Recombinant:
Article Title: Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins
Article Snippet: AMPK activity was determined by measurement of the phosphorylation of the ULight-SAMS peptide (TRF0118, PerkinElmer). .. In brief, active recombinant human AMPK α1β1γ1 (02-113, Carna Biosciences, Kobe, Japan) or α2β1γ1 (02-114, Carna Biosciences) (0.3 nM) was incubated with indicated concentrations of RX-375 or Compound B, or 100 μM AMP in 10 μL of kinase buffer (50 mM HEPES-NaOH [pH 7.5], 1 mM EGTA, 10 mM MgCl 2 , 2 mM dithiothreitol, and 0.01% Tween 20) containing 50 nM ULight-SAMS peptide and 10 μM ATP in white OptiPlate 384-well microplates (6007290, PerkinElmer) at room temperature for 30 min. After the addition of 10 μL of detection mix (CR97–100, PerkinElmer) containing 40 mM EDTA and 2 nM Eu-conjugated antibodies to phospho-ACC (TRF0208, PerkinElmer) and incubation for an additional 1 h, phosphorylation of the peptide was determined by time-resolved fluorescence resonance energy transfer (excitation at 320 nm, emission at 615 and 665 nm). .. The medium was rapidly aspirated from HepG2 cells treated with Compound B or metformin in six-well plates, and the cells were rapidly washed with 5 mL of ice-cold phosphate-buffered saline before the addition of ice-cold 70% perchloric acid (244252, Sigma-Aldrich).
Incubation:
Article Title: Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins
Article Snippet: AMPK activity was determined by measurement of the phosphorylation of the ULight-SAMS peptide (TRF0118, PerkinElmer). .. In brief, active recombinant human AMPK α1β1γ1 (02-113, Carna Biosciences, Kobe, Japan) or α2β1γ1 (02-114, Carna Biosciences) (0.3 nM) was incubated with indicated concentrations of RX-375 or Compound B, or 100 μM AMP in 10 μL of kinase buffer (50 mM HEPES-NaOH [pH 7.5], 1 mM EGTA, 10 mM MgCl 2 , 2 mM dithiothreitol, and 0.01% Tween 20) containing 50 nM ULight-SAMS peptide and 10 μM ATP in white OptiPlate 384-well microplates (6007290, PerkinElmer) at room temperature for 30 min. After the addition of 10 μL of detection mix (CR97–100, PerkinElmer) containing 40 mM EDTA and 2 nM Eu-conjugated antibodies to phospho-ACC (TRF0208, PerkinElmer) and incubation for an additional 1 h, phosphorylation of the peptide was determined by time-resolved fluorescence resonance energy transfer (excitation at 320 nm, emission at 615 and 665 nm). .. The medium was rapidly aspirated from HepG2 cells treated with Compound B or metformin in six-well plates, and the cells were rapidly washed with 5 mL of ice-cold phosphate-buffered saline before the addition of ice-cold 70% perchloric acid (244252, Sigma-Aldrich).
Phospho-proteomics:
Article Title: Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins
Article Snippet: AMPK activity was determined by measurement of the phosphorylation of the ULight-SAMS peptide (TRF0118, PerkinElmer). .. In brief, active recombinant human AMPK α1β1γ1 (02-113, Carna Biosciences, Kobe, Japan) or α2β1γ1 (02-114, Carna Biosciences) (0.3 nM) was incubated with indicated concentrations of RX-375 or Compound B, or 100 μM AMP in 10 μL of kinase buffer (50 mM HEPES-NaOH [pH 7.5], 1 mM EGTA, 10 mM MgCl 2 , 2 mM dithiothreitol, and 0.01% Tween 20) containing 50 nM ULight-SAMS peptide and 10 μM ATP in white OptiPlate 384-well microplates (6007290, PerkinElmer) at room temperature for 30 min. After the addition of 10 μL of detection mix (CR97–100, PerkinElmer) containing 40 mM EDTA and 2 nM Eu-conjugated antibodies to phospho-ACC (TRF0208, PerkinElmer) and incubation for an additional 1 h, phosphorylation of the peptide was determined by time-resolved fluorescence resonance energy transfer (excitation at 320 nm, emission at 615 and 665 nm). .. The medium was rapidly aspirated from HepG2 cells treated with Compound B or metformin in six-well plates, and the cells were rapidly washed with 5 mL of ice-cold phosphate-buffered saline before the addition of ice-cold 70% perchloric acid (244252, Sigma-Aldrich).
Fluorescence:
Article Title: Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins
Article Snippet: AMPK activity was determined by measurement of the phosphorylation of the ULight-SAMS peptide (TRF0118, PerkinElmer). .. In brief, active recombinant human AMPK α1β1γ1 (02-113, Carna Biosciences, Kobe, Japan) or α2β1γ1 (02-114, Carna Biosciences) (0.3 nM) was incubated with indicated concentrations of RX-375 or Compound B, or 100 μM AMP in 10 μL of kinase buffer (50 mM HEPES-NaOH [pH 7.5], 1 mM EGTA, 10 mM MgCl 2 , 2 mM dithiothreitol, and 0.01% Tween 20) containing 50 nM ULight-SAMS peptide and 10 μM ATP in white OptiPlate 384-well microplates (6007290, PerkinElmer) at room temperature for 30 min. After the addition of 10 μL of detection mix (CR97–100, PerkinElmer) containing 40 mM EDTA and 2 nM Eu-conjugated antibodies to phospho-ACC (TRF0208, PerkinElmer) and incubation for an additional 1 h, phosphorylation of the peptide was determined by time-resolved fluorescence resonance energy transfer (excitation at 320 nm, emission at 615 and 665 nm). .. The medium was rapidly aspirated from HepG2 cells treated with Compound B or metformin in six-well plates, and the cells were rapidly washed with 5 mL of ice-cold phosphate-buffered saline before the addition of ice-cold 70% perchloric acid (244252, Sigma-Aldrich).
Förster Resonance Energy Transfer:
Article Title: Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins
Article Snippet: AMPK activity was determined by measurement of the phosphorylation of the ULight-SAMS peptide (TRF0118, PerkinElmer). .. In brief, active recombinant human AMPK α1β1γ1 (02-113, Carna Biosciences, Kobe, Japan) or α2β1γ1 (02-114, Carna Biosciences) (0.3 nM) was incubated with indicated concentrations of RX-375 or Compound B, or 100 μM AMP in 10 μL of kinase buffer (50 mM HEPES-NaOH [pH 7.5], 1 mM EGTA, 10 mM MgCl 2 , 2 mM dithiothreitol, and 0.01% Tween 20) containing 50 nM ULight-SAMS peptide and 10 μM ATP in white OptiPlate 384-well microplates (6007290, PerkinElmer) at room temperature for 30 min. After the addition of 10 μL of detection mix (CR97–100, PerkinElmer) containing 40 mM EDTA and 2 nM Eu-conjugated antibodies to phospho-ACC (TRF0208, PerkinElmer) and incubation for an additional 1 h, phosphorylation of the peptide was determined by time-resolved fluorescence resonance energy transfer (excitation at 320 nm, emission at 615 and 665 nm). .. The medium was rapidly aspirated from HepG2 cells treated with Compound B or metformin in six-well plates, and the cells were rapidly washed with 5 mL of ice-cold phosphate-buffered saline before the addition of ice-cold 70% perchloric acid (244252, Sigma-Aldrich).
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