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  • 99
    New England Biolabs datp
    tRNA quantification using OTTER ( A ) Schematic drawing of tRNA fluorescence labeling reaction in OTTER. A target tRNA is first hybridized with a specifically-designed antisense oligo DNA with the 5’-extension of dAdTdTdTdT (Type 1 oligo DNA). The overhang in the tRNA/DNA hybrid was filled by Klenow fragment (3’-5’ exo − ) with <t>dATP</t> and fluorescence-labeled dUTP (such as <t>TMR-dUTP;</t> marked by star) as substrates. ( B ) An example for tRNA-Arg UCU quantification. Typical reactions of OTTER for tRNA-Arg UCU were analyzed by urea-PAGE and scanned with a fluorescence scanner. The fluorescence-labeled tRNA-Arg UCU (closed triangle) as well as the fluorescence-labeled template oligo DNA by a weak reverse transcription activity of the Klenow fragment (asterisk) were detected. Since the 3’ end of the oligo DNA falls on the TΨC region rather conserved even among different tRNA species, unrelated tRNAs also acted as templates to produce the strong signal. Three replicates of the reaction were analyzed. The amounts of the standard TMR-oligo DNA on the gel (open triangle) were 0.500, 0.250, 0.100, 0.050, and 0.020 pmol/lane. ( C ) The three OTTER reaction products for tRNA-Arg UCU shown in ( B ) (“+” lanes) were subjected to Northern blotting with a reaction without the template oligo DNA (“−” lane).
    Datp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3884 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher datp
    tRNA quantification using OTTER ( A ) Schematic drawing of tRNA fluorescence labeling reaction in OTTER. A target tRNA is first hybridized with a specifically-designed antisense oligo DNA with the 5’-extension of dAdTdTdTdT (Type 1 oligo DNA). The overhang in the tRNA/DNA hybrid was filled by Klenow fragment (3’-5’ exo − ) with <t>dATP</t> and fluorescence-labeled dUTP (such as <t>TMR-dUTP;</t> marked by star) as substrates. ( B ) An example for tRNA-Arg UCU quantification. Typical reactions of OTTER for tRNA-Arg UCU were analyzed by urea-PAGE and scanned with a fluorescence scanner. The fluorescence-labeled tRNA-Arg UCU (closed triangle) as well as the fluorescence-labeled template oligo DNA by a weak reverse transcription activity of the Klenow fragment (asterisk) were detected. Since the 3’ end of the oligo DNA falls on the TΨC region rather conserved even among different tRNA species, unrelated tRNAs also acted as templates to produce the strong signal. Three replicates of the reaction were analyzed. The amounts of the standard TMR-oligo DNA on the gel (open triangle) were 0.500, 0.250, 0.100, 0.050, and 0.020 pmol/lane. ( C ) The three OTTER reaction products for tRNA-Arg UCU shown in ( B ) (“+” lanes) were subjected to Northern blotting with a reaction without the template oligo DNA (“−” lane).
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    95
    Millipore datp
    tRNA quantification using OTTER ( A ) Schematic drawing of tRNA fluorescence labeling reaction in OTTER. A target tRNA is first hybridized with a specifically-designed antisense oligo DNA with the 5’-extension of dAdTdTdTdT (Type 1 oligo DNA). The overhang in the tRNA/DNA hybrid was filled by Klenow fragment (3’-5’ exo − ) with <t>dATP</t> and fluorescence-labeled dUTP (such as <t>TMR-dUTP;</t> marked by star) as substrates. ( B ) An example for tRNA-Arg UCU quantification. Typical reactions of OTTER for tRNA-Arg UCU were analyzed by urea-PAGE and scanned with a fluorescence scanner. The fluorescence-labeled tRNA-Arg UCU (closed triangle) as well as the fluorescence-labeled template oligo DNA by a weak reverse transcription activity of the Klenow fragment (asterisk) were detected. Since the 3’ end of the oligo DNA falls on the TΨC region rather conserved even among different tRNA species, unrelated tRNAs also acted as templates to produce the strong signal. Three replicates of the reaction were analyzed. The amounts of the standard TMR-oligo DNA on the gel (open triangle) were 0.500, 0.250, 0.100, 0.050, and 0.020 pmol/lane. ( C ) The three OTTER reaction products for tRNA-Arg UCU shown in ( B ) (“+” lanes) were subjected to Northern blotting with a reaction without the template oligo DNA (“−” lane).
    Datp, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 1397 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs 200um datp
    tRNA quantification using OTTER ( A ) Schematic drawing of tRNA fluorescence labeling reaction in OTTER. A target tRNA is first hybridized with a specifically-designed antisense oligo DNA with the 5’-extension of dAdTdTdTdT (Type 1 oligo DNA). The overhang in the tRNA/DNA hybrid was filled by Klenow fragment (3’-5’ exo − ) with <t>dATP</t> and fluorescence-labeled dUTP (such as <t>TMR-dUTP;</t> marked by star) as substrates. ( B ) An example for tRNA-Arg UCU quantification. Typical reactions of OTTER for tRNA-Arg UCU were analyzed by urea-PAGE and scanned with a fluorescence scanner. The fluorescence-labeled tRNA-Arg UCU (closed triangle) as well as the fluorescence-labeled template oligo DNA by a weak reverse transcription activity of the Klenow fragment (asterisk) were detected. Since the 3’ end of the oligo DNA falls on the TΨC region rather conserved even among different tRNA species, unrelated tRNAs also acted as templates to produce the strong signal. Three replicates of the reaction were analyzed. The amounts of the standard TMR-oligo DNA on the gel (open triangle) were 0.500, 0.250, 0.100, 0.050, and 0.020 pmol/lane. ( C ) The three OTTER reaction products for tRNA-Arg UCU shown in ( B ) (“+” lanes) were subjected to Northern blotting with a reaction without the template oligo DNA (“−” lane).
