Structured Review

PerkinElmer alpha 32 p atp
A BiP ATPase mutant is not sufficient to protect cells during oxidative stress. ( A ) <t>ATP</t> hydrolysis was assessed by determining the fraction of [alpha- 32 P]ATP converted to [alpha- 32 P]ADP as described in the 'Materials and methods'. Data represent the means ± SD of three independent assays. ( B ) CSY278 containing plasmids pCS681, pCS774, pCS687, or empty vector were spotted on SMM-leu or SMM Gal-leu plates, and plates were incubated at 37°C for 3 d. ( C ) CSY275 containing a UPRE- lacZ reporter (pCS852) and plasmids pCS681, pCS802, pCS774, pCS687, pCS688, or pCS750 were cultured in SMM-ura-leu at 24°C to log-phase and shifted to 37°C (with or without 2 mM DTT) for 90 min prior to harvest. Three independent transformants of each strain were grown and assayed for beta–galactosidase activity in duplicate. Data represent the mean of averaged values for the three transformants ± SD. DOI: http://dx.doi.org/10.7554/eLife.03496.009
Alpha 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Redox signaling via the molecular chaperone BiP protects cells against endoplasmic reticulum-derived oxidative stress"

Article Title: Redox signaling via the molecular chaperone BiP protects cells against endoplasmic reticulum-derived oxidative stress

Journal: eLife

doi: 10.7554/eLife.03496

A BiP ATPase mutant is not sufficient to protect cells during oxidative stress. ( A ) ATP hydrolysis was assessed by determining the fraction of [alpha- 32 P]ATP converted to [alpha- 32 P]ADP as described in the 'Materials and methods'. Data represent the means ± SD of three independent assays. ( B ) CSY278 containing plasmids pCS681, pCS774, pCS687, or empty vector were spotted on SMM-leu or SMM Gal-leu plates, and plates were incubated at 37°C for 3 d. ( C ) CSY275 containing a UPRE- lacZ reporter (pCS852) and plasmids pCS681, pCS802, pCS774, pCS687, pCS688, or pCS750 were cultured in SMM-ura-leu at 24°C to log-phase and shifted to 37°C (with or without 2 mM DTT) for 90 min prior to harvest. Three independent transformants of each strain were grown and assayed for beta–galactosidase activity in duplicate. Data represent the mean of averaged values for the three transformants ± SD. DOI: http://dx.doi.org/10.7554/eLife.03496.009
Figure Legend Snippet: A BiP ATPase mutant is not sufficient to protect cells during oxidative stress. ( A ) ATP hydrolysis was assessed by determining the fraction of [alpha- 32 P]ATP converted to [alpha- 32 P]ADP as described in the 'Materials and methods'. Data represent the means ± SD of three independent assays. ( B ) CSY278 containing plasmids pCS681, pCS774, pCS687, or empty vector were spotted on SMM-leu or SMM Gal-leu plates, and plates were incubated at 37°C for 3 d. ( C ) CSY275 containing a UPRE- lacZ reporter (pCS852) and plasmids pCS681, pCS802, pCS774, pCS687, pCS688, or pCS750 were cultured in SMM-ura-leu at 24°C to log-phase and shifted to 37°C (with or without 2 mM DTT) for 90 min prior to harvest. Three independent transformants of each strain were grown and assayed for beta–galactosidase activity in duplicate. Data represent the mean of averaged values for the three transformants ± SD. DOI: http://dx.doi.org/10.7554/eLife.03496.009

Techniques Used: Mutagenesis, Plasmid Preparation, Incubation, Cell Culture, Activity Assay

Replacement of the BiP cysteine with aspartic acid, phenylalanine, tyrosine, or tryptophan results in decreased BiP function. ( A ) CSY214 containing the plasmids pCS681, pCS802, pCS687, pCS688, pCS750 or empty vector were spotted onto SMM plates with or without 5-fluoroorotic acid (5-FOA) and incubated for 2 d at 30°C. ( B ) CSY289, 290, 368, 292, and 293 were spotted onto YPD plates and incubated at 24°, 30°, and 37°C. ( C ) Strains from B were cultured at 24°C to log-phase in YPD and shifted to 37°C for 90 min prior to harvest. Accumulation of unprocessed untranslocated forms of the proteins Kar2, PDI, and Gas1 were detected by western blotting. ( D ) Strains from B containing an UPRE- lacZ reporter plasmid (pJC8) were cultured in SMM-ura at 24°C to log-phase and shifted to 37°C (with or without 2 mM DTT) for 90 min prior to harvest. Samples were assayed for beta–galactosidase activity. Three independent transformants of each strain were grown and assayed in duplicate. Data represent the mean of averaged values for the three transformants ± SD. ( E – G ) ATP hydrolysis was assessed by determining the fraction of [alpha- 32 P]ATP converted to [alpha- 32 P]ADP as described in the 'Materials and methods'. Panels F and G show CHP and NEM-treated samples, respectively. Samples without chemical additions in panels F and G were mock treated to match the CHP or NEM-treatment. Data represent the means ± SD of three independent assays. DOI: http://dx.doi.org/10.7554/eLife.03496.007
Figure Legend Snippet: Replacement of the BiP cysteine with aspartic acid, phenylalanine, tyrosine, or tryptophan results in decreased BiP function. ( A ) CSY214 containing the plasmids pCS681, pCS802, pCS687, pCS688, pCS750 or empty vector were spotted onto SMM plates with or without 5-fluoroorotic acid (5-FOA) and incubated for 2 d at 30°C. ( B ) CSY289, 290, 368, 292, and 293 were spotted onto YPD plates and incubated at 24°, 30°, and 37°C. ( C ) Strains from B were cultured at 24°C to log-phase in YPD and shifted to 37°C for 90 min prior to harvest. Accumulation of unprocessed untranslocated forms of the proteins Kar2, PDI, and Gas1 were detected by western blotting. ( D ) Strains from B containing an UPRE- lacZ reporter plasmid (pJC8) were cultured in SMM-ura at 24°C to log-phase and shifted to 37°C (with or without 2 mM DTT) for 90 min prior to harvest. Samples were assayed for beta–galactosidase activity. Three independent transformants of each strain were grown and assayed in duplicate. Data represent the mean of averaged values for the three transformants ± SD. ( E – G ) ATP hydrolysis was assessed by determining the fraction of [alpha- 32 P]ATP converted to [alpha- 32 P]ADP as described in the 'Materials and methods'. Panels F and G show CHP and NEM-treated samples, respectively. Samples without chemical additions in panels F and G were mock treated to match the CHP or NEM-treatment. Data represent the means ± SD of three independent assays. DOI: http://dx.doi.org/10.7554/eLife.03496.007

Techniques Used: Plasmid Preparation, Incubation, Cell Culture, Western Blot, Activity Assay

Related Articles

RNA Extraction:

Article Title: Proteotoxicity from aberrant ribosome biogenesis compromises cell fitness
Article Snippet: Paragraph title: Total RNA extraction and northern blotting ... Probes were made in a reaction containing 50 ng of a PCR product as template, random hexamer primers, Klenow (exo-), and 5 µl alpha-32 P-ATP (PerkinElmer).

In Vitro:

Article Title: A Conserved Cysteine within the ATPase Domain of the Endoplasmic Reticulum Chaperone BiP is Necessary for a Complete Complement of BiP Activities
Article Snippet: Paragraph title: In vitro protein assays ... To measure intrinsic ATPase activity, 1 μM Kar2 was mixed with 50 μM cold ATP and 0.01 μCi/μl of hot [alpha-32 P]ATP (Perkin-Elmer, Waltham, MA) in ATPase buffer (50 mM Tris-HCl, pH 7.4, 50 mM KCl, 5 mM MgCl2 , 1 mM DTT) at room temperature.

Northern Blot:

Article Title: Proteotoxicity from aberrant ribosome biogenesis compromises cell fitness
Article Snippet: Paragraph title: Total RNA extraction and northern blotting ... Probes were made in a reaction containing 50 ng of a PCR product as template, random hexamer primers, Klenow (exo-), and 5 µl alpha-32 P-ATP (PerkinElmer).

Random Hexamer Labeling:

Article Title: Proteotoxicity from aberrant ribosome biogenesis compromises cell fitness
Article Snippet: .. Probes were made in a reaction containing 50 ng of a PCR product as template, random hexamer primers, Klenow (exo-), and 5 µl alpha-32 P-ATP (PerkinElmer). .. Denatured probes were hybridized overnight and washed twice in 2X SSC/0.5% SDS at 65°C for 30 min before exposure on a phosphor screen and imaging on a Typhoon scanner.

Concentration Assay:

Article Title: Structural basis of transcription: role of the trigger loop in substrate specificity and catalysis
Article Snippet: Complexes were incubated with 50 μCi alpha-32 P-ATP (3000 Ci/mmol)(Perkin Elmer) to label active elongation complexes for 5 min at room temperature. .. The rNTP or 2’-dNTP specified for incorporation at the subsequent template position was then added at a final concentration of 200 μM for 5 min at room temperature.

Article Title: A Conserved Cysteine within the ATPase Domain of the Endoplasmic Reticulum Chaperone BiP is Necessary for a Complete Complement of BiP Activities
Article Snippet: The column was subsequently washed with (i) 10 cv of wash buffer (50 mM HEPES, pH 7.4, 0.3 M NaCl, 5% glycerol, 10 mM imidazole) supplemented with 1% Triton-X-100, (ii) 10 cv of wash buffer with a final concentration of 1 M NaCl, (iii) 10 cv of wash buffer lacking glycerol and containing 10 mM MgCl2 and 5 mM ATP, and (iv) 10 cv of wash buffer with 0.5 M Tris-HCl, pH 7.4. .. To measure intrinsic ATPase activity, 1 μM Kar2 was mixed with 50 μM cold ATP and 0.01 μCi/μl of hot [alpha-32 P]ATP (Perkin-Elmer, Waltham, MA) in ATPase buffer (50 mM Tris-HCl, pH 7.4, 50 mM KCl, 5 mM MgCl2 , 1 mM DTT) at room temperature.

Incubation:

Article Title: Structural basis of transcription: role of the trigger loop in substrate specificity and catalysis
Article Snippet: .. Complexes were incubated with 50 μCi alpha-32 P-ATP (3000 Ci/mmol)(Perkin Elmer) to label active elongation complexes for 5 min at room temperature. .. The rNTP or 2’-dNTP specified for incorporation at the subsequent template position was then added at a final concentration of 200 μM for 5 min at room temperature.

Article Title: Redox signaling via the molecular chaperone BiP protects cells against endoplasmic reticulum-derived oxidative stress
Article Snippet: .. ATPase activity To measure ATPase activity, 1 µM C-terminally tagged Kar2 and 2.5 µM GST-Sec63J were incubated with 0.1 mM of cold ATP and 0.45 µCi of [alpha-32 P]ATP (Perkin–Elmer, Waltham, MA) in ATPase buffer (50 mM Tris–HCl, pH 7.4, 50 mM KCl, 5 mM MgCl2 , 1 mM DTT) in a total volume of 45 µl at room temperature. .. For peroxide-treated Kar2 samples, 3 µM Kar2 and 5 µM GST-Sec63J were incubated in assay buffer lacking DTT (to prevent Kar2 reduction).

Thin Layer Chromatography:

Article Title: A Conserved Cysteine within the ATPase Domain of the Endoplasmic Reticulum Chaperone BiP is Necessary for a Complete Complement of BiP Activities
Article Snippet: To measure intrinsic ATPase activity, 1 μM Kar2 was mixed with 50 μM cold ATP and 0.01 μCi/μl of hot [alpha-32 P]ATP (Perkin-Elmer, Waltham, MA) in ATPase buffer (50 mM Tris-HCl, pH 7.4, 50 mM KCl, 5 mM MgCl2 , 1 mM DTT) at room temperature. .. Samples were spotted onto polyethyleneimine cellulose TLC plates, and plates were developed in 1 M formic acid and 0.5 M LiCl.

Article Title: Redox signaling via the molecular chaperone BiP protects cells against endoplasmic reticulum-derived oxidative stress
Article Snippet: ATPase activity To measure ATPase activity, 1 µM C-terminally tagged Kar2 and 2.5 µM GST-Sec63J were incubated with 0.1 mM of cold ATP and 0.45 µCi of [alpha-32 P]ATP (Perkin–Elmer, Waltham, MA) in ATPase buffer (50 mM Tris–HCl, pH 7.4, 50 mM KCl, 5 mM MgCl2 , 1 mM DTT) in a total volume of 45 µl at room temperature. .. Samples (1–2 µl) were spotted on polyethyleneimine cellulose TLC plates (Sigma-Aldrich, St. Louis, MO), and plates were developed in 1 M formic acid and 0.5 M LiCl.

Activity Assay:

Article Title: A Conserved Cysteine within the ATPase Domain of the Endoplasmic Reticulum Chaperone BiP is Necessary for a Complete Complement of BiP Activities
Article Snippet: .. To measure intrinsic ATPase activity, 1 μM Kar2 was mixed with 50 μM cold ATP and 0.01 μCi/μl of hot [alpha-32 P]ATP (Perkin-Elmer, Waltham, MA) in ATPase buffer (50 mM Tris-HCl, pH 7.4, 50 mM KCl, 5 mM MgCl2 , 1 mM DTT) at room temperature. .. To measure stimulation of Kar2 activity, the reaction was supplemented with a final concentration of 1.5 μM GST-Sec63J and 1 μM Lhs1.

Article Title: Redox signaling via the molecular chaperone BiP protects cells against endoplasmic reticulum-derived oxidative stress
Article Snippet: .. ATPase activity To measure ATPase activity, 1 µM C-terminally tagged Kar2 and 2.5 µM GST-Sec63J were incubated with 0.1 mM of cold ATP and 0.45 µCi of [alpha-32 P]ATP (Perkin–Elmer, Waltham, MA) in ATPase buffer (50 mM Tris–HCl, pH 7.4, 50 mM KCl, 5 mM MgCl2 , 1 mM DTT) in a total volume of 45 µl at room temperature. .. For peroxide-treated Kar2 samples, 3 µM Kar2 and 5 µM GST-Sec63J were incubated in assay buffer lacking DTT (to prevent Kar2 reduction).

Imaging:

Article Title: Proteotoxicity from aberrant ribosome biogenesis compromises cell fitness
Article Snippet: Probe was hybridized overnight and washed twice in 2X SSC/0.5% SDS at 42°C for 30 min before exposure on a phosphor screen and imaging on a Typhoon. .. Probes were made in a reaction containing 50 ng of a PCR product as template, random hexamer primers, Klenow (exo-), and 5 µl alpha-32 P-ATP (PerkinElmer).

BIA-KA:

Article Title: A Conserved Cysteine within the ATPase Domain of the Endoplasmic Reticulum Chaperone BiP is Necessary for a Complete Complement of BiP Activities
Article Snippet: Protein concentrations were determined using a BCA protein assay (Thermo Fisher Scientific, Waltham, MA) with bovine serum albumin as a standard. .. To measure intrinsic ATPase activity, 1 μM Kar2 was mixed with 50 μM cold ATP and 0.01 μCi/μl of hot [alpha-32 P]ATP (Perkin-Elmer, Waltham, MA) in ATPase buffer (50 mM Tris-HCl, pH 7.4, 50 mM KCl, 5 mM MgCl2 , 1 mM DTT) at room temperature.

Polymerase Chain Reaction:

Article Title: Proteotoxicity from aberrant ribosome biogenesis compromises cell fitness
Article Snippet: .. Probes were made in a reaction containing 50 ng of a PCR product as template, random hexamer primers, Klenow (exo-), and 5 µl alpha-32 P-ATP (PerkinElmer). .. Denatured probes were hybridized overnight and washed twice in 2X SSC/0.5% SDS at 65°C for 30 min before exposure on a phosphor screen and imaging on a Typhoon scanner.

Lysis:

Article Title: A Conserved Cysteine within the ATPase Domain of the Endoplasmic Reticulum Chaperone BiP is Necessary for a Complete Complement of BiP Activities
Article Snippet: Insoluble material was removed by centrifugation at 16,000 × g for 30 min. Soluble material was loaded onto a HiTrap chelating column charged with nickel, and the column was washed with 10 column volumes (cv) of lysis buffer. .. To measure intrinsic ATPase activity, 1 μM Kar2 was mixed with 50 μM cold ATP and 0.01 μCi/μl of hot [alpha-32 P]ATP (Perkin-Elmer, Waltham, MA) in ATPase buffer (50 mM Tris-HCl, pH 7.4, 50 mM KCl, 5 mM MgCl2 , 1 mM DTT) at room temperature.

Centrifugation:

Article Title: A Conserved Cysteine within the ATPase Domain of the Endoplasmic Reticulum Chaperone BiP is Necessary for a Complete Complement of BiP Activities
Article Snippet: Insoluble material was removed by centrifugation at 16,000 × g for 30 min. Soluble material was loaded onto a HiTrap chelating column charged with nickel, and the column was washed with 10 column volumes (cv) of lysis buffer. .. To measure intrinsic ATPase activity, 1 μM Kar2 was mixed with 50 μM cold ATP and 0.01 μCi/μl of hot [alpha-32 P]ATP (Perkin-Elmer, Waltham, MA) in ATPase buffer (50 mM Tris-HCl, pH 7.4, 50 mM KCl, 5 mM MgCl2 , 1 mM DTT) at room temperature.

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    PerkinElmer α 32 p atp
    Labeling and biochemical characterization of T7 RNAP activity. ( A ) Synthesis of Cy5-conjugated HaloTag Ligand ( S3 ) used to label RNAP from Cy5 acid S1 and HaloTag ligand-amine S2 . See ‘Materials and methods’ for details. ( B ) SDS-PAGE gel images of the unlabeled (‘RNAP’: no HaloTag; ‘Halo-RNAP’: with HaloTag) and the Cy5-labeled Halo-RNAP (‘Cy5-Halo-RNAP’) T7 RNAP. All RNAPs have a 6(His) tag at the N-terminus used for purification. Top: image stained with Coomassie Brilliant Blue. Bottom: the same gel scanned for Cy5 fluorescence (prior to Coomassie staining). ( C ) In vitro transcription activity of RNAP. The concentrations of RNAP derivatives were as indicated. The DNA template (a PCR fragment spanning from −75 to +295 of the concensus T7 RNAP promoter), was used at 10 nM. RNA products were labeled by incorporation of α 32 <t>P-ATP,</t> resolved on a denaturing polyacrylamide gel, and imaged by autoradiography. The major bands at ∼295 nt are the expected run-off products. DOI: http://dx.doi.org/10.7554/eLife.01775.009
    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
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    Labeling and biochemical characterization of T7 RNAP activity. ( A ) Synthesis of Cy5-conjugated HaloTag Ligand ( S3 ) used to label RNAP from Cy5 acid S1 and HaloTag ligand-amine S2 . See ‘Materials and methods’ for details. ( B ) SDS-PAGE gel images of the unlabeled (‘RNAP’: no HaloTag; ‘Halo-RNAP’: with HaloTag) and the Cy5-labeled Halo-RNAP (‘Cy5-Halo-RNAP’) T7 RNAP. All RNAPs have a 6(His) tag at the N-terminus used for purification. Top: image stained with Coomassie Brilliant Blue. Bottom: the same gel scanned for Cy5 fluorescence (prior to Coomassie staining). ( C ) In vitro transcription activity of RNAP. The concentrations of RNAP derivatives were as indicated. The DNA template (a PCR fragment spanning from −75 to +295 of the concensus T7 RNAP promoter), was used at 10 nM. RNA products were labeled by incorporation of α 32 P-ATP, resolved on a denaturing polyacrylamide gel, and imaged by autoradiography. The major bands at ∼295 nt are the expected run-off products. DOI: http://dx.doi.org/10.7554/eLife.01775.009

    Journal: eLife

    Article Title: Single-molecule tracking of the transcription cycle by sub-second RNA detection

    doi: 10.7554/eLife.01775

    Figure Lengend Snippet: Labeling and biochemical characterization of T7 RNAP activity. ( A ) Synthesis of Cy5-conjugated HaloTag Ligand ( S3 ) used to label RNAP from Cy5 acid S1 and HaloTag ligand-amine S2 . See ‘Materials and methods’ for details. ( B ) SDS-PAGE gel images of the unlabeled (‘RNAP’: no HaloTag; ‘Halo-RNAP’: with HaloTag) and the Cy5-labeled Halo-RNAP (‘Cy5-Halo-RNAP’) T7 RNAP. All RNAPs have a 6(His) tag at the N-terminus used for purification. Top: image stained with Coomassie Brilliant Blue. Bottom: the same gel scanned for Cy5 fluorescence (prior to Coomassie staining). ( C ) In vitro transcription activity of RNAP. The concentrations of RNAP derivatives were as indicated. The DNA template (a PCR fragment spanning from −75 to +295 of the concensus T7 RNAP promoter), was used at 10 nM. RNA products were labeled by incorporation of α 32 P-ATP, resolved on a denaturing polyacrylamide gel, and imaged by autoradiography. The major bands at ∼295 nt are the expected run-off products. DOI: http://dx.doi.org/10.7554/eLife.01775.009

    Article Snippet: In ensemble measurements, 10 nM DNA template (same PCR fragment as used in single-molecule assays), 0.17 μCi/μl α-32 P-ATP (Perkin Elmer, Waltham, MA) and different concentrations of RNAP were also included in the reaction.

    Techniques: Labeling, Activity Assay, SDS Page, Purification, Staining, Fluorescence, In Vitro, Polymerase Chain Reaction, Autoradiography

    Characterization of h-mtRNAP-catalyzed pyropho sphorolysis. (a) Experimental design. h-mtRNAP (1 μ ) (0.5 μ M) and [α- 32 P]ATP (0.45 μ M) for 5

    Journal: Biochemistry

    Article Title: Human Mitochondrial RNA Polymerase: Evaluation of the Single-Nucleotide-Addition Cycle on Synthetic RNA/DNA Scaffolds

    doi: 10.1021/bi200350d

    Figure Lengend Snippet: Characterization of h-mtRNAP-catalyzed pyropho sphorolysis. (a) Experimental design. h-mtRNAP (1 μ ) (0.5 μ M) and [α- 32 P]ATP (0.45 μ M) for 5

    Article Snippet: [α-32 P]GTP (3000 Ci/mmol) and [α-32 P]ATP (3000 Ci/mmol) were from Perkin-Elmer.

    Techniques:

    Exchange of the intracellular radiolabeled substrates. A : E. coli cells harboring the vector containing HvBT1 and the control vector were incubated with 1 µM [α- 32 P] ADP-Glc at 30°C for 5 min. The assay buffer was diluted with non-labeled ATP, ADP, AMP, and ADP-Glc for indicated time points. The cells were filtered and washed under vacuum, and then measured for radioactivity. The data presented here are the mean ± SE of three independent experiments, each with three replicates. B : the procedures for ADP efflux assay was performed as described for ADP-Glc in (A) with ADP and ADP-Glc dilutions. C : E. coli C43 cells harboring the vector containing HvBT1 and the control vector were preloaded with nucleotides at a final concentration of 1 mM, and then the cells were incubated at 30°C for 5 min. The cells were centrifuged and re-suspended in potassium phosphate buffer (50 mM, pH 7.2) with [α- 32 P] ADP-Glc at concentration of 100 µM at 30°C for 8 min. The data presented are the mean ± SE of three independent experiments, each with three replicates.

    Journal: PLoS ONE

    Article Title: Biochemical and Molecular Characterization of Barley Plastidial ADP-Glucose Transporter (HvBT1)

    doi: 10.1371/journal.pone.0098524

    Figure Lengend Snippet: Exchange of the intracellular radiolabeled substrates. A : E. coli cells harboring the vector containing HvBT1 and the control vector were incubated with 1 µM [α- 32 P] ADP-Glc at 30°C for 5 min. The assay buffer was diluted with non-labeled ATP, ADP, AMP, and ADP-Glc for indicated time points. The cells were filtered and washed under vacuum, and then measured for radioactivity. The data presented here are the mean ± SE of three independent experiments, each with three replicates. B : the procedures for ADP efflux assay was performed as described for ADP-Glc in (A) with ADP and ADP-Glc dilutions. C : E. coli C43 cells harboring the vector containing HvBT1 and the control vector were preloaded with nucleotides at a final concentration of 1 mM, and then the cells were incubated at 30°C for 5 min. The cells were centrifuged and re-suspended in potassium phosphate buffer (50 mM, pH 7.2) with [α- 32 P] ADP-Glc at concentration of 100 µM at 30°C for 8 min. The data presented are the mean ± SE of three independent experiments, each with three replicates.

    Article Snippet: [α-32 P]ADP was enzymatically synthesized from [α-32 P] ATP (3000 Ci [111 TBq]/mmol) (PerkinElmer).

    Techniques: Plasmid Preparation, Incubation, Gas Chromatography, Labeling, Radioactivity, Concentration Assay

    Recognition of Ca tRNA Ser by representative bacterial, yeast, human and archaeal LeuRSs. ( A ) A representative graph showing aminoacylation of [ 32 P] Ca tRNA Leu with Leu by various LeuRSs. The generated Leu-[ 32 P] Ca tRNA Leu or free [ 32 P] Ca tRNA Leu was separated by TLC. Known amounts of [α- 32 P]ATP were serially diluted and loaded onto the TLC plate after separation for quantification. ( B ) Quantitative analysis of Leu-[ 32 P] Ca tRNA Ser generated by Ca LeuRS (black circle), Ca LeuRS-D422A (black square), Sc LeuRS (black up-pointing triangle) and hcLeuRS (black down-pointing triangle). No charged [ 32 P] Ca tRNA Ser was catalyzed by Ca mtLeuRS, Ec LeuRS and Ph LeuRS.

    Journal: Nucleic Acids Research

    Article Title: Aminoacylation and translational quality control strategy employed by leucyl-tRNA synthetase from a human pathogen with genetic code ambiguity

    doi: 10.1093/nar/gkt741

    Figure Lengend Snippet: Recognition of Ca tRNA Ser by representative bacterial, yeast, human and archaeal LeuRSs. ( A ) A representative graph showing aminoacylation of [ 32 P] Ca tRNA Leu with Leu by various LeuRSs. The generated Leu-[ 32 P] Ca tRNA Leu or free [ 32 P] Ca tRNA Leu was separated by TLC. Known amounts of [α- 32 P]ATP were serially diluted and loaded onto the TLC plate after separation for quantification. ( B ) Quantitative analysis of Leu-[ 32 P] Ca tRNA Ser generated by Ca LeuRS (black circle), Ca LeuRS-D422A (black square), Sc LeuRS (black up-pointing triangle) and hcLeuRS (black down-pointing triangle). No charged [ 32 P] Ca tRNA Ser was catalyzed by Ca mtLeuRS, Ec LeuRS and Ph LeuRS.

    Article Snippet: [3 H]Leu, [32 P]tetrasodium pyrophosphate and [α-32 P]ATP were obtained from PerkinElmer Life Sciences (Boston, MA, USA).

    Techniques: Generated, Thin Layer Chromatography

    Mis-charging of [ 32 P] Ca tRNA Ser with Nva and post-transfer editing of Nva-[ 32 P] Ca tRNA Ser by Ca LeuRS and Ca LeuRS-D422A. ( A ) A representative graph showing mis-charging of [ 32 P] Ca tRNA Ser with non-cognate Nva. Free [ 32 P] Ca tRNA Ser and mis-charged [ 32 P] Ca tRNA Ser are represented by [ 32 P]AMP and Nva-[ 32 P]AMP after digestion of nuclease S1. Known amounts of [α- 32 P]ATP were serially diluted and loaded onto the TLC plate after separation for quantification. ( B ) Quantitative analysis of Nva-[ 32 P] Ca tRNA Ser generated by Ca LeuRS (black square) and Ca LeuRS-D422A (black up-pointing triangle) in (A). ( C ) A representative graph showing hydrolysis of Nva-[ 32 P] Ca tRNA Ser by Ca LeuRS and Ca LeuRS-D422A. A control reaction represented the spontaneous hydrolysis of Nva-[ 32 P] Ca tRNA Ser without the addition of enzyme. ( D ) Analysis of post-transfer editing of Nva-[ 32 P] Ca tRNA Ser by Ca LeuRS (black square) and Ca LeuRS-D422A (black up-pointing triangle) in (C).

    Journal: Nucleic Acids Research

    Article Title: Aminoacylation and translational quality control strategy employed by leucyl-tRNA synthetase from a human pathogen with genetic code ambiguity

    doi: 10.1093/nar/gkt741

    Figure Lengend Snippet: Mis-charging of [ 32 P] Ca tRNA Ser with Nva and post-transfer editing of Nva-[ 32 P] Ca tRNA Ser by Ca LeuRS and Ca LeuRS-D422A. ( A ) A representative graph showing mis-charging of [ 32 P] Ca tRNA Ser with non-cognate Nva. Free [ 32 P] Ca tRNA Ser and mis-charged [ 32 P] Ca tRNA Ser are represented by [ 32 P]AMP and Nva-[ 32 P]AMP after digestion of nuclease S1. Known amounts of [α- 32 P]ATP were serially diluted and loaded onto the TLC plate after separation for quantification. ( B ) Quantitative analysis of Nva-[ 32 P] Ca tRNA Ser generated by Ca LeuRS (black square) and Ca LeuRS-D422A (black up-pointing triangle) in (A). ( C ) A representative graph showing hydrolysis of Nva-[ 32 P] Ca tRNA Ser by Ca LeuRS and Ca LeuRS-D422A. A control reaction represented the spontaneous hydrolysis of Nva-[ 32 P] Ca tRNA Ser without the addition of enzyme. ( D ) Analysis of post-transfer editing of Nva-[ 32 P] Ca tRNA Ser by Ca LeuRS (black square) and Ca LeuRS-D422A (black up-pointing triangle) in (C).

    Article Snippet: [3 H]Leu, [32 P]tetrasodium pyrophosphate and [α-32 P]ATP were obtained from PerkinElmer Life Sciences (Boston, MA, USA).

    Techniques: Thin Layer Chromatography, Generated

    Post-transfer editing and mis-aminoacylation of Ca tRNA Leu by Ca LeuRS and Ca LeuRS-D422A. ( A ) A representative graph showing hydrolysis of Nva-[ 32 P] Ca tRNA Leu by Ca LeuRS and Ca LeuRS-D422A. Nuclease S1-generated Nva-[ 32 P]AMP (reflecting Nva-[ 32 P] Ca tRNA Leu ) and [ 32 P]AMP (reflecting free [ 32 P] Ca tRNA Leu ) were separated by TLC. A control reaction represented the spontaneous hydrolysis of Nva-[ 32 P] Ca tRNA Leu without the addition of enzyme. ( B ) Analysis of post-transfer editing of Nva-[ 32 P] Ca tRNA Leu in (A). ( C ) A representative graph showing mis-charging of [ 32 P] Ca tRNA Leu with non-cognate Nva (left) or ABA (right). Free [ 32 P] Ca tRNA Leu and mis-charged [ 32 P] Ca tRNA Leu are represented by [ 32 P]AMP and Nva-[ 32 P]AMP or ABA-[ 32 P]AMP, respectively. Known amounts of [α- 32 P]ATP were serially diluted and loaded onto the TLC plate after separation for quantification. ( D ) Quantitative analysis of Nva-[ 32 P] Ca tRNA Leu or ABA-[ 32 P] Ca tRNA Leu generated by Ca LeuRS (black square) and Ca LeuRS-D422A (black up-pointing triangle) or ABA-[ 32 P] Ca tRNA Leu by Ca LeuRS (white up-pointing triangle) and Ca LeuRS-D422A (white down-pointing triangle) in (C).

    Journal: Nucleic Acids Research

    Article Title: Aminoacylation and translational quality control strategy employed by leucyl-tRNA synthetase from a human pathogen with genetic code ambiguity

    doi: 10.1093/nar/gkt741

    Figure Lengend Snippet: Post-transfer editing and mis-aminoacylation of Ca tRNA Leu by Ca LeuRS and Ca LeuRS-D422A. ( A ) A representative graph showing hydrolysis of Nva-[ 32 P] Ca tRNA Leu by Ca LeuRS and Ca LeuRS-D422A. Nuclease S1-generated Nva-[ 32 P]AMP (reflecting Nva-[ 32 P] Ca tRNA Leu ) and [ 32 P]AMP (reflecting free [ 32 P] Ca tRNA Leu ) were separated by TLC. A control reaction represented the spontaneous hydrolysis of Nva-[ 32 P] Ca tRNA Leu without the addition of enzyme. ( B ) Analysis of post-transfer editing of Nva-[ 32 P] Ca tRNA Leu in (A). ( C ) A representative graph showing mis-charging of [ 32 P] Ca tRNA Leu with non-cognate Nva (left) or ABA (right). Free [ 32 P] Ca tRNA Leu and mis-charged [ 32 P] Ca tRNA Leu are represented by [ 32 P]AMP and Nva-[ 32 P]AMP or ABA-[ 32 P]AMP, respectively. Known amounts of [α- 32 P]ATP were serially diluted and loaded onto the TLC plate after separation for quantification. ( D ) Quantitative analysis of Nva-[ 32 P] Ca tRNA Leu or ABA-[ 32 P] Ca tRNA Leu generated by Ca LeuRS (black square) and Ca LeuRS-D422A (black up-pointing triangle) or ABA-[ 32 P] Ca tRNA Leu by Ca LeuRS (white up-pointing triangle) and Ca LeuRS-D422A (white down-pointing triangle) in (C).

    Article Snippet: [3 H]Leu, [32 P]tetrasodium pyrophosphate and [α-32 P]ATP were obtained from PerkinElmer Life Sciences (Boston, MA, USA).

    Techniques: Generated, Thin Layer Chromatography