus the input fraction (INP) was detected by Western blotting.

    Journal: mBio

    Article Title: RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling

    doi: 10.1128/mBio.00833-16

    Figure Lengend Snippet: RIG-I:RNA conformation as probed by limited trypsin digestion time course. (A to D) RIG-I fragments were detected by Western blotting with a monoclonal antibody to the helicase domain. Cell extracts were incubated with trypsin in the absence (A [–RNA]) or presence of RNA ligands, including nonbinding control yeast tRNA (B [tRNA]), polyI:C (C), or polyU/UC (D), and with 1 mM ATP or AMP-PNP. Aliquots were removed from the reaction at various time points (minutes) post-addition of trypsin. Additional data are provided in Fig. S6 in the supplemental material. (E) Biotinylated RNAs were used in pulldown experiments to test for RNA binding by the 80-kDa and 55-kDa RIG-I fragments. Trypsin digests (+ tryp) and control lysates (− tryp) were incubated without RNA (-RNA), with nonbiotinylated polyU/UC RNA (-btn), with biotinylated polyU/UC RNA (can), or with biotinylated X RNA (X) in the presence of AMP-PNP. After 1.5 h, trypsin digestions were quenched by adding protease inhibitor and incubated with streptavidin paramagnetic beads. RIG-I present in the bead-bound fraction (BND) versus the input fraction (INP) was detected by Western blotting.

    Article Snippet: The reaction volume was 500 µl and included 1 mg to 2 mg total lysate protein, RNA (33 nM to 1 µM), and 2 mM AMP-PNP (Roche) or ATP (New England Biolabs).

    Techniques: Western Blot, Incubation, RNA Binding Assay, Protease Inhibitor

    RNA mod and RIG-I trypsin sensitivity. (A to D) Digestion of 293T cell lysate for 2 h in the presence of polyU/UC RNA with the indicated modifications or canonical nucleotides (can), at increasing polyU/UC RNA concentrations (0, 12.5, 25, 50, 100, 200, 400, 800, and 1,600 nM), in the presence of 2 mM ATP or AMP-PNP. Data represent results of Western blotting for 55-kDa and 80-kDa fragments of RIG-I with anti-helicase antibody. (E) Digestions of 293T cell lysate performed for 1.5 h in the presence of AMP-PNP and polyU/UC RNA with the indicated modifications, at increasing concentrations (0, 33, 100, 300, and 900 nM), or mass equivalent of polyI:C.

    Journal: mBio

    Article Title: RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling

    doi: 10.1128/mBio.00833-16

    Figure Lengend Snippet: RNA mod and RIG-I trypsin sensitivity. (A to D) Digestion of 293T cell lysate for 2 h in the presence of polyU/UC RNA with the indicated modifications or canonical nucleotides (can), at increasing polyU/UC RNA concentrations (0, 12.5, 25, 50, 100, 200, 400, 800, and 1,600 nM), in the presence of 2 mM ATP or AMP-PNP. Data represent results of Western blotting for 55-kDa and 80-kDa fragments of RIG-I with anti-helicase antibody. (E) Digestions of 293T cell lysate performed for 1.5 h in the presence of AMP-PNP and polyU/UC RNA with the indicated modifications, at increasing concentrations (0, 33, 100, 300, and 900 nM), or mass equivalent of polyI:C.

    Article Snippet: The reaction volume was 500 µl and included 1 mg to 2 mg total lysate protein, RNA (33 nM to 1 µM), and 2 mM AMP-PNP (Roche) or ATP (New England Biolabs).

    Techniques: Western Blot

    Detecting backbone damage in a DNA duplex. (a–b) Current histograms showing the measured blockade currents for 14 µM solutions of the (a) nicked and (b) repaired duplexes, respectively. (c) Current histogram of a mixture of the same amount (7 µM) of nicked and repaired duplexes. (d) Sample current-time traces generated during dsDNA residence events, showing the difference in blockade currents, I N and I R , for nicked DNA and repaired DNA, respectively. The trace was post-filtered at 1 kHz for presentation. An expanded view of the same I-t trace is also shown in (d) to present the difference in blockade current of the two types of events. The expanded I-t trace was post-filtered at 0.1 kHz for presentation. Experiments were carried out at 20.0 °C in a 100 mM KCl (7.5% PEG, 5 mM MgCl 2 , 1 mM ATP) solution buffered to pH 7.6 by 66 mM Tris-HCl. Counts indicate the number of individual dsDNA unzipping events.