    200um Datp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher biotin 14 datp
    tRNA quantification using OTTER ( A ) Schematic drawing of tRNA fluorescence labeling reaction in OTTER. A target tRNA is first hybridized with a specifically-designed antisense oligo DNA with the 5’-extension of dAdTdTdTdT (Type 1 oligo DNA). The overhang in the tRNA/DNA hybrid was filled by Klenow fragment (3’-5’ exo − ) with <t>dATP</t> and fluorescence-labeled dUTP (such as <t>TMR-dUTP;</t> marked by star) as substrates. ( B ) An example for tRNA-Arg UCU quantification. Typical reactions of OTTER for tRNA-Arg UCU were analyzed by urea-PAGE and scanned with a fluorescence scanner. The fluorescence-labeled tRNA-Arg UCU (closed triangle) as well as the fluorescence-labeled template oligo DNA by a weak reverse transcription activity of the Klenow fragment (asterisk) were detected. Since the 3’ end of the oligo DNA falls on the TΨC region rather conserved even among different tRNA species, unrelated tRNAs also acted as templates to produce the strong signal. Three replicates of the reaction were analyzed. The amounts of the standard TMR-oligo DNA on the gel (open triangle) were 0.500, 0.250, 0.100, 0.050, and 0.020 pmol/lane. ( C ) The three OTTER reaction products for tRNA-Arg UCU shown in ( B ) (“+” lanes) were subjected to Northern blotting with a reaction without the template oligo DNA (“−” lane).
    Biotin 14 Datp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 1836 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    New England Biolabs bst dna polymerase lg frag
    tRNA quantification using OTTER ( A ) Schematic drawing of tRNA fluorescence labeling reaction in OTTER. A target tRNA is first hybridized with a specifically-designed antisense oligo DNA with the 5’-extension of dAdTdTdTdT (Type 1 oligo DNA). The overhang in the tRNA/DNA hybrid was filled by Klenow fragment (3’-5’ exo − ) with <t>dATP</t> and fluorescence-labeled dUTP (such as <t>TMR-dUTP;</t> marked by star) as substrates. ( B ) An example for tRNA-Arg UCU quantification. Typical reactions of OTTER for tRNA-Arg UCU were analyzed by urea-PAGE and scanned with a fluorescence scanner. The fluorescence-labeled tRNA-Arg UCU (closed triangle) as well as the fluorescence-labeled template oligo DNA by a weak reverse transcription activity of the Klenow fragment (asterisk) were detected. Since the 3’ end of the oligo DNA falls on the TΨC region rather conserved even among different tRNA species, unrelated tRNAs also acted as templates to produce the strong signal. Three replicates of the reaction were analyzed. The amounts of the standard TMR-oligo DNA on the gel (open triangle) were 0.500, 0.250, 0.100, 0.050, and 0.020 pmol/lane. ( C ) The three OTTER reaction products for tRNA-Arg UCU shown in ( B ) (“+” lanes) were subjected to Northern blotting with a reaction without the template oligo DNA (“−” lane).
    Bst Dna Polymerase Lg Frag, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs terminal deoxynucleotidyl transferase
    Schematic representation of MGK. (a) Mixed library is grown in a reference and selective condition, and genomic DNA is isolated from each population. (b) Using genomic DNA as template, single-stranded DNA flanks are generated by linear extension of outward-facing insertion cassette-specific biotinylated primers (blue arrows). (c) The biotinylated flanks are separated from the template using streptavidin-coated magnetic beads, and polyadenylated at the 3'-ends using terminal <t>deoxynucleotidyl</t> transferase in the presence of dATP. (d) Microarray targets are PCR-amplified using an oligo d(T) primer (red arrows) and a nested Km r -specific primer (black arrows). Amino-allyl dUTP is incorporated during this step. (e) Fluorescent dyes are conjugated to microarray targets. (f) Differentially labeled targets are mixed and hybridized to a custom DNA microarray. Km r , kanamycin resistance; MGK, Monitoring of Gene Knockouts; PCR, polymerase chain reaction.
    Terminal Deoxynucleotidyl Transferase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 184 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    tRNA quantification using OTTER ( A ) Schematic drawing of tRNA fluorescence labeling reaction in OTTER. A target tRNA is first hybridized with a specifically-designed antisense oligo DNA with the 5’-extension of dAdTdTdTdT (Type 1 oligo DNA). The overhang in the tRNA/DNA hybrid was filled by Klenow fragment (3’-5’ exo − ) with dATP and fluorescence-labeled dUTP (such as TMR-dUTP; marked by star) as substrates. ( B ) An example for tRNA-Arg UCU quantification. Typical reactions of OTTER for tRNA-Arg UCU were analyzed by urea-PAGE and scanned with a fluorescence scanner. The fluorescence-labeled tRNA-Arg UCU (closed triangle) as well as the fluorescence-labeled template oligo DNA by a weak reverse transcription activity of the Klenow fragment (asterisk) were detected. Since the 3’ end of the oligo DNA falls on the TΨC region rather conserved even among different tRNA species, unrelated tRNAs also acted as templates to produce the strong signal. Three replicates of the reaction were analyzed. The amounts of the standard TMR-oligo DNA on the gel (open triangle) were 0.500, 0.250, 0.100, 0.050, and 0.020 pmol/lane. ( C ) The three OTTER reaction products for tRNA-Arg UCU shown in ( B ) (“+” lanes) were subjected to Northern blotting with a reaction without the template oligo DNA (“−” lane).

