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PerkinElmer m dtt
Kinetics of phosphotransfer from AtsR to AtsT. A , 5 μmol of AtsR were preincubated with 5 μCi ([γ- 33 P]ATP) in a standard phosphorylation mixture (100 m m Tris-HCl, pH 8, 50 m m <t>KCl,</t> 5 m m MgCl 2 , 1 m m <t>DTT)</t> for 15 min, and then 5 μmol
M Dtt, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 92/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/m dtt/product/PerkinElmer
Average 92 stars, based on 35 article reviews
Price from $9.99 to $1999.99
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92/100 stars

Images

1) Product Images from "Characterization of the AtsR Hybrid Sensor Kinase Phosphorelay Pathway and Identification of Its Response Regulator in Burkholderia cenocepacia *"

Article Title: Characterization of the AtsR Hybrid Sensor Kinase Phosphorelay Pathway and Identification of Its Response Regulator in Burkholderia cenocepacia *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.489914

Kinetics of phosphotransfer from AtsR to AtsT. A , 5 μmol of AtsR were preincubated with 5 μCi ([γ- 33 P]ATP) in a standard phosphorylation mixture (100 m m Tris-HCl, pH 8, 50 m m KCl, 5 m m MgCl 2 , 1 m m DTT) for 15 min, and then 5 μmol
Figure Legend Snippet: Kinetics of phosphotransfer from AtsR to AtsT. A , 5 μmol of AtsR were preincubated with 5 μCi ([γ- 33 P]ATP) in a standard phosphorylation mixture (100 m m Tris-HCl, pH 8, 50 m m KCl, 5 m m MgCl 2 , 1 m m DTT) for 15 min, and then 5 μmol

Techniques Used:

Kinetics of phosphotransfer from AtsR and AtsR D536A to AtsT. After 10 min of preincubation of 5 μmol AtsR ( A ) or AtsR D536A ( B ) in a standard phosphorylation mixture (100 m m Tris-HCl, pH 8, 50 m m KCl, 5 m m MgCl 2 , 1 m m DTT, 5 μCi [γ-
Figure Legend Snippet: Kinetics of phosphotransfer from AtsR and AtsR D536A to AtsT. After 10 min of preincubation of 5 μmol AtsR ( A ) or AtsR D536A ( B ) in a standard phosphorylation mixture (100 m m Tris-HCl, pH 8, 50 m m KCl, 5 m m MgCl 2 , 1 m m DTT, 5 μCi [γ-

Techniques Used:

Phosphotransfer from AtsR and its derivatives to the AtsT response regulator. Five μmol of AtsR, AtsR H245A , and AtsR-HK was preincubated in individual standard phosphorylation mixtures (100 m m Tris-HCl pH 8, 50 m m KCl, 5 m m MgCl 2 , 1 m m DTT, 5
Figure Legend Snippet: Phosphotransfer from AtsR and its derivatives to the AtsT response regulator. Five μmol of AtsR, AtsR H245A , and AtsR-HK was preincubated in individual standard phosphorylation mixtures (100 m m Tris-HCl pH 8, 50 m m KCl, 5 m m MgCl 2 , 1 m m DTT, 5

Techniques Used:

2) Product Images from "Mycobacterium smegmatis Lhr Is a DNA-dependent ATPase and a 3?-to-5? DNA Translocase and Helicase That Prefers to Unwind 3?-Tailed RNA:DNA Hybrids *"

Article Title: Mycobacterium smegmatis Lhr Is a DNA-dependent ATPase and a 3?-to-5? DNA Translocase and Helicase That Prefers to Unwind 3?-Tailed RNA:DNA Hybrids *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.466854

Helicase activity of Lhr truncations on a 3′-tailed DNA:DNA or RNA:DNA duplex substrate. Helicase reaction mixtures (10 μl) contained 20 m m Tris-HCl, pH 7.0, 1 m m DTT, 5 m m CaCl 2 , either 50 n m of 5′ 32 P-labeled 3′-tailed
Figure Legend Snippet: Helicase activity of Lhr truncations on a 3′-tailed DNA:DNA or RNA:DNA duplex substrate. Helicase reaction mixtures (10 μl) contained 20 m m Tris-HCl, pH 7.0, 1 m m DTT, 5 m m CaCl 2 , either 50 n m of 5′ 32 P-labeled 3′-tailed