    Journal: ACS nano

    Article Title: Kinetics of T3-DNA Ligase-Catalyzed Phosphodiester Bond Formation Measured using the α-Hemolysin Nanopore

    doi: 10.1021/acsnano.6b05995

    Figure Lengend Snippet: Detecting backbone damage in a DNA duplex. (a–b) Current histograms showing the measured blockade currents for 14 µM solutions of the (a) nicked and (b) repaired duplexes, respectively. (c) Current histogram of a mixture of the same amount (7 µM) of nicked and repaired duplexes. (d) Sample current-time traces generated during dsDNA residence events, showing the difference in blockade currents, I N and I R , for nicked DNA and repaired DNA, respectively. The trace was post-filtered at 1 kHz for presentation. An expanded view of the same I-t trace is also shown in (d) to present the difference in blockade current of the two types of events. The expanded I-t trace was post-filtered at 0.1 kHz for presentation. Experiments were carried out at 20.0 °C in a 100 mM KCl (7.5% PEG, 5 mM MgCl 2 , 1 mM ATP) solution buffered to pH 7.6 by 66 mM Tris-HCl. Counts indicate the number of individual dsDNA unzipping events.

    Article Snippet: T3-DNA ligase at 3 × 106 units/mL and its cofactor ATP were purchased from New England Biolabs, Ipswich, MA.

    Techniques: Generated

    Phosphorylation of E6AP at T485 confers its interaction with 14-3-3γ. (Left) PKA and 14-3-3 phosphoconsensus motifs. (Right) Direct interaction assay with purified 14-3-3γ. Purified GST fusion proteins were either untreated or subjected to phosphorylation (indicated as “P”) with PKA in the presence of nonradiolabeled ATP. They were then incubated with purified recombinant 14-3-3γ. (Upper) After extensive washing, the bound protein was detected by Western blotting using anti-14-3-3γ antibody. (Lower) Ponceau staining of the nitrocellulose membrane.

    Journal: Journal of Virology

    Article Title: Human Papillomavirus 16 (HPV-16), HPV-18, and HPV-31 E6 Override the Normal Phosphoregulation of E6AP Enzymatic Activity

    doi: 10.1128/JVI.01390-17

    Figure Lengend Snippet: Phosphorylation of E6AP at T485 confers its interaction with 14-3-3γ. (Left) PKA and 14-3-3 phosphoconsensus motifs. (Right) Direct interaction assay with purified 14-3-3γ. Purified GST fusion proteins were either untreated or subjected to phosphorylation (indicated as “P”) with PKA in the presence of nonradiolabeled ATP. They were then incubated with purified recombinant 14-3-3γ. (Upper) After extensive washing, the bound protein was detected by Western blotting using anti-14-3-3γ antibody. (Lower) Ponceau staining of the nitrocellulose membrane.

    Article Snippet: In vitro phosphorylation of the GST fusion proteins was carried out as described above in the presence of 10 μM nonradiolabeled ATP (NEB).

    Techniques: Purification, Incubation, Recombinant, Western Blot, Staining

    Amino acid 128–165 is responsible for direct binding with Hsp70 to prevent Apaf-1 binding and to aid in caspase-9 processing induced by cytochrome c /ATP. ( a ) His-tagged M1or M1Δ128–165 (10 μ g) was immobilized on Ni +2 overnight in HBS at 4 °C, and then washed and incubated with recombinant Hsp70 (10 μ g) for 4 h at 4 °C. Associated Hsp70 was detected by immunoblotting. Wild-type Hsp70 associated with native M1 but not with M1Δ128–165. ( b ) Jurkat cell extracts were Hsp70 immunodepleted, before recombinant Hsp70 and M1 or M1Δ128–165 proteins were added to it incubated and Apaf-1 was immunoprecipitated. Hsp70 did not associate with Apaf-1 when native M1 was present, but did associate in the absence of M1 or in the presence of M1Δ128–165 protein. ( c ) Co-addition of recombinant M1 enhances processing of procaspase-9 induced by dATP or ATP and cytochrome c in Jurkat cell extracts in the presence of wild-type Hsp70, whereas in the presence of M1Δ128–165 protein, caspase-9 activation is inhibited