    Journal: bioRxiv

    Article Title: OTTER, a new method quantifying absolute amounts of tRNAs

    doi: 10.1101/2020.05.18.101501

    Figure Lengend Snippet: tRNA quantification using OTTER ( A ) Schematic drawing of tRNA fluorescence labeling reaction in OTTER. A target tRNA is first hybridized with a specifically-designed antisense oligo DNA with the 5’-extension of dAdTdTdTdT (Type 1 oligo DNA). The overhang in the tRNA/DNA hybrid was filled by Klenow fragment (3’-5’ exo − ) with dATP and fluorescence-labeled dUTP (such as TMR-dUTP; marked by star) as substrates. ( B ) An example for tRNA-Arg UCU quantification. Typical reactions of OTTER for tRNA-Arg UCU were analyzed by urea-PAGE and scanned with a fluorescence scanner. The fluorescence-labeled tRNA-Arg UCU (closed triangle) as well as the fluorescence-labeled template oligo DNA by a weak reverse transcription activity of the Klenow fragment (asterisk) were detected. Since the 3’ end of the oligo DNA falls on the TΨC region rather conserved even among different tRNA species, unrelated tRNAs also acted as templates to produce the strong signal. Three replicates of the reaction were analyzed. The amounts of the standard TMR-oligo DNA on the gel (open triangle) were 0.500, 0.250, 0.100, 0.050, and 0.020 pmol/lane. ( C ) The three OTTER reaction products for tRNA-Arg UCU shown in ( B ) (“+” lanes) were subjected to Northern blotting with a reaction without the template oligo DNA (“−” lane).

    Article Snippet: After adding final concentration of 10 μM TMR-dUTP (Roche Diagnostics, Basel, Switzerland), 250 μM dATP, 10 mM MgCl2 , and 2.0 units of Klenow Fragment (3’-5’ exo− )(New England Biolabs, Ipswich, Massachusetts, USA), total 10 μl of the mixture was incubate at 37°C for 90 min.

    Techniques: Fluorescence, Labeling, Polyacrylamide Gel Electrophoresis, Activity Assay, Northern Blot

    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: ATP (NEB, P0756S) or dATP (NEB, N0440S) was used at 1 mM/ml.

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

    Schematic representation of MGK. (a) Mixed library is grown in a reference and selective condition, and genomic DNA is isolated from each population. (b) Using genomic DNA as template, single-stranded DNA flanks are generated by linear extension of outward-facing insertion cassette-specific biotinylated primers (blue arrows). (c) The biotinylated flanks are separated from the template using streptavidin-coated magnetic beads, and polyadenylated at the 3'-ends using terminal deoxynucleotidyl transferase in the presence of dATP. (d) Microarray targets are PCR-amplified using an oligo d(T) primer (red arrows) and a nested Km r -specific primer (black arrows). Amino-allyl dUTP is incorporated during this step. (e) Fluorescent dyes are conjugated to microarray targets. (f) Differentially labeled targets are mixed and hybridized to a custom DNA microarray. Km r , kanamycin resistance; MGK, Monitoring of Gene Knockouts; PCR, polymerase chain reaction.

    Journal: Genome Biology

    Article Title: Monitoring of gene knockouts: genome-wide profiling of conditionally essential genes

    doi: 10.1186/gb-2007-8-5-r87

    Figure Lengend Snippet: Schematic representation of MGK. (a) Mixed library is grown in a reference and selective condition, and genomic DNA is isolated from each population. (b) Using genomic DNA as template, single-stranded DNA flanks are generated by linear extension of outward-facing insertion cassette-specific biotinylated primers (blue arrows). (c) The biotinylated flanks are separated from the template using streptavidin-coated magnetic beads, and polyadenylated at the 3'-ends using terminal deoxynucleotidyl transferase in the presence of dATP. (d) Microarray targets are PCR-amplified using an oligo d(T) primer (red arrows) and a nested Km r -specific primer (black arrows). Amino-allyl dUTP is incorporated during this step. (e) Fluorescent dyes are conjugated to microarray targets. (f) Differentially labeled targets are mixed and hybridized to a custom DNA microarray. Km r , kanamycin resistance; MGK, Monitoring of Gene Knockouts; PCR, polymerase chain reaction.

    Article Snippet: Polyadenylation of the 3'-ends of single-stranded DNA The reaction was carried out in a total volume of 50 μl of buffer no. 4 (New England Biolabs, Ipswich, MA, USA; 50 mmol/l potassium acetate, 20 mmol/l Tris-acetate, 10 mmol/l magnesium acetate, and 1 mmol/l dithiothreitol) containing 0.25 mmol/l CoCl2 , 60 μmol/l dATP, 20 U terminal deoxynucleotidyl transferase (New England Biolabs), and 20 μl of bead suspension from the previous step.

    Techniques: Isolation, Generated, Magnetic Beads, Microarray, Polymerase Chain Reaction, Amplification, Labeling

    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: In vitro procaspase-9 activation in the presence of recombinant Hsp70, M1 and mutants Cyt c (10 μ g) and dATP/ATP (1 mM) were incubated in the presence or absence of Hsp70 (5 μ g) or Hsp70AAAA (5 μ g) or Hsp70 (5 μ g) and M1 (10 μ g) or Hsp70 (5 μ g) and M1Δ128–165(10 μ g) in cytosolic Jurkat cell extracts (50 μ g protein equivalent) at 37 °C.

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