Techniques Used: Activity Assay, Labeling

Single strand DNA length requirements for ATPase and helicase activity. A , ATPase reaction mixtures (10 μl) containing 20 m m Tris-HCl, pH 7.0, 1 m m DTT, 1 m m CaCl 2 , 1 m m [α- 32 P]ATP, 100 n m Lhr, and increasing amounts of 59-mer, 24-mer,
Figure Legend Snippet: Single strand DNA length requirements for ATPase and helicase activity. A , ATPase reaction mixtures (10 μl) containing 20 m m Tris-HCl, pH 7.0, 1 m m DTT, 1 m m CaCl 2 , 1 m m [α- 32 P]ATP, 100 n m Lhr, and increasing amounts of 59-mer, 24-mer,

Techniques Used: Activity Assay

Lhr ATPase and helicase activity with DNA and RNA substrates. A , ATPase reaction mixtures (10 μl) containing 20 m m Tris-HCl, pH 7.0, 1 m m DTT, 1 m m CaCl 2 , 1 m m [α- 32 P]ATP, 100 n m Lhr, and the indicated amount of 24-mer DNA or RNA were
Figure Legend Snippet: Lhr ATPase and helicase activity with DNA and RNA substrates. A , ATPase reaction mixtures (10 μl) containing 20 m m Tris-HCl, pH 7.0, 1 m m DTT, 1 m m CaCl 2 , 1 m m [α- 32 P]ATP, 100 n m Lhr, and the indicated amount of 24-mer DNA or RNA were

Techniques Used: Activity Assay

Lhr is a 3′-to-5′ DNA helicase. A , helicase reaction mixtures (10 μl) contained 20 m m Tris-HCl, pH 7.0, 1 m m DTT, 5 m m CaCl 2 , 100 n m of the indicated DNA substrate (depicted at the bottom , with the 5′ 32 P label denoted
Figure Legend Snippet: Lhr is a 3′-to-5′ DNA helicase. A , helicase reaction mixtures (10 μl) contained 20 m m Tris-HCl, pH 7.0, 1 m m DTT, 5 m m CaCl 2 , 100 n m of the indicated DNA substrate (depicted at the bottom , with the 5′ 32 P label denoted

Techniques Used:

Related Articles

In Vitro:

Article Title: Ser/Thr Phosphorylation Regulates the Fatty Acyl-AMP Ligase Activity of FadD32, an Essential Enzyme in Mycolic Acid Biosynthesis *
Article Snippet: .. In vitro phosphorylation was performed with 4 μg of wild-type FadD32 in 20 μl of buffer P (25 m m Tris-HCl (pH 7.0), 1 m m DTT, 5 m m MgCl2 , 1 m m EDTA, 50 μ m ATP), 200 μCi ml−1 (65 n m ) [γ-33 P]ATP (PerkinElmer Life Sciences, 3000 Ci mmol−1 ), and 0.5 μg of kinase to obtain the optimal autophosphorylation activity for each mycobacterial kinase for 30 min at 37 °C. .. Each reaction mixture was stopped by addition of an equal volume of 5× Laemmli buffer, and the mixture was heated at 100 °C for 5 min. After electrophoresis, gels were soaked in 16% TCA for 10 min at 90 °C, and dried.

Article Title: Disruption of a Nuclear NFATc2 Protein Stabilization Loop Confers Breast and Pancreatic Cancer Growth Suppression by Zoledronic Acid *
Article Snippet: .. To perform in vitro kinase assays, protein G-agarose-bound HA-NFATc2 proteins were incubated with recombinant GSK-3β (500,000 units/ml; New England Biolabs) for 30 min at 30 °C in a buffer containing 20 m m Tris HCL, pH 7.5, 10 m m MgCl2 , 5 m m DTT, 200 μ m ATP (cold), and 3 μCi of [γ-32 P]ATP (PerkinElmer Life Sciences). .. The beads were spun down, resuspended in an equal volume of 2× SDS-PAGE loading buffer, and boiled for 5 min.

Article Title: Cell Cycle-dependent Phosphorylation and Ubiquitination of a G Protein ? Subunit *
Article Snippet: .. In vitro kinase assays were conducted by incubating 0.08–0.16 pmol of purified GST-Elm1 (3.2–6.4 n m final concentration) and 12.5–25 pmol of recombinant purified Gpa1 or indicated mutants (0.5–1 μ m final concentration) in 1× kinase reaction buffer previously described for in vitro Elm1 kinase assays and containing 50 m m Tris-HCl (pH 7.5), 10 m m MgCl2 , 5 m m DTT, 2 m m EGTA, 200 m m sodium orthovanadate, and 10 μCi of [γ-32 P]ATP (PerkinElmer Life Sciences) for 1 h at 30 °C ( ). .. Reactions were stopped by addition of 6× SDS-PAGE loading buffer and immediately subjected to SDS-PAGE.

Mutagenesis:

Article Title: Crystal Structure of the Human Primase *
Article Snippet: .. The de novo activity of primase was tested in a 20-μl reaction containing 30 m m Hepes-KOH (pH 7.9), 50 m m KCl, 1 m m DTT, 2 m m MnCl2 , 50 μ m UTP, 50 μ m CTP, 0.15 μ m [γ-33 P]ATP (5000 Ci/mmol; PerkinElmer Life Sciences), 2 μ m template, 0.3 μ m primase (wild type or mutant), and 0.4 μ m p70-p180C . .. Reactions were assembled on ice and incubated at 35 °C for 1–20 min in a thermal cycler (Thermolyne Amplitron I).

Purification:

Article Title: Cell Cycle-dependent Phosphorylation and Ubiquitination of a G Protein ? Subunit *
Article Snippet: .. In vitro kinase assays were conducted by incubating 0.08–0.16 pmol of purified GST-Elm1 (3.2–6.4 n m final concentration) and 12.5–25 pmol of recombinant purified Gpa1 or indicated mutants (0.5–1 μ m final concentration) in 1× kinase reaction buffer previously described for in vitro Elm1 kinase assays and containing 50 m m Tris-HCl (pH 7.5), 10 m m MgCl2 , 5 m m DTT, 2 m m EGTA, 200 m m sodium orthovanadate, and 10 μCi of [γ-32 P]ATP (PerkinElmer Life Sciences) for 1 h at 30 °C ( ). .. Reactions were stopped by addition of 6× SDS-PAGE loading buffer and immediately subjected to SDS-PAGE.

Enzyme-linked Immunosorbent Assay:

Article Title: Interferon-inducible LY6E Protein Promotes HIV-1 Infection
Article Snippet: .. Briefly, 10 μl of cell supernatants was mixed with 40 μl of reaction mixture (50 m m Tris-HCl (pH 7.9), 5 m m MgCl2 , 0.5 m m EGTA, 0.05% Triton X-100, 2% (v/v) ethylene glycol, 150 m m KCl, 5 m m DTT, 0.3 m m GSH (reduced glutathione), 0.5 units/ml poly(rA)·oligo(dT), and 0.1 μCi/μl 3 H-labeled dTTP (PerkinElmer Life Sciences)) and incubated at 37 °C for 3 h. Samples were then placed on ice for 30 min with 150 μl of prechilled 10% (mass/volume (m/v)) TCA and filtered onto an ELISA plate (Merck Millipore), and the RT activity was measured in a Microbeta counter (Beckman Coulter). .. HIV-1 infectivity was determined by infecting indicator HeLa-TZM cells.

Concentration Assay:

Article Title: Cell Cycle-dependent Phosphorylation and Ubiquitination of a G Protein ? Subunit *
Article Snippet: .. In vitro kinase assays were conducted by incubating 0.08–0.16 pmol of purified GST-Elm1 (3.2–6.4 n m final concentration) and 12.5–25 pmol of recombinant purified Gpa1 or indicated mutants (0.5–1 μ m final concentration) in 1× kinase reaction buffer previously described for in vitro Elm1 kinase assays and containing 50 m m Tris-HCl (pH 7.5), 10 m m MgCl2 , 5 m m DTT, 2 m m EGTA, 200 m m sodium orthovanadate, and 10 μCi of [γ-32 P]ATP (PerkinElmer Life Sciences) for 1 h at 30 °C ( ). .. Reactions were stopped by addition of 6× SDS-PAGE loading buffer and immediately subjected to SDS-PAGE.

Incubation:

Article Title: Disruption of a Nuclear NFATc2 Protein Stabilization Loop Confers Breast and Pancreatic Cancer Growth Suppression by Zoledronic Acid *
Article Snippet: .. To perform in vitro kinase assays, protein G-agarose-bound HA-NFATc2 proteins were incubated with recombinant GSK-3β (500,000 units/ml; New England Biolabs) for 30 min at 30 °C in a buffer containing 20 m m Tris HCL, pH 7.5, 10 m m MgCl2 , 5 m m DTT, 200 μ m ATP (cold), and 3 μCi of [γ-32 P]ATP (PerkinElmer Life Sciences). .. The beads were spun down, resuspended in an equal volume of 2× SDS-PAGE loading buffer, and boiled for 5 min.

Article Title: Interferon-inducible LY6E Protein Promotes HIV-1 Infection
Article Snippet: .. Briefly, 10 μl of cell supernatants was mixed with 40 μl of reaction mixture (50 m m Tris-HCl (pH 7.9), 5 m m MgCl2 , 0.5 m m EGTA, 0.05% Triton X-100, 2% (v/v) ethylene glycol, 150 m m KCl, 5 m m DTT, 0.3 m m GSH (reduced glutathione), 0.5 units/ml poly(rA)·oligo(dT), and 0.1 μCi/μl 3 H-labeled dTTP (PerkinElmer Life Sciences)) and incubated at 37 °C for 3 h. Samples were then placed on ice for 30 min with 150 μl of prechilled 10% (mass/volume (m/v)) TCA and filtered onto an ELISA plate (Merck Millipore), and the RT activity was measured in a Microbeta counter (Beckman Coulter). .. HIV-1 infectivity was determined by infecting indicator HeLa-TZM cells.

Article Title: Mycobacterium smegmatis Lhr Is a DNA-dependent ATPase and a 3?-to-5? DNA Translocase and Helicase That Prefers to Unwind 3?-Tailed RNA:DNA Hybrids *
Article Snippet: .. Reaction mixtures containing (per 10 μl) 20 m m Tris-HCl, pH 7.0, 1 m m DTT, 1 m m CaCl2 , 1 m m [α-32 P]ATP (Perkin-Elmer Life Sciences), and DNA and Lhr as specified were incubated for 30 min at 37 °C. ..

Article Title: Phosphatase of Regenerating Liver 2 (PRL2) Is Essential for Placental Development by Down-regulating PTEN (Phosphatase and Tensin Homologue Deleted on Chromosome 10) and Activating Akt Protein *
Article Snippet: .. To analyze the PI3K activity of the immunoprecipitants, the beads were incubated in class I PI3K reaction buffer (Echelon) with 5 m m DTT, 10 μ m phosphatidylinositol 4,5-bisphosphate diC8 (Echelon), 100 μ m ATP, and 10 μCi of [γ-32 P]ATP (3,000 Ci/mmol) (PerkinElmer Life Sciences) at 37 °C for 3 h. The reaction was terminated by the addition of EDTA to 5 m m , and the phospholipids were separated on a TLC plate as described ( ) followed by exposing to x-ray film. .. The position of phosphatidylinositol 3,4,5-trisphosphate diC8 on the TLC plate was determined by phosphatidylinositol 3,4,5-trisphosphate diC8 standard (Echelon).

Thin Layer Chromatography:

Article Title: Phosphatase of Regenerating Liver 2 (PRL2) Is Essential for Placental Development by Down-regulating PTEN (Phosphatase and Tensin Homologue Deleted on Chromosome 10) and Activating Akt Protein *
Article Snippet: .. To analyze the PI3K activity of the immunoprecipitants, the beads were incubated in class I PI3K reaction buffer (Echelon) with 5 m m DTT, 10 μ m phosphatidylinositol 4,5-bisphosphate diC8 (Echelon), 100 μ m ATP, and 10 μCi of [γ-32 P]ATP (3,000 Ci/mmol) (PerkinElmer Life Sciences) at 37 °C for 3 h. The reaction was terminated by the addition of EDTA to 5 m m , and the phospholipids were separated on a TLC plate as described ( ) followed by exposing to x-ray film. .. The position of phosphatidylinositol 3,4,5-trisphosphate diC8 on the TLC plate was determined by phosphatidylinositol 3,4,5-trisphosphate diC8 standard (Echelon).

Activity Assay:

Article Title: Ser/Thr Phosphorylation Regulates the Fatty Acyl-AMP Ligase Activity of FadD32, an Essential Enzyme in Mycolic Acid Biosynthesis *
Article Snippet: .. In vitro phosphorylation was performed with 4 μg of wild-type FadD32 in 20 μl of buffer P (25 m m Tris-HCl (pH 7.0), 1 m m DTT, 5 m m MgCl2 , 1 m m EDTA, 50 μ m ATP), 200 μCi ml−1 (65 n m ) [γ-33 P]ATP (PerkinElmer Life Sciences, 3000 Ci mmol−1 ), and 0.5 μg of kinase to obtain the optimal autophosphorylation activity for each mycobacterial kinase for 30 min at 37 °C. .. Each reaction mixture was stopped by addition of an equal volume of 5× Laemmli buffer, and the mixture was heated at 100 °C for 5 min. After electrophoresis, gels were soaked in 16% TCA for 10 min at 90 °C, and dried.

Article Title: Interferon-inducible LY6E Protein Promotes HIV-1 Infection
Article Snippet: .. Briefly, 10 μl of cell supernatants was mixed with 40 μl of reaction mixture (50 m m Tris-HCl (pH 7.9), 5 m m MgCl2 , 0.5 m m EGTA, 0.05% Triton X-100, 2% (v/v) ethylene glycol, 150 m m KCl, 5 m m DTT, 0.3 m m GSH (reduced glutathione), 0.5 units/ml poly(rA)·oligo(dT), and 0.1 μCi/μl 3 H-labeled dTTP (PerkinElmer Life Sciences)) and incubated at 37 °C for 3 h. Samples were then placed on ice for 30 min with 150 μl of prechilled 10% (mass/volume (m/v)) TCA and filtered onto an ELISA plate (Merck Millipore), and the RT activity was measured in a Microbeta counter (Beckman Coulter). .. HIV-1 infectivity was determined by infecting indicator HeLa-TZM cells.

Article Title: Crystal Structure of the Human Primase *
Article Snippet: .. The de novo activity of primase was tested in a 20-μl reaction containing 30 m m Hepes-KOH (pH 7.9), 50 m m KCl, 1 m m DTT, 2 m m MnCl2 , 50 μ m UTP, 50 μ m CTP, 0.15 μ m [γ-33 P]ATP (5000 Ci/mmol; PerkinElmer Life Sciences), 2 μ m template, 0.3 μ m primase (wild type or mutant), and 0.4 μ m p70-p180C . .. Reactions were assembled on ice and incubated at 35 °C for 1–20 min in a thermal cycler (Thermolyne Amplitron I).

Article Title: Phosphatase of Regenerating Liver 2 (PRL2) Is Essential for Placental Development by Down-regulating PTEN (Phosphatase and Tensin Homologue Deleted on Chromosome 10) and Activating Akt Protein *
Article Snippet: .. To analyze the PI3K activity of the immunoprecipitants, the beads were incubated in class I PI3K reaction buffer (Echelon) with 5 m m DTT, 10 μ m phosphatidylinositol 4,5-bisphosphate diC8 (Echelon), 100 μ m ATP, and 10 μCi of [γ-32 P]ATP (3,000 Ci/mmol) (PerkinElmer Life Sciences) at 37 °C for 3 h. The reaction was terminated by the addition of EDTA to 5 m m , and the phospholipids were separated on a TLC plate as described ( ) followed by exposing to x-ray film. .. The position of phosphatidylinositol 3,4,5-trisphosphate diC8 on the TLC plate was determined by phosphatidylinositol 3,4,5-trisphosphate diC8 standard (Echelon).

Article Title: Characterization of the AtsR Hybrid Sensor Kinase Phosphorelay Pathway and Identification of Its Response Regulator in Burkholderia cenocepacia *
Article Snippet: .. For autophosphorylation and phosphotransfer reactions, 5 μmol of each protein was added to the phosphorylation buffer containing 50 m m Tris-HCl, pH 8.0, 50 m m KCl, 5 m m MgCl2 , 1 m m DTT, and 5 μCi of [γ-33 P]ATP (specific activity of 3000 Ci/mmol; 3.3 μ m stock solution) (PerkinElmer Life Sciences) in a final volume of 10 μl. ..

Recombinant:

Article Title: Disruption of a Nuclear NFATc2 Protein Stabilization Loop Confers Breast and Pancreatic Cancer Growth Suppression by Zoledronic Acid *
Article Snippet: .. To perform in vitro kinase assays, protein G-agarose-bound HA-NFATc2 proteins were incubated with recombinant GSK-3β (500,000 units/ml; New England Biolabs) for 30 min at 30 °C in a buffer containing 20 m m Tris HCL, pH 7.5, 10 m m MgCl2 , 5 m m DTT, 200 μ m ATP (cold), and 3 μCi of [γ-32 P]ATP (PerkinElmer Life Sciences). .. The beads were spun down, resuspended in an equal volume of 2× SDS-PAGE loading buffer, and boiled for 5 min.

Article Title: Cell Cycle-dependent Phosphorylation and Ubiquitination of a G Protein ? Subunit *
Article Snippet: .. In vitro kinase assays were conducted by incubating 0.08–0.16 pmol of purified GST-Elm1 (3.2–6.4 n m final concentration) and 12.5–25 pmol of recombinant purified Gpa1 or indicated mutants (0.5–1 μ m final concentration) in 1× kinase reaction buffer previously described for in vitro Elm1 kinase assays and containing 50 m m Tris-HCl (pH 7.5), 10 m m MgCl2 , 5 m m DTT, 2 m m EGTA, 200 m m sodium orthovanadate, and 10 μCi of [γ-32 P]ATP (PerkinElmer Life Sciences) for 1 h at 30 °C ( ). .. Reactions were stopped by addition of 6× SDS-PAGE loading buffer and immediately subjected to SDS-PAGE.

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    PerkinElmer m dtt
    Kinetics of phosphotransfer from AtsR to AtsT. A , 5 μmol of AtsR were preincubated with 5 μCi ([γ- 33 P]ATP) in a standard phosphorylation mixture (100 m m Tris-HCl, pH 8, 50 m m <t>KCl,</t> 5 m m MgCl 2 , 1 m m <t>DTT)</t> for 15 min, and then 5 μmol
    M Dtt, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 92/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m dtt/product/PerkinElmer
    Average 92 stars, based on 35 article reviews
    Price from $9.99 to $1999.99
    m dtt - by Bioz Stars, 2020-09
    92/100 stars
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    Kinetics of phosphotransfer from AtsR to AtsT. A , 5 μmol of AtsR were preincubated with 5 μCi ([γ- 33 P]ATP) in a standard phosphorylation mixture (100 m m Tris-HCl, pH 8, 50 m m KCl, 5 m m MgCl 2 , 1 m m DTT) for 15 min, and then 5 μmol

    Journal: The Journal of Biological Chemistry

    Article Title: Characterization of the AtsR Hybrid Sensor Kinase Phosphorelay Pathway and Identification of Its Response Regulator in Burkholderia cenocepacia *

    doi: 10.1074/jbc.M113.489914

    Figure Lengend Snippet: Kinetics of phosphotransfer from AtsR to AtsT. A , 5 μmol of AtsR were preincubated with 5 μCi ([γ- 33 P]ATP) in a standard phosphorylation mixture (100 m m Tris-HCl, pH 8, 50 m m KCl, 5 m m MgCl 2 , 1 m m DTT) for 15 min, and then 5 μmol

    Article Snippet: For autophosphorylation and phosphotransfer reactions, 5 μmol of each protein was added to the phosphorylation buffer containing 50 m m Tris-HCl, pH 8.0, 50 m m KCl, 5 m m MgCl2 , 1 m m DTT, and 5 μCi of [γ-33 P]ATP (specific activity of 3000 Ci/mmol; 3.3 μ m stock solution) (PerkinElmer Life Sciences) in a final volume of 10 μl.

    Techniques:

    Kinetics of phosphotransfer from AtsR and AtsR D536A to AtsT. After 10 min of preincubation of 5 μmol AtsR ( A ) or AtsR D536A ( B ) in a standard phosphorylation mixture (100 m m Tris-HCl, pH 8, 50 m m KCl, 5 m m MgCl 2 , 1 m m DTT, 5 μCi [γ-

    Journal: The Journal of Biological Chemistry

    Article Title: Characterization of the AtsR Hybrid Sensor Kinase Phosphorelay Pathway and Identification of Its Response Regulator in Burkholderia cenocepacia *

    doi: 10.1074/jbc.M113.489914

    Figure Lengend Snippet: Kinetics of phosphotransfer from AtsR and AtsR D536A to AtsT. After 10 min of preincubation of 5 μmol AtsR ( A ) or AtsR D536A ( B ) in a standard phosphorylation mixture (100 m m Tris-HCl, pH 8, 50 m m KCl, 5 m m MgCl 2 , 1 m m DTT, 5 μCi [γ-

    Article Snippet: For autophosphorylation and phosphotransfer reactions, 5 μmol of each protein was added to the phosphorylation buffer containing 50 m m Tris-HCl, pH 8.0, 50 m m KCl, 5 m m MgCl2 , 1 m m DTT, and 5 μCi of [γ-33 P]ATP (specific activity of 3000 Ci/mmol; 3.3 μ m stock solution) (PerkinElmer Life Sciences) in a final volume of 10 μl.

    Techniques:

    Phosphotransfer from AtsR and its derivatives to the AtsT response regulator. Five μmol of AtsR, AtsR H245A , and AtsR-HK was preincubated in individual standard phosphorylation mixtures (100 m m Tris-HCl pH 8, 50 m m KCl, 5 m m MgCl 2 , 1 m m DTT, 5

    Journal: The Journal of Biological Chemistry

    Article Title: Characterization of the AtsR Hybrid Sensor Kinase Phosphorelay Pathway and Identification of Its Response Regulator in Burkholderia cenocepacia *

    doi: 10.1074/jbc.M113.489914

    Figure Lengend Snippet: Phosphotransfer from AtsR and its derivatives to the AtsT response regulator. Five μmol of AtsR, AtsR H245A , and AtsR-HK was preincubated in individual standard phosphorylation mixtures (100 m m Tris-HCl pH 8, 50 m m KCl, 5 m m MgCl 2 , 1 m m DTT, 5

    Article Snippet: For autophosphorylation and phosphotransfer reactions, 5 μmol of each protein was added to the phosphorylation buffer containing 50 m m Tris-HCl, pH 8.0, 50 m m KCl, 5 m m MgCl2 , 1 m m DTT, and 5 μCi of [γ-33 P]ATP (specific activity of 3000 Ci/mmol; 3.3 μ m stock solution) (PerkinElmer Life Sciences) in a final volume of 10 μl.

    Techniques:

    Helicase activity of Lhr truncations on a 3′-tailed DNA:DNA or RNA:DNA duplex substrate. Helicase reaction mixtures (10 μl) contained 20 m m Tris-HCl, pH 7.0, 1 m m DTT, 5 m m CaCl 2 , either 50 n m of 5′ 32 P-labeled 3′-tailed

    Journal: The Journal of Biological Chemistry

    Article Title: Mycobacterium smegmatis Lhr Is a DNA-dependent ATPase and a 3?-to-5? DNA Translocase and Helicase That Prefers to Unwind 3?-Tailed RNA:DNA Hybrids *

    doi: 10.1074/jbc.M113.466854

    Figure Lengend Snippet: Helicase activity of Lhr truncations on a 3′-tailed DNA:DNA or RNA:DNA duplex substrate. Helicase reaction mixtures (10 μl) contained 20 m m Tris-HCl, pH 7.0, 1 m m DTT, 5 m m CaCl 2 , either 50 n m of 5′ 32 P-labeled 3′-tailed

    Article Snippet: Reaction mixtures containing (per 10 μl) 20 m m Tris-HCl, pH 7.0, 1 m m DTT, 1 m m CaCl2 , 1 m m [α-32 P]ATP (Perkin-Elmer Life Sciences), and DNA and Lhr as specified were incubated for 30 min at 37 °C.

    Techniques: Activity Assay, Labeling

    Single strand DNA length requirements for ATPase and helicase activity. A , ATPase reaction mixtures (10 μl) containing 20 m m Tris-HCl, pH 7.0, 1 m m DTT, 1 m m CaCl 2 , 1 m m [α- 32 P]ATP, 100 n m Lhr, and increasing amounts of 59-mer, 24-mer,

    Journal: The Journal of Biological Chemistry

    Article Title: Mycobacterium smegmatis Lhr Is a DNA-dependent ATPase and a 3?-to-5? DNA Translocase and Helicase That Prefers to Unwind 3?-Tailed RNA:DNA Hybrids *

    doi: 10.1074/jbc.M113.466854

    Figure Lengend Snippet: Single strand DNA length requirements for ATPase and helicase activity. A , ATPase reaction mixtures (10 μl) containing 20 m m Tris-HCl, pH 7.0, 1 m m DTT, 1 m m CaCl 2 , 1 m m [α- 32 P]ATP, 100 n m Lhr, and increasing amounts of 59-mer, 24-mer,

    Article Snippet: Reaction mixtures containing (per 10 μl) 20 m m Tris-HCl, pH 7.0, 1 m m DTT, 1 m m CaCl2 , 1 m m [α-32 P]ATP (Perkin-Elmer Life Sciences), and DNA and Lhr as specified were incubated for 30 min at 37 °C.

    Techniques: Activity Assay

    Lhr ATPase and helicase activity with DNA and RNA substrates. A , ATPase reaction mixtures (10 μl) containing 20 m m Tris-HCl, pH 7.0, 1 m m DTT, 1 m m CaCl 2 , 1 m m [α- 32 P]ATP, 100 n m Lhr, and the indicated amount of 24-mer DNA or RNA were

    Journal: The Journal of Biological Chemistry

    Article Title: Mycobacterium smegmatis Lhr Is a DNA-dependent ATPase and a 3?-to-5? DNA Translocase and Helicase That Prefers to Unwind 3?-Tailed RNA:DNA Hybrids *

    doi: 10.1074/jbc.M113.466854

    Figure Lengend Snippet: Lhr ATPase and helicase activity with DNA and RNA substrates. A , ATPase reaction mixtures (10 μl) containing 20 m m Tris-HCl, pH 7.0, 1 m m DTT, 1 m m CaCl 2 , 1 m m [α- 32 P]ATP, 100 n m Lhr, and the indicated amount of 24-mer DNA or RNA were

    Article Snippet: Reaction mixtures containing (per 10 μl) 20 m m Tris-HCl, pH 7.0, 1 m m DTT, 1 m m CaCl2 , 1 m m [α-32 P]ATP (Perkin-Elmer Life Sciences), and DNA and Lhr as specified were incubated for 30 min at 37 °C.

    Techniques: Activity Assay

    Lhr is a 3′-to-5′ DNA helicase. A , helicase reaction mixtures (10 μl) contained 20 m m Tris-HCl, pH 7.0, 1 m m DTT, 5 m m CaCl 2 , 100 n m of the indicated DNA substrate (depicted at the bottom , with the 5′ 32 P label denoted

    Journal: The Journal of Biological Chemistry

    Article Title: Mycobacterium smegmatis Lhr Is a DNA-dependent ATPase and a 3?-to-5? DNA Translocase and Helicase That Prefers to Unwind 3?-Tailed RNA:DNA Hybrids *

    doi: 10.1074/jbc.M113.466854

    Figure Lengend Snippet: Lhr is a 3′-to-5′ DNA helicase. A , helicase reaction mixtures (10 μl) contained 20 m m Tris-HCl, pH 7.0, 1 m m DTT, 5 m m CaCl 2 , 100 n m of the indicated DNA substrate (depicted at the bottom , with the 5′ 32 P label denoted

    Article Snippet: Reaction mixtures containing (per 10 μl) 20 m m Tris-HCl, pH 7.0, 1 m m DTT, 1 m m CaCl2 , 1 m m [α-32 P]ATP (Perkin-Elmer Life Sciences), and DNA and Lhr as specified were incubated for 30 min at 37 °C.

    Techniques: