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PerkinElmer α 32 p atp labeled
α 32 P Atp Labeled, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
α 32 p atp labeled - by Bioz Stars, 2020-04
86/100 stars

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Selection:

Article Title: Inhibition of Cell Proliferation by an Anti-EGFR Aptamer
Article Snippet: .. Some 10 nM [α-32 P]-ATP-labeled (3000Ci/mmol, 10mCi/ml, Perkin Elmer, Waltham, MA) Aptamers E03, E04, and E07 were incubated with 100 nM hEGFR (with or without DTT treatment) for 30 min at 25°C in Selection Buffer. .. To assess binding specificity, about 10 nM of [α-32 P]-ATP-labeled Aptamers E03, E04, and E07 were also incubated with 100 nM hIgG and mEGFR (mouse EGFR) with or without DTT treatment for 30 min at 25°C in Selection Buffer.

Produced:

Article Title: Inhibition of Cell Proliferation by an Anti-EGFR Aptamer
Article Snippet: To confirm that the monomer had been produced the solution was mixed with 4X loading dye and loaded onto a 4–12% NuPAGE gel (Invitrogen) with 1X MOPS running buffer alongside marker proteins. .. Some 10 nM [α-32 P]-ATP-labeled (3000Ci/mmol, 10mCi/ml, Perkin Elmer, Waltham, MA) Aptamers E03, E04, and E07 were incubated with 100 nM hEGFR (with or without DTT treatment) for 30 min at 25°C in Selection Buffer.

Incubation:

Article Title: Inhibition of Cell Proliferation by an Anti-EGFR Aptamer
Article Snippet: .. Some 10 nM [α-32 P]-ATP-labeled (3000Ci/mmol, 10mCi/ml, Perkin Elmer, Waltham, MA) Aptamers E03, E04, and E07 were incubated with 100 nM hEGFR (with or without DTT treatment) for 30 min at 25°C in Selection Buffer. .. To assess binding specificity, about 10 nM of [α-32 P]-ATP-labeled Aptamers E03, E04, and E07 were also incubated with 100 nM hIgG and mEGFR (mouse EGFR) with or without DTT treatment for 30 min at 25°C in Selection Buffer.

Marker:

Article Title: Inhibition of Cell Proliferation by an Anti-EGFR Aptamer
Article Snippet: To confirm that the monomer had been produced the solution was mixed with 4X loading dye and loaded onto a 4–12% NuPAGE gel (Invitrogen) with 1X MOPS running buffer alongside marker proteins. .. Some 10 nM [α-32 P]-ATP-labeled (3000Ci/mmol, 10mCi/ml, Perkin Elmer, Waltham, MA) Aptamers E03, E04, and E07 were incubated with 100 nM hEGFR (with or without DTT treatment) for 30 min at 25°C in Selection Buffer.

Staining:

Article Title: Inhibition of Cell Proliferation by an Anti-EGFR Aptamer
Article Snippet: The gel was developed at 200 V for 1 hour, and stained as previously described (Supplementary Material, ). .. Some 10 nM [α-32 P]-ATP-labeled (3000Ci/mmol, 10mCi/ml, Perkin Elmer, Waltham, MA) Aptamers E03, E04, and E07 were incubated with 100 nM hEGFR (with or without DTT treatment) for 30 min at 25°C in Selection Buffer.

Binding Assay:

Article Title: Inhibition of Cell Proliferation by an Anti-EGFR Aptamer
Article Snippet: Paragraph title: Binding specificities and dissociation constants ... Some 10 nM [α-32 P]-ATP-labeled (3000Ci/mmol, 10mCi/ml, Perkin Elmer, Waltham, MA) Aptamers E03, E04, and E07 were incubated with 100 nM hEGFR (with or without DTT treatment) for 30 min at 25°C in Selection Buffer.

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  • 93
    PerkinElmer α 32 p ctp
    SI3 and the jaw do not modulate the active site through their putative downstream DNA contacts. (A) His pause escape on a promoter-driven his pause template ( 29 ) for wild-type, ΔSI3, Δjaw, and ΔSI3Δjaw RNAPs. Preformed [α- 32 <t>P]-CTP-labeled</t> A29 complexes were incubated with 20 μM GTP, 150 μM ATP, CTP and UTP, together with 50 μg heparin/ml. Position of pause and run-off (RO) transcripts are indicated. Samples were taken at the times indicated (ch, chase). (B) Bar graph showing the average pause half-life values of wild-type, ΔSI3, Δjaw, and ΔSI3Δjaw RNAPs at his pause site derived from the experiment shown in (A) and two additional experiments shown as means ± SD. Numbers above the bars indicate the fold-increase in pause escape rate. (C) Pausing for wild-type, ΔSI3, Δjaw, and ΔSI3Δjaw RNAPs on templates with different amounts of downstream sequence. Top : schematic of paused elongation complex illustrating the progressive downstream-DNA truncations. The RNAP (gray spacefill), DNA (gray) and RNA (red) are shown. Positions of duplex DNA truncations are indicated on the schematic drawing by green lines and numbers indicating the number of base pairs downstream of the pause site, where the pause site with RNAP in pre-translocated register is denoted as the +1 position. Bottom : normalized pause half-lives of wild-type, ΔSI3, Δjaw, and ΔSI3Δjaw RNAPs on truncation templates. The half-life of wild-type RNAP on DS+47 (47 bp of downstream DNA duplex) was normalized to 1 and all other pause half-life values were normalized accordingly. Data are means ± SD of experimental triplicates.
    α 32 P Ctp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 93/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α 32 p ctp/product/PerkinElmer
    Average 93 stars, based on 23 article reviews
    Price from $9.99 to $1999.99
    α 32 p ctp - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

    89
    PerkinElmer α 32 p labeled atp
    Substrate specificity of IbpA-Fic2. A , the Fic domains of IbpA, PfhB2 and HYPE target Tyr-32 of Cdc42, whereas VopS targets Thr-35. Bacterially expressed GST-tagged IbpA-Fic2, IbpA-Fic1, PfhB2-Fic2, VopS, or HYPE-Fic was incubated with wild type ( W ), Y32F ( Y ) or T35A ( T ) versions of Cdc42 expressed as GST fusion proteins in bacteria in an in vitro adenylylation assay using [α- 32 <t>P]ATP.</t> Samples were separated on SDS-PAGE and visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The position of Cdc42 on the gel is indicated by arrows . The Fic domains of IbpA, PfhB2, and HYPE adenylylate wild type Cdc42 and Cdc42-T35A but not Cdc42-Y32F, indicating their specificity for the switch 1 tyrosine. In contrast, VopS fails to adenylylate only Cdc42-T35A, indicating its specificity for the switch 1 threonine. B , IbpA-Fic2 targets both the active and the inactive forms of Rho GTPases. Bacterially expressed untagged Cdc42, Rac, and RhoA loaded with GDP or GMP-PNP (as described under “Experimental Procedures”) were incubated with IbpA-Fic2 in an in vitro adenylylation assay. The protein load was visualized by Coomassie Blue staining, and the amount of adenylylation was visualized by autoradiography. The nucleotide status of the GTPases was confirmed prior to adenylylation by incubation with GST-Pak (Cdc42 and Rac) or GST-Rhotekin (RhoA) followed by separation on SDS-PAGE and Western analysis using antibodies directed against the individual GTPases. C , IbpA-Fic2 is active against the Cdc42-RhoGDI complex. HA-tagged Cdc42 was expressed in HEK293A cells. Bacterially expressed His 6 -SUMO-RhoGDI bound to nickel-agarose beads was incubated with the HEK239A cell extract treated for GDP loading of Rho GTPases (as described under “Experimental Procedures.”) to allow Cdc42-RhoGDI complex formation. After washing, the beads were subjected to the in vitro adenylylation reaction in the presence or absence of GST-tagged IbpA-Fic2. The supernatant ( supe ) and bead eluate were separated on SDS-PAGE and visualized by autoradiography. The protein load was monitored by Ponceau S staining.
    α 32 P Labeled Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α 32 p labeled atp/product/PerkinElmer
    Average 89 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    α 32 p labeled atp - by Bioz Stars, 2020-04
    89/100 stars
      Buy from Supplier

    95
    PerkinElmer α 32 p atp
    TATase activity of Δ97nsP4 and purified nsP4. Polymerase was exposed to template RNA in the presence of [α- 32 P] <t>ATP,</t> and products were resolved on 1.5% agarose-phosphate gels. Signal intensity was quantified by phosphoimager, with nsP4 labeling of Wt(+) established as baseline (100%). RNA substrates were either Wt+ genome analog (lanes 2, 3, 5, and 7) or Wt+ without a poly(A) tail (noA; lanes 4 and 6), or Wt+ on which the 3′-OH has been blocked (oxid; lane 8).
    α 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 95/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α 32 p atp/product/PerkinElmer
    Average 95 stars, based on 31 article reviews
    Price from $9.99 to $1999.99
    α 32 p atp - by Bioz Stars, 2020-04
    95/100 stars
      Buy from Supplier

    Image Search Results


    SI3 and the jaw do not modulate the active site through their putative downstream DNA contacts. (A) His pause escape on a promoter-driven his pause template ( 29 ) for wild-type, ΔSI3, Δjaw, and ΔSI3Δjaw RNAPs. Preformed [α- 32 P]-CTP-labeled A29 complexes were incubated with 20 μM GTP, 150 μM ATP, CTP and UTP, together with 50 μg heparin/ml. Position of pause and run-off (RO) transcripts are indicated. Samples were taken at the times indicated (ch, chase). (B) Bar graph showing the average pause half-life values of wild-type, ΔSI3, Δjaw, and ΔSI3Δjaw RNAPs at his pause site derived from the experiment shown in (A) and two additional experiments shown as means ± SD. Numbers above the bars indicate the fold-increase in pause escape rate. (C) Pausing for wild-type, ΔSI3, Δjaw, and ΔSI3Δjaw RNAPs on templates with different amounts of downstream sequence. Top : schematic of paused elongation complex illustrating the progressive downstream-DNA truncations. The RNAP (gray spacefill), DNA (gray) and RNA (red) are shown. Positions of duplex DNA truncations are indicated on the schematic drawing by green lines and numbers indicating the number of base pairs downstream of the pause site, where the pause site with RNAP in pre-translocated register is denoted as the +1 position. Bottom : normalized pause half-lives of wild-type, ΔSI3, Δjaw, and ΔSI3Δjaw RNAPs on truncation templates. The half-life of wild-type RNAP on DS+47 (47 bp of downstream DNA duplex) was normalized to 1 and all other pause half-life values were normalized accordingly. Data are means ± SD of experimental triplicates.

    Journal: Nucleic Acids Research

    Article Title: Trigger-helix folding pathway and SI3 mediate catalysis and hairpin-stabilized pausing by Escherichia coli RNA polymerase

    doi: 10.1093/nar/gku997

    Figure Lengend Snippet: SI3 and the jaw do not modulate the active site through their putative downstream DNA contacts. (A) His pause escape on a promoter-driven his pause template ( 29 ) for wild-type, ΔSI3, Δjaw, and ΔSI3Δjaw RNAPs. Preformed [α- 32 P]-CTP-labeled A29 complexes were incubated with 20 μM GTP, 150 μM ATP, CTP and UTP, together with 50 μg heparin/ml. Position of pause and run-off (RO) transcripts are indicated. Samples were taken at the times indicated (ch, chase). (B) Bar graph showing the average pause half-life values of wild-type, ΔSI3, Δjaw, and ΔSI3Δjaw RNAPs at his pause site derived from the experiment shown in (A) and two additional experiments shown as means ± SD. Numbers above the bars indicate the fold-increase in pause escape rate. (C) Pausing for wild-type, ΔSI3, Δjaw, and ΔSI3Δjaw RNAPs on templates with different amounts of downstream sequence. Top : schematic of paused elongation complex illustrating the progressive downstream-DNA truncations. The RNAP (gray spacefill), DNA (gray) and RNA (red) are shown. Positions of duplex DNA truncations are indicated on the schematic drawing by green lines and numbers indicating the number of base pairs downstream of the pause site, where the pause site with RNAP in pre-translocated register is denoted as the +1 position. Bottom : normalized pause half-lives of wild-type, ΔSI3, Δjaw, and ΔSI3Δjaw RNAPs on truncation templates. The half-life of wild-type RNAP on DS+47 (47 bp of downstream DNA duplex) was normalized to 1 and all other pause half-life values were normalized accordingly. Data are means ± SD of experimental triplicates.

    Article Snippet: The 3′deoxyG13 RNA was obtained from Thermo Fisher Scientific Biosciences Inc. [γ-32 P]ATP, [α-32 P]CTP and [α-32 P]GTP were from PerkinElmer Life Sciences; rNTPs were from Promega (Madison, WI, USA).

    Techniques: Labeling, Incubation, Derivative Assay, Sequencing

    Substrate specificity of IbpA-Fic2. A , the Fic domains of IbpA, PfhB2 and HYPE target Tyr-32 of Cdc42, whereas VopS targets Thr-35. Bacterially expressed GST-tagged IbpA-Fic2, IbpA-Fic1, PfhB2-Fic2, VopS, or HYPE-Fic was incubated with wild type ( W ), Y32F ( Y ) or T35A ( T ) versions of Cdc42 expressed as GST fusion proteins in bacteria in an in vitro adenylylation assay using [α- 32 P]ATP. Samples were separated on SDS-PAGE and visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The position of Cdc42 on the gel is indicated by arrows . The Fic domains of IbpA, PfhB2, and HYPE adenylylate wild type Cdc42 and Cdc42-T35A but not Cdc42-Y32F, indicating their specificity for the switch 1 tyrosine. In contrast, VopS fails to adenylylate only Cdc42-T35A, indicating its specificity for the switch 1 threonine. B , IbpA-Fic2 targets both the active and the inactive forms of Rho GTPases. Bacterially expressed untagged Cdc42, Rac, and RhoA loaded with GDP or GMP-PNP (as described under “Experimental Procedures”) were incubated with IbpA-Fic2 in an in vitro adenylylation assay. The protein load was visualized by Coomassie Blue staining, and the amount of adenylylation was visualized by autoradiography. The nucleotide status of the GTPases was confirmed prior to adenylylation by incubation with GST-Pak (Cdc42 and Rac) or GST-Rhotekin (RhoA) followed by separation on SDS-PAGE and Western analysis using antibodies directed against the individual GTPases. C , IbpA-Fic2 is active against the Cdc42-RhoGDI complex. HA-tagged Cdc42 was expressed in HEK293A cells. Bacterially expressed His 6 -SUMO-RhoGDI bound to nickel-agarose beads was incubated with the HEK239A cell extract treated for GDP loading of Rho GTPases (as described under “Experimental Procedures.”) to allow Cdc42-RhoGDI complex formation. After washing, the beads were subjected to the in vitro adenylylation reaction in the presence or absence of GST-tagged IbpA-Fic2. The supernatant ( supe ) and bead eluate were separated on SDS-PAGE and visualized by autoradiography. The protein load was monitored by Ponceau S staining.

    Journal: The Journal of Biological Chemistry

    Article Title: Comparative Analysis of Histophilus somni Immunoglobulin-binding Protein A (IbpA) with Other Fic Domain-containing Enzymes Reveals Differences in Substrate and Nucleotide Specificities *

    doi: 10.1074/jbc.M111.227603

    Figure Lengend Snippet: Substrate specificity of IbpA-Fic2. A , the Fic domains of IbpA, PfhB2 and HYPE target Tyr-32 of Cdc42, whereas VopS targets Thr-35. Bacterially expressed GST-tagged IbpA-Fic2, IbpA-Fic1, PfhB2-Fic2, VopS, or HYPE-Fic was incubated with wild type ( W ), Y32F ( Y ) or T35A ( T ) versions of Cdc42 expressed as GST fusion proteins in bacteria in an in vitro adenylylation assay using [α- 32 P]ATP. Samples were separated on SDS-PAGE and visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The position of Cdc42 on the gel is indicated by arrows . The Fic domains of IbpA, PfhB2, and HYPE adenylylate wild type Cdc42 and Cdc42-T35A but not Cdc42-Y32F, indicating their specificity for the switch 1 tyrosine. In contrast, VopS fails to adenylylate only Cdc42-T35A, indicating its specificity for the switch 1 threonine. B , IbpA-Fic2 targets both the active and the inactive forms of Rho GTPases. Bacterially expressed untagged Cdc42, Rac, and RhoA loaded with GDP or GMP-PNP (as described under “Experimental Procedures”) were incubated with IbpA-Fic2 in an in vitro adenylylation assay. The protein load was visualized by Coomassie Blue staining, and the amount of adenylylation was visualized by autoradiography. The nucleotide status of the GTPases was confirmed prior to adenylylation by incubation with GST-Pak (Cdc42 and Rac) or GST-Rhotekin (RhoA) followed by separation on SDS-PAGE and Western analysis using antibodies directed against the individual GTPases. C , IbpA-Fic2 is active against the Cdc42-RhoGDI complex. HA-tagged Cdc42 was expressed in HEK293A cells. Bacterially expressed His 6 -SUMO-RhoGDI bound to nickel-agarose beads was incubated with the HEK239A cell extract treated for GDP loading of Rho GTPases (as described under “Experimental Procedures.”) to allow Cdc42-RhoGDI complex formation. After washing, the beads were subjected to the in vitro adenylylation reaction in the presence or absence of GST-tagged IbpA-Fic2. The supernatant ( supe ) and bead eluate were separated on SDS-PAGE and visualized by autoradiography. The protein load was monitored by Ponceau S staining.

    Article Snippet: For nucleotide specificity assays, in vitro reactions were conducted as above with α-32 P-labeled ATP, GTP, CTP, UTP, or dTTP (PerkinElmer Life Sciences) containing 1 mm of each respective cold dNTP.

    Techniques: Incubation, In Vitro, SDS Page, Autoradiography, Staining, Western Blot

    Nucleotide specificity of IbpA-Fic2. A , GST-tagged and purified IbpA-Fic1, IbpA-Fic2, PfhB2-Fic1, PfhB2-Fic2, and VopS and His 6 -SUMO-tagged HYPE-Fic were incubated with Cdc42 1–179 Q61L in an in vitro reaction using [α- 32 P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples separated by SDS-PAGE were visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The ability of the indicated Fic enzymes to utilize different nucleotides for post-translationally modifying Cdc42 is shown. All the panels were given equal exposure times for autoradiography. The dotted line represents a break in the gels. B , reactions with His 6 -SUMO-tagged HYPE-Fic displayed in panel A were rerun on SDS-PAGE and visualized by longer exposures for autoradiography ( upper panel ) and Coomassie Blue staining ( bottom panel ). HYPE-Fic efficiently uses ATP, and CTP to a lesser degree, to modify Cdc42. C , point mutations in the IbpA-Fic2 Fic motif did not alter its affinity for nucleotides. GST-tagged and purified Pro-3718 to Gly (IbpA_Fic2-P/G) and Glu-3271 to Asp (IbpA_Fic2-E/D) mutants of IbpA-Fic2, as well as wild type IbpA-Fic2 and VopS, were incubated with Cdc42-Q61L using [α- 32 P]ATP and -GTP in an in vitro reaction. Samples were separated on SDS-PAGE and visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). Conversion of the IbpA-Fic2 Fic motif sequence to match the corresponding residues in the Fic motif of VopS did not confer specificity for nucleotides. D , comparison of IbpA-Fic2 and VopS to target switch 1 Tyr-32 and Thr-35 mutants of Cdc42 using different nucleotides. GST-tagged IbpA-Fic2 and VopS were incubated with wild type ( W ), Y32F ( Y ), or T35A ( T ) versions of Cdc42 expressed as GST fusion proteins in bacteria in an in vitro assay using [α- 32 P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples were assessed by autoradiography ( top panel ) with exposure times adjusted for optimal visualization and by Coomassie Blue staining ( lower panel ). Mutation of T35A in Cdc42 did not alter the ability of IbpA-Fic2 to target the switch 1 Tyr-32 for modification. In contrast, the Y32F mutation in Cdc42 severely impaired VopS in modifying Thr-35 using the different nucleotide sources.

    Journal: The Journal of Biological Chemistry

    Article Title: Comparative Analysis of Histophilus somni Immunoglobulin-binding Protein A (IbpA) with Other Fic Domain-containing Enzymes Reveals Differences in Substrate and Nucleotide Specificities *

    doi: 10.1074/jbc.M111.227603

    Figure Lengend Snippet: Nucleotide specificity of IbpA-Fic2. A , GST-tagged and purified IbpA-Fic1, IbpA-Fic2, PfhB2-Fic1, PfhB2-Fic2, and VopS and His 6 -SUMO-tagged HYPE-Fic were incubated with Cdc42 1–179 Q61L in an in vitro reaction using [α- 32 P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples separated by SDS-PAGE were visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The ability of the indicated Fic enzymes to utilize different nucleotides for post-translationally modifying Cdc42 is shown. All the panels were given equal exposure times for autoradiography. The dotted line represents a break in the gels. B , reactions with His 6 -SUMO-tagged HYPE-Fic displayed in panel A were rerun on SDS-PAGE and visualized by longer exposures for autoradiography ( upper panel ) and Coomassie Blue staining ( bottom panel ). HYPE-Fic efficiently uses ATP, and CTP to a lesser degree, to modify Cdc42. C , point mutations in the IbpA-Fic2 Fic motif did not alter its affinity for nucleotides. GST-tagged and purified Pro-3718 to Gly (IbpA_Fic2-P/G) and Glu-3271 to Asp (IbpA_Fic2-E/D) mutants of IbpA-Fic2, as well as wild type IbpA-Fic2 and VopS, were incubated with Cdc42-Q61L using [α- 32 P]ATP and -GTP in an in vitro reaction. Samples were separated on SDS-PAGE and visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). Conversion of the IbpA-Fic2 Fic motif sequence to match the corresponding residues in the Fic motif of VopS did not confer specificity for nucleotides. D , comparison of IbpA-Fic2 and VopS to target switch 1 Tyr-32 and Thr-35 mutants of Cdc42 using different nucleotides. GST-tagged IbpA-Fic2 and VopS were incubated with wild type ( W ), Y32F ( Y ), or T35A ( T ) versions of Cdc42 expressed as GST fusion proteins in bacteria in an in vitro assay using [α- 32 P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples were assessed by autoradiography ( top panel ) with exposure times adjusted for optimal visualization and by Coomassie Blue staining ( lower panel ). Mutation of T35A in Cdc42 did not alter the ability of IbpA-Fic2 to target the switch 1 Tyr-32 for modification. In contrast, the Y32F mutation in Cdc42 severely impaired VopS in modifying Thr-35 using the different nucleotide sources.

    Article Snippet: For nucleotide specificity assays, in vitro reactions were conducted as above with α-32 P-labeled ATP, GTP, CTP, UTP, or dTTP (PerkinElmer Life Sciences) containing 1 mm of each respective cold dNTP.

    Techniques: Purification, Incubation, In Vitro, SDS Page, Autoradiography, Staining, Sequencing, Mutagenesis, Modification

    TATase activity of Δ97nsP4 and purified nsP4. Polymerase was exposed to template RNA in the presence of [α- 32 P] ATP, and products were resolved on 1.5% agarose-phosphate gels. Signal intensity was quantified by phosphoimager, with nsP4 labeling of Wt(+) established as baseline (100%). RNA substrates were either Wt+ genome analog (lanes 2, 3, 5, and 7) or Wt+ without a poly(A) tail (noA; lanes 4 and 6), or Wt+ on which the 3′-OH has been blocked (oxid; lane 8).

    Journal: Virology

    Article Title: Characterization of purified Sindbis Virus nsP4 RNA-dependent RNA Polymerase activity in vitro

    doi: 10.1016/j.virol.2008.10.030

    Figure Lengend Snippet: TATase activity of Δ97nsP4 and purified nsP4. Polymerase was exposed to template RNA in the presence of [α- 32 P] ATP, and products were resolved on 1.5% agarose-phosphate gels. Signal intensity was quantified by phosphoimager, with nsP4 labeling of Wt(+) established as baseline (100%). RNA substrates were either Wt+ genome analog (lanes 2, 3, 5, and 7) or Wt+ without a poly(A) tail (noA; lanes 4 and 6), or Wt+ on which the 3′-OH has been blocked (oxid; lane 8).

    Article Snippet: Standard reaction mixtures (total volume 25 μL) contained 50 mM Tris-HCl (pH 8.5), 75 mM KCl, 5.0 mM MgCl2 , 10 mM dithiothreitol, 10 μg/mL actinomycin-D,1 mCi/mL [α-32 P]-ATP (800 Ci/mmol, Perkin Elmer), 800 units/mL RNasin, 0.5 μg template RNA (1 pmol), 400 nM purified nsP4 or 4 μM Δ97nsP4.

    Techniques: Activity Assay, Purification, Labeling

    (A) A schematic drawing of the tRNA 75C substrate for A76 addition, using ATP as the nucleotide donor. The tRNA is labeled during transcription by incorporation of [α- 32 P]AMP, indicated by red dots. (B) Representative time courses of A76 addition

    Journal: Journal of molecular biology

    Article Title: tRNA Integrity Is A Prerequisite for Rapid CCA Addition: Implication for Quality Control

    doi: 10.1016/j.jmb.2008.04.005

    Figure Lengend Snippet: (A) A schematic drawing of the tRNA 75C substrate for A76 addition, using ATP as the nucleotide donor. The tRNA is labeled during transcription by incorporation of [α- 32 P]AMP, indicated by red dots. (B) Representative time courses of A76 addition

    Article Snippet: The full-length tRNAs and 3′ fragments of the ASL-nicked substrates were labeled internally by using α-32 P-ATP (3000 Ci/mmol, Perkin Elmer) during the T7 transcription and were purified by 12% PAGE/7M urea.

    Techniques: Labeling