    Journal: Cell Death & Disease

    Article Title: Cell death regulation during influenza A virus infection by matrix (M1) protein: a model of viral control over the cellular survival pathway

    doi: 10.1038/cddis.2011.75

    Figure Lengend Snippet: Amino acid 128–165 is responsible for direct binding with Hsp70 to prevent Apaf-1 binding and to aid in caspase-9 processing induced by cytochrome c /ATP. ( a ) His-tagged M1or M1Δ128–165 (10 μ g) was immobilized on Ni +2 overnight in HBS at 4 °C, and then washed and incubated with recombinant Hsp70 (10 μ g) for 4 h at 4 °C. Associated Hsp70 was detected by immunoblotting. Wild-type Hsp70 associated with native M1 but not with M1Δ128–165. ( b ) Jurkat cell extracts were Hsp70 immunodepleted, before recombinant Hsp70 and M1 or M1Δ128–165 proteins were added to it incubated and Apaf-1 was immunoprecipitated. Hsp70 did not associate with Apaf-1 when native M1 was present, but did associate in the absence of M1 or in the presence of M1Δ128–165 protein. ( c ) Co-addition of recombinant M1 enhances processing of procaspase-9 induced by dATP or ATP and cytochrome c in Jurkat cell extracts in the presence of wild-type Hsp70, whereas in the presence of M1Δ128–165 protein, caspase-9 activation is inhibited

    Article Snippet: Endogenous caspase-9 was activated by adding 1 mM dATP/ATP (NEB) and 10 μ M horse-heart cyt c (Sigma) to cytosolic extracts (300 μ g protein equivalent) and incubating at 37 °C for respective times.

    Techniques: Binding Assay, Incubation, Recombinant, Immunoprecipitation, Activation Assay

    General outline of the chemiluminescence detection of nitric oxide. Soluble guanylyl cyclase converts GTP to cGMP and pyrophosphate (PP i ) in reaction accelerated by NO. The product of the reaction, PP i , is converted to ATP by ATP-sulfurylase. Finally,

    Journal: Biochemical and biophysical research communications

    Article Title: Detection of nitric oxide production in cell cultures by luciferin–luciferase chemiluminescence

    doi: 10.1016/j.bbrc.2015.08.001

    Figure Lengend Snippet: General outline of the chemiluminescence detection of nitric oxide. Soluble guanylyl cyclase converts GTP to cGMP and pyrophosphate (PP i ) in reaction accelerated by NO. The product of the reaction, PP i , is converted to ATP by ATP-sulfurylase. Finally,

    Article Snippet: Guanylyl cyclase (ALX-202-039) was from Alexis Biochemicals; ATP sulfurylase (M0394L) was from New England Biolabs; inorganic pyrophosphatase (I1891), firefly luciferase (L9506), superoxide dismutase (S9697), BSA (A7906) were from Sigma–Aldrich.

    Techniques:

    Extending the half-life of siRNA duplexes prolongs the persistence of RNA interference in vivo. ( A ) Comparing the stability of unmodified siRNAs with siRNAs containing 2′-fluoro-uridine and 2′-fluoro-cytidine (2′-FU, 2′-FC) modifications ( a ) and thioate linkage (P–S) modifications ( b ). Unmodified or modified EGFP antisense strand siRNAs (AS) were 5′-labeled with [γ- 32 P]ATP by T4 polynucleotide kinases. Duplex siRNAs were formed by annealing equal molar ratios of sense-strand (SS) siRNAs with the 5′- 32 P-labeled antisense strand. To analyze siRNA stability in HeLa cell extract, 50 pmole of siRNA was incubated with 500 μg of HeLa cell extract in 50 μL of reaction mixture containing 20 mM HEPES (pH 7.9), 100 mM KCl, 10 mM NaCl, 2 mM MgCl 2 , and 10% glycerol. At various time points, siRNAs were extracted and analyzed on 20% polyacrylamide gels containing 7 M urea followed by phosphorimage analysis (Fugi). ( B ) Kinetics of RNAi effects of duplex siRNA with 2′-fluoro-uridine and 2′-fluoro-cytidine modification in HeLa cells over a 144-h time course. The fluorescence intensity ratio of target (GFP) to control (RFP) protein was determined in the presence of unmodified dsRNA (blue bars) and duplex siRNA with 2′-fluoro-uridine and -cytidine modifications (DS-2′-FU, 2′-FC, cyan bar) and normalized to the ratio observed in the presence of mock-treated cells (red bars). Normalized ratios at

    Journal: RNA

    Article Title: siRNA function in RNAi: A chemical modification analysis

    doi: 10.1261/rna.5103703

    Figure Lengend Snippet: Extending the half-life of siRNA duplexes prolongs the persistence of RNA interference in vivo. ( A ) Comparing the stability of unmodified siRNAs with siRNAs containing 2′-fluoro-uridine and 2′-fluoro-cytidine (2′-FU, 2′-FC) modifications ( a ) and thioate linkage (P–S) modifications ( b ). Unmodified or modified EGFP antisense strand siRNAs (AS) were 5′-labeled with [γ- 32 P]ATP by T4 polynucleotide kinases. Duplex siRNAs were formed by annealing equal molar ratios of sense-strand (SS) siRNAs with the 5′- 32 P-labeled antisense strand. To analyze siRNA stability in HeLa cell extract, 50 pmole of siRNA was incubated with 500 μg of HeLa cell extract in 50 μL of reaction mixture containing 20 mM HEPES (pH 7.9), 100 mM KCl, 10 mM NaCl, 2 mM MgCl 2 , and 10% glycerol. At various time points, siRNAs were extracted and analyzed on 20% polyacrylamide gels containing 7 M urea followed by phosphorimage analysis (Fugi). ( B ) Kinetics of RNAi effects of duplex siRNA with 2′-fluoro-uridine and 2′-fluoro-cytidine modification in HeLa cells over a 144-h time course. The fluorescence intensity ratio of target (GFP) to control (RFP) protein was determined in the presence of unmodified dsRNA (blue bars) and duplex siRNA with 2′-fluoro-uridine and -cytidine modifications (DS-2′-FU, 2′-FC, cyan bar) and normalized to the ratio observed in the presence of mock-treated cells (red bars). Normalized ratios at

    Article Snippet: Unmodified or modified EGFP antisense strand siRNAs were 5′-labeled with [γ-32 P]ATP (3000 Ci/mM; ICN) by T4 polynucleotide kinases (New England Biolabs) at 37°C for 1 h and chase-kinased by adding 1 mM ATP at 37°C for 15 min. Free ATP and kinases were removed by the QIAGEN nucleotide removal kit.

    Techniques: In Vivo, Modification, Labeling, Incubation, Fluorescence

    BACE1 mRNA in human brain and pancreas contains the full-length 5′ UTR. ( A ) Schematic representation of the riboprobe. The riboprobe was designed to protect most of the transcript leader and 166 nt of the ORF. A full-length BACE1 5′ UTR was expected to protect a fragment of 536 nt (from 85 to 621), while the putative alternatively spliced variant was expected to protect a fragment of 403 nt (from 218 to 621). Arrows represent uAUGs, while black boxes represent uORFs. ( B ) Total RNAs from human brain (Br) and pancreas (Pan) have similar quality. Equal amounts of total RNAs were separate on agarose gel. ( C ) RNase protection assay. 10 μg of total RNA either from human brain or pancreas were incubated with 32 P-labeled riboprobe for BACE1 alone, or BACE1 and GAPDH together. Protected signal for BACE1 (∼500 bp) and GAPDH (∼240 bp) are indicated with arrows. Cont − RNA = control reaction without RNA, Br = human brain, Pan = human pancreas, cont + RNA = control reaction with yeast RNA, ladder is γ-ATP labeled 50 bp DNA marker. ( D ) qPCR analysis. The values were normalized on L19 expression (L19 ratio brain/pancreas = 0.6).

    Journal: Nucleic Acids Research

    Article Title: Complex translational regulation of BACE1 involves upstream AUGs and stimulatory elements within the 5? untranslated region

    doi: 10.1093/nar/gkm191

    Figure Lengend Snippet: BACE1 mRNA in human brain and pancreas contains the full-length 5′ UTR. ( A ) Schematic representation of the riboprobe. The riboprobe was designed to protect most of the transcript leader and 166 nt of the ORF. A full-length BACE1 5′ UTR was expected to protect a fragment of 536 nt (from 85 to 621), while the putative alternatively spliced variant was expected to protect a fragment of 403 nt (from 218 to 621). Arrows represent uAUGs, while black boxes represent uORFs. ( B ) Total RNAs from human brain (Br) and pancreas (Pan) have similar quality. Equal amounts of total RNAs were separate on agarose gel. ( C ) RNase protection assay. 10 μg of total RNA either from human brain or pancreas were incubated with 32 P-labeled riboprobe for BACE1 alone, or BACE1 and GAPDH together. Protected signal for BACE1 (∼500 bp) and GAPDH (∼240 bp) are indicated with arrows. Cont − RNA = control reaction without RNA, Br = human brain, Pan = human pancreas, cont + RNA = control reaction with yeast RNA, ladder is γ-ATP labeled 50 bp DNA marker. ( D ) qPCR analysis. The values were normalized on L19 expression (L19 ratio brain/pancreas = 0.6).

    Article Snippet: A [γ-32 P]-ATP labeled 50 bp DNA ladder (New England Biolabs, Beverly, MA, USA) was used as a size reference on the gel.

    Techniques: Variant Assay, Agarose Gel Electrophoresis, Rnase Protection Assay, Incubation, Labeling, Marker, Real-time Polymerase Chain Reaction, Expressing

    CK2-phosphorylated NS5A-D2D3: phosphorylation sites, kinetics, and conformational changes

    Journal: Biophysical Journal

    Article Title: The Disordered Region of the HCV Protein NS5A: Conformational Dynamics, SH3 Binding, and Phosphorylation

    doi: 10.1016/j.bpj.2015.06.040

    Figure Lengend Snippet: CK2-phosphorylated NS5A-D2D3: phosphorylation sites, kinetics, and conformational changes

    Article Snippet: ATP was added just before the start of the reaction to a final concentration of 5 mM, and 4000 U CK2 kinase (New England BioLabs, Ipswich, MA) was added to the 300 μ L sample.

    Techniques:

    Phosphorylation study of NS5A-D2D3 by CK2. ( a ) 1 H- 15 N correlation spectra of NS5A-D2D3 ( green ) measured 380 min after adding CK2 kinase. The spectral region, displaying cross peaks of phosphoserine and phosphothreonine residues (annotated in the

    Journal: Biophysical Journal

    Article Title: The Disordered Region of the HCV Protein NS5A: Conformational Dynamics, SH3 Binding, and Phosphorylation

    doi: 10.1016/j.bpj.2015.06.040

    Figure Lengend Snippet: Phosphorylation study of NS5A-D2D3 by CK2. ( a ) 1 H- 15 N correlation spectra of NS5A-D2D3 ( green ) measured 380 min after adding CK2 kinase. The spectral region, displaying cross peaks of phosphoserine and phosphothreonine residues (annotated in the

    Article Snippet: ATP was added just before the start of the reaction to a final concentration of 5 mM, and 4000 U CK2 kinase (New England BioLabs, Ipswich, MA) was added to the 300 μ L sample.

    Techniques:

    Mitotic phosphorylation of RCC1 is essential in vivo. ( A ) RCC1 and RCC1S2,11A have the same GEF activity in vitro. ( B ) Phosphorylation of RCC1 does not affect its GEF activity in vitro. Purified 6His-RCC1 or 6His-RCC1S2,11A was phosphorylated by Cdc2

    Journal: Genes & Development

    Article Title: Phosphorylation of RCC1 in mitosis is essential for producing a high RanGTP concentration on chromosomes and for spindle assembly in mammalian cells

    doi: 10.1101/gad.1177304

    Figure Lengend Snippet: Mitotic phosphorylation of RCC1 is essential in vivo. ( A ) RCC1 and RCC1S2,11A have the same GEF activity in vitro. ( B ) Phosphorylation of RCC1 does not affect its GEF activity in vitro. Purified 6His-RCC1 or 6His-RCC1S2,11A was phosphorylated by Cdc2

    Article Snippet: For GEF assays, 0.44 nM purified 6His-RCC1 or 6His-RCC1S2,11A was treated with buffer or Cdc2 kinase (New England BioLab), and then used to catalyze the nucleotide release of 1 μM RanGDP[H3 ] as described ( ).

    Techniques: In Vivo, Activity Assay, In Vitro, Purification

    T4 DNA relaxation within nanoslits after electrokinetic loading. Plots show the relaxation kinetics (0.4 mM ionic strength, New England Biolabs buffer 4) (see Materials and Methods ) of two DNA molecules [the first (black) enters nanoslits in a folded

    Journal:

    Article Title: A single-molecule barcoding system using nanoslits for DNA analysis

    doi: 10.1073/pnas.0611151104

    Figure Lengend Snippet: T4 DNA relaxation within nanoslits after electrokinetic loading. Plots show the relaxation kinetics (0.4 mM ionic strength, New England Biolabs buffer 4) (see Materials and Methods ) of two DNA molecules [the first (black) enters nanoslits in a folded

    Article Snippet: Preexisting nicks were repaired by using 2 units of T4 DNA ligase (1 mM ATP) at 16°C for 2 h, with a total volume of 17.5 μl of New England Biolabs buffer 4 or New England Biolabs buffer 2.

    Techniques: