Structured Review

PerkinElmer γ p atp
γ P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/γ p atp/product/PerkinElmer
Average 99 stars, based on 6 article reviews
Price from $9.99 to $1999.99
γ p atp - by Bioz Stars, 2020-02
99/100 stars

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Related Articles

Synthesized:

Article Title: Resveratrol Prevents Impairment in Activation of Retinoic Acid Receptors and MAP Kinases in the Embryos of a Rodent Model of Diabetic Embryopathy
Article Snippet: Previously published RAR and RXR probes were selected and synthesized from integrated DNA technologies. .. The oligonucleotides were labeled with [γ- P]ATP (PerkinElmer, Waltham, Massachusetts) using T4 polynucleotide kinase.

Article Title: The bacterial transcription termination factor Rho coordinates Mg2+ homeostasis with translational signals
Article Snippet: RNA was synthesized from these templates using the Megascript T7 kit (Ambion) according to the manufacturer’s instructions. .. Dephosphorylated RNA was labeled with [γ- P]-ATP (Perkin Elmer) using T4 polynucleotide kinase (NEB) according to the manufacturer’s instructions for 30 min at 37°C.

Incubation:

Article Title: Resveratrol Prevents Impairment in Activation of Retinoic Acid Receptors and MAP Kinases in the Embryos of a Rodent Model of Diabetic Embryopathy
Article Snippet: Ten micrograms of total embryonic protein extract was incubated with 2 µg of poly(dI-dC) and radiolabeled probes (~10 000 cpm) in 20 µL of 10 mmol/L HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (pH 7.9), 50 mmol/L NaCl, 1 mmol/L EDTA, and 10% glycerol for 30 minutes at 25°C. .. The oligonucleotides were labeled with [γ- P]ATP (PerkinElmer, Waltham, Massachusetts) using T4 polynucleotide kinase.

Article Title: Autophosphorylation of CaMKK2 generates autonomous activity that is disrupted by a T85S mutation linked to anxiety and bipolar disorder
Article Snippet: .. For a standard 30 μl assay, 10 μl of recombinant CaMKK2 immobilised on anti-Flag agarose beads (50% v/v) was incubated in assay buffer (50 mM HEPES [pH 7.4], 1 mM DTT, 0.02% [v/v] Brij-35) containing 200 μM peptide substrate, 10 or 50 μM CaCl2 , 1 μM calmodulin (Sigma), 200 μM [γ- P]-ATP (Perkin Elmer) and 5 mM MgCl2 . .. Reactions were incubated at 30 °C for 10 min, after which they were terminated by spotting 15 μl onto P81 phosphocellulose paper and washing extensively in 1% phosphoric acid.

Article Title: Structure of a class II preQ1 riboswitch reveals ligand recognition by a new fold
Article Snippet: RNA was prepared by in vitro transcription (as described above for ITC), dephosphorylated with alkaline phosphatase, and radiolabeled with [γ P]ATP (PerkinElmer) and T4 polynucleotide kinase (New England Biolabs), which was then PAGE purified. .. In-line probing reactions each contained 1.0 × 106 CPM and were incubated at 25 °C for ~40 h. Reaction products were separated by denaturing 7.5% PAGE, the gel was dried, and then exposed to a phosphor storage screen for ~40 h. Imaging was carried out using a GE Storm 860, and gel quantification was conducted by use of SAFA with data normalized against invariant nucleotides selected by SAFA ; the nucleotides chosen as invariant bases were: 34, 40, 50, 53 and 54.

Article Title: The bacterial transcription termination factor Rho coordinates Mg2+ homeostasis with translational signals
Article Snippet: 1 µg of template DNA was used and reactions were incubated at 37°C overnight. .. Dephosphorylated RNA was labeled with [γ- P]-ATP (Perkin Elmer) using T4 polynucleotide kinase (NEB) according to the manufacturer’s instructions for 30 min at 37°C.

Article Title: DAPK2 is a novel regulator of mTORC1 activity and autophagy
Article Snippet: .. In vitro DAPK2 kinase assay Hundred nanograms of DAPK2 was incubated with 750 ng–1.5 μ g substrate in a kinase assay buffer (50 mM HEPES, pH 7.5, 20 mM MgCl2 , 50 μ M ATP, 5 μ Ci [γ - P]ATP or [γ - P]ATP (Perkin-Elmer, Waltham, MA, USA), protease and phosphatase inhibitors, 500 mM EGTA or 0.5 mM CaCl2 , 1 μ M CaM) at 30 °C for 30 min. ..

Activity Assay:

Article Title: Autophosphorylation of CaMKK2 generates autonomous activity that is disrupted by a T85S mutation linked to anxiety and bipolar disorder
Article Snippet: Paragraph title: CaMKK2 activity assay ... For a standard 30 μl assay, 10 μl of recombinant CaMKK2 immobilised on anti-Flag agarose beads (50% v/v) was incubated in assay buffer (50 mM HEPES [pH 7.4], 1 mM DTT, 0.02% [v/v] Brij-35) containing 200 μM peptide substrate, 10 or 50 μM CaCl2 , 1 μM calmodulin (Sigma), 200 μM [γ- P]-ATP (Perkin Elmer) and 5 mM MgCl2 .

Expressing:

Article Title: Autophosphorylation of CaMKK2 generates autonomous activity that is disrupted by a T85S mutation linked to anxiety and bipolar disorder
Article Snippet: For a standard 30 μl assay, 10 μl of recombinant CaMKK2 immobilised on anti-Flag agarose beads (50% v/v) was incubated in assay buffer (50 mM HEPES [pH 7.4], 1 mM DTT, 0.02% [v/v] Brij-35) containing 200 μM peptide substrate, 10 or 50 μM CaCl2 , 1 μM calmodulin (Sigma), 200 μM [γ- P]-ATP (Perkin Elmer) and 5 mM MgCl2 . .. Activity was corrected for variations in CaMKK2 expression between samples by immunoblotting using an anti-Flag antibody.

Western Blot:

Article Title: DAPK2 is a novel regulator of mTORC1 activity and autophagy
Article Snippet: In vitro DAPK2 kinase assay Hundred nanograms of DAPK2 was incubated with 750 ng–1.5 μ g substrate in a kinase assay buffer (50 mM HEPES, pH 7.5, 20 mM MgCl2 , 50 μ M ATP, 5 μ Ci [γ - P]ATP or [γ - P]ATP (Perkin-Elmer, Waltham, MA, USA), protease and phosphatase inhibitors, 500 mM EGTA or 0.5 mM CaCl2 , 1 μ M CaM) at 30 °C for 30 min. .. For reactions in which radioactive ATP was not used, the concentration of ATP in the assay was increased to 100 μ M, and the samples were transferred to nitrocellulose membrane and subjected to western blot using phospho-specific Abs.

Kinase Assay:

Article Title: DAPK2 is a novel regulator of mTORC1 activity and autophagy
Article Snippet: .. In vitro DAPK2 kinase assay Hundred nanograms of DAPK2 was incubated with 750 ng–1.5 μ g substrate in a kinase assay buffer (50 mM HEPES, pH 7.5, 20 mM MgCl2 , 50 μ M ATP, 5 μ Ci [γ - P]ATP or [γ - P]ATP (Perkin-Elmer, Waltham, MA, USA), protease and phosphatase inhibitors, 500 mM EGTA or 0.5 mM CaCl2 , 1 μ M CaM) at 30 °C for 30 min. ..

Gel Purification:

Article Title: The yeast transcription elongation factor Spt4/5 is a sequence‐specific RNA binding protein
Article Snippet: DNA pentaprobes were 5′ end‐labeled with [γ‐ P] ATP (PerkinElmer) using T4 polynucleotide kinase. .. Unincorporated [32 P] nucleotides were removed from DNA pentaprobe labeling reactions using Sephadex® G‐25 Quick Spin™ columns and gel purification for the RNA pentaprobes.

Activation Assay:

Article Title: Autophosphorylation of CaMKK2 generates autonomous activity that is disrupted by a T85S mutation linked to anxiety and bipolar disorder
Article Snippet: CaMKK2 activity assay CaMKK2 activity was measured by its ability to phosphorylate a synthetic peptide substrate (LSNLYHQGKFLQTFCGAPLYRRR) corresponding to the activation loop residues 196–215 of human NuaK2, except that serine-212 was substituted with an alanine to prevent phosphorylation of this residue by proline-directed kinases. .. For a standard 30 μl assay, 10 μl of recombinant CaMKK2 immobilised on anti-Flag agarose beads (50% v/v) was incubated in assay buffer (50 mM HEPES [pH 7.4], 1 mM DTT, 0.02% [v/v] Brij-35) containing 200 μM peptide substrate, 10 or 50 μM CaCl2 , 1 μM calmodulin (Sigma), 200 μM [γ- P]-ATP (Perkin Elmer) and 5 mM MgCl2 .

Footprinting:

Article Title: IL-17 receptor-associated adaptor Act1 directly stabilizes mRNAs to mediate IL-17 inflammatory signaling
Article Snippet: For RNase footprinting experiments, cold synthetic transcripts were dephosphorylated with SuperSAP (Affymetrix), purified, and resuspended in nuclease-free water. .. Dephosphorylated transcripts were end-labeled in the presence of [γ P] ATP (3000 Ci/mmole; Perkin Elmer Easy Tides) and T4 PNK (NEB) at 20 units/pmole RNA.

Generated:

Article Title: The bacterial transcription termination factor Rho coordinates Mg2+ homeostasis with translational signals
Article Snippet: T7 promoter-driven transcription templates corresponding to the corA leader were generated by PCR using plasmid pYS1040 or mutant derivatives and primers 8445 and 8444. .. Dephosphorylated RNA was labeled with [γ- P]-ATP (Perkin Elmer) using T4 polynucleotide kinase (NEB) according to the manufacturer’s instructions for 30 min at 37°C.

Imaging:

Article Title: Structure of a class II preQ1 riboswitch reveals ligand recognition by a new fold
Article Snippet: RNA was prepared by in vitro transcription (as described above for ITC), dephosphorylated with alkaline phosphatase, and radiolabeled with [γ P]ATP (PerkinElmer) and T4 polynucleotide kinase (New England Biolabs), which was then PAGE purified. .. In-line probing reactions each contained 1.0 × 106 CPM and were incubated at 25 °C for ~40 h. Reaction products were separated by denaturing 7.5% PAGE, the gel was dried, and then exposed to a phosphor storage screen for ~40 h. Imaging was carried out using a GE Storm 860, and gel quantification was conducted by use of SAFA with data normalized against invariant nucleotides selected by SAFA ; the nucleotides chosen as invariant bases were: 34, 40, 50, 53 and 54.

Sequencing:

Article Title: Structure of a class II preQ1 riboswitch reveals ligand recognition by a new fold
Article Snippet: In-line probing of the L. rhamnosis preQ1 -II riboswitch The in-line probing reaction was carried out on an extended preQ1 -II sequence ( ) essentially as described . .. RNA was prepared by in vitro transcription (as described above for ITC), dephosphorylated with alkaline phosphatase, and radiolabeled with [γ P]ATP (PerkinElmer) and T4 polynucleotide kinase (New England Biolabs), which was then PAGE purified.

Recombinant:

Article Title: Autophosphorylation of CaMKK2 generates autonomous activity that is disrupted by a T85S mutation linked to anxiety and bipolar disorder
Article Snippet: .. For a standard 30 μl assay, 10 μl of recombinant CaMKK2 immobilised on anti-Flag agarose beads (50% v/v) was incubated in assay buffer (50 mM HEPES [pH 7.4], 1 mM DTT, 0.02% [v/v] Brij-35) containing 200 μM peptide substrate, 10 or 50 μM CaCl2 , 1 μM calmodulin (Sigma), 200 μM [γ- P]-ATP (Perkin Elmer) and 5 mM MgCl2 . .. Reactions were incubated at 30 °C for 10 min, after which they were terminated by spotting 15 μl onto P81 phosphocellulose paper and washing extensively in 1% phosphoric acid.

Radioactivity:

Article Title: Autophosphorylation of CaMKK2 generates autonomous activity that is disrupted by a T85S mutation linked to anxiety and bipolar disorder
Article Snippet: For a standard 30 μl assay, 10 μl of recombinant CaMKK2 immobilised on anti-Flag agarose beads (50% v/v) was incubated in assay buffer (50 mM HEPES [pH 7.4], 1 mM DTT, 0.02% [v/v] Brij-35) containing 200 μM peptide substrate, 10 or 50 μM CaCl2 , 1 μM calmodulin (Sigma), 200 μM [γ- P]-ATP (Perkin Elmer) and 5 mM MgCl2 . .. Radioactivity was quantified by scintillation counting.

Mutagenesis:

Article Title: The bacterial transcription termination factor Rho coordinates Mg2+ homeostasis with translational signals
Article Snippet: T7 promoter-driven transcription templates corresponding to the corA leader were generated by PCR using plasmid pYS1040 or mutant derivatives and primers 8445 and 8444. .. Dephosphorylated RNA was labeled with [γ- P]-ATP (Perkin Elmer) using T4 polynucleotide kinase (NEB) according to the manufacturer’s instructions for 30 min at 37°C.

Labeling:

Article Title: The yeast transcription elongation factor Spt4/5 is a sequence‐specific RNA binding protein
Article Snippet: DNA pentaprobes were 5′ end‐labeled with [γ‐ P] ATP (PerkinElmer) using T4 polynucleotide kinase. .. Unincorporated [32 P] nucleotides were removed from DNA pentaprobe labeling reactions using Sephadex® G‐25 Quick Spin™ columns and gel purification for the RNA pentaprobes.

Article Title: Resveratrol Prevents Impairment in Activation of Retinoic Acid Receptors and MAP Kinases in the Embryos of a Rodent Model of Diabetic Embryopathy
Article Snippet: .. The oligonucleotides were labeled with [γ- P]ATP (PerkinElmer, Waltham, Massachusetts) using T4 polynucleotide kinase. .. One negative control (without protein) was included with each gel shift assay to evaluate the background.

Article Title: The bacterial transcription termination factor Rho coordinates Mg2+ homeostasis with translational signals
Article Snippet: .. Dephosphorylated RNA was labeled with [γ- P]-ATP (Perkin Elmer) using T4 polynucleotide kinase (NEB) according to the manufacturer’s instructions for 30 min at 37°C. .. Labeled RNA was purified on a 6% TBE urea gel and ethanol precipitated.

Purification:

Article Title: Structure of a class II preQ1 riboswitch reveals ligand recognition by a new fold
Article Snippet: .. RNA was prepared by in vitro transcription (as described above for ITC), dephosphorylated with alkaline phosphatase, and radiolabeled with [γ P]ATP (PerkinElmer) and T4 polynucleotide kinase (New England Biolabs), which was then PAGE purified. ..

Article Title: IL-17 receptor-associated adaptor Act1 directly stabilizes mRNAs to mediate IL-17 inflammatory signaling
Article Snippet: For RNase footprinting experiments, cold synthetic transcripts were dephosphorylated with SuperSAP (Affymetrix), purified, and resuspended in nuclease-free water. .. Dephosphorylated transcripts were end-labeled in the presence of [γ P] ATP (3000 Ci/mmole; Perkin Elmer Easy Tides) and T4 PNK (NEB) at 20 units/pmole RNA.

Article Title: The bacterial transcription termination factor Rho coordinates Mg2+ homeostasis with translational signals
Article Snippet: RNAs were phenol/chloroform extracted, purified on 6% TBE urea gels (Life Technologies) and ethanol precipitated. .. Dephosphorylated RNA was labeled with [γ- P]-ATP (Perkin Elmer) using T4 polynucleotide kinase (NEB) according to the manufacturer’s instructions for 30 min at 37°C.

Polymerase Chain Reaction:

Article Title: The bacterial transcription termination factor Rho coordinates Mg2+ homeostasis with translational signals
Article Snippet: T7 promoter-driven transcription templates corresponding to the corA leader were generated by PCR using plasmid pYS1040 or mutant derivatives and primers 8445 and 8444. .. Dephosphorylated RNA was labeled with [γ- P]-ATP (Perkin Elmer) using T4 polynucleotide kinase (NEB) according to the manufacturer’s instructions for 30 min at 37°C.

Electrophoretic Mobility Shift Assay:

Article Title: Resveratrol Prevents Impairment in Activation of Retinoic Acid Receptors and MAP Kinases in the Embryos of a Rodent Model of Diabetic Embryopathy
Article Snippet: Paragraph title: Electrophoretic Mobility Shift Assay ... The oligonucleotides were labeled with [γ- P]ATP (PerkinElmer, Waltham, Massachusetts) using T4 polynucleotide kinase.

Polyacrylamide Gel Electrophoresis:

Article Title: Structure of a class II preQ1 riboswitch reveals ligand recognition by a new fold
Article Snippet: .. RNA was prepared by in vitro transcription (as described above for ITC), dephosphorylated with alkaline phosphatase, and radiolabeled with [γ P]ATP (PerkinElmer) and T4 polynucleotide kinase (New England Biolabs), which was then PAGE purified. ..

Plasmid Preparation:

Article Title: The bacterial transcription termination factor Rho coordinates Mg2+ homeostasis with translational signals
Article Snippet: T7 promoter-driven transcription templates corresponding to the corA leader were generated by PCR using plasmid pYS1040 or mutant derivatives and primers 8445 and 8444. .. Dephosphorylated RNA was labeled with [γ- P]-ATP (Perkin Elmer) using T4 polynucleotide kinase (NEB) according to the manufacturer’s instructions for 30 min at 37°C.

Negative Control:

Article Title: Resveratrol Prevents Impairment in Activation of Retinoic Acid Receptors and MAP Kinases in the Embryos of a Rodent Model of Diabetic Embryopathy
Article Snippet: The oligonucleotides were labeled with [γ- P]ATP (PerkinElmer, Waltham, Massachusetts) using T4 polynucleotide kinase. .. One negative control (without protein) was included with each gel shift assay to evaluate the background.

Binding Assay:

Article Title: Resveratrol Prevents Impairment in Activation of Retinoic Acid Receptors and MAP Kinases in the Embryos of a Rodent Model of Diabetic Embryopathy
Article Snippet: The oligonucleotides were labeled with [γ- P]ATP (PerkinElmer, Waltham, Massachusetts) using T4 polynucleotide kinase. .. Binding reactions were resolved on 4.5% native polyacrylamide gels containing 0.5× TBE (Tris/Borate/EDTA) buffer (45 mmol/L Tris base, 45 mmol/L boric acid, 1 mmol/L EDTA, pH 8.0) at 4°C for 2 hours at 150 V. The gels were then dried using gel drying film (Promega, Madison, Wisconsin) soaked in 40% methanol, 10% glycerol, and 7.5% acetic acid for 3 to 5 minutes.

Article Title: Autophosphorylation of CaMKK2 generates autonomous activity that is disrupted by a T85S mutation linked to anxiety and bipolar disorder
Article Snippet: The peptide also contained three additional arginine residues at the C-terminus to promote binding of the peptide to P81 phosphocellulose paper. .. For a standard 30 μl assay, 10 μl of recombinant CaMKK2 immobilised on anti-Flag agarose beads (50% v/v) was incubated in assay buffer (50 mM HEPES [pH 7.4], 1 mM DTT, 0.02% [v/v] Brij-35) containing 200 μM peptide substrate, 10 or 50 μM CaCl2 , 1 μM calmodulin (Sigma), 200 μM [γ- P]-ATP (Perkin Elmer) and 5 mM MgCl2 .

In Vitro:

Article Title: The yeast transcription elongation factor Spt4/5 is a sequence‐specific RNA binding protein
Article Snippet: DNA pentaprobes were 5′ end‐labeled with [γ‐ P] ATP (PerkinElmer) using T4 polynucleotide kinase. .. [α‐ P] UTP was incorporated into RNA pentaprobes during in‐vitro transcription.

Article Title: Structure of a class II preQ1 riboswitch reveals ligand recognition by a new fold
Article Snippet: .. RNA was prepared by in vitro transcription (as described above for ITC), dephosphorylated with alkaline phosphatase, and radiolabeled with [γ P]ATP (PerkinElmer) and T4 polynucleotide kinase (New England Biolabs), which was then PAGE purified. ..

Article Title: IL-17 receptor-associated adaptor Act1 directly stabilizes mRNAs to mediate IL-17 inflammatory signaling
Article Snippet: Paragraph title: In vitro transcription and cap-labeling: ... Dephosphorylated transcripts were end-labeled in the presence of [γ P] ATP (3000 Ci/mmole; Perkin Elmer Easy Tides) and T4 PNK (NEB) at 20 units/pmole RNA.

Article Title: DAPK2 is a novel regulator of mTORC1 activity and autophagy
Article Snippet: .. In vitro DAPK2 kinase assay Hundred nanograms of DAPK2 was incubated with 750 ng–1.5 μ g substrate in a kinase assay buffer (50 mM HEPES, pH 7.5, 20 mM MgCl2 , 50 μ M ATP, 5 μ Ci [γ - P]ATP or [γ - P]ATP (Perkin-Elmer, Waltham, MA, USA), protease and phosphatase inhibitors, 500 mM EGTA or 0.5 mM CaCl2 , 1 μ M CaM) at 30 °C for 30 min. ..

Concentration Assay:

Article Title: DAPK2 is a novel regulator of mTORC1 activity and autophagy
Article Snippet: In vitro DAPK2 kinase assay Hundred nanograms of DAPK2 was incubated with 750 ng–1.5 μ g substrate in a kinase assay buffer (50 mM HEPES, pH 7.5, 20 mM MgCl2 , 50 μ M ATP, 5 μ Ci [γ - P]ATP or [γ - P]ATP (Perkin-Elmer, Waltham, MA, USA), protease and phosphatase inhibitors, 500 mM EGTA or 0.5 mM CaCl2 , 1 μ M CaM) at 30 °C for 30 min. .. For reactions in which radioactive ATP was not used, the concentration of ATP in the assay was increased to 100 μ M, and the samples were transferred to nitrocellulose membrane and subjected to western blot using phospho-specific Abs.

Chick Chorioallantoic Membrane Assay:

Article Title: DAPK2 is a novel regulator of mTORC1 activity and autophagy
Article Snippet: .. In vitro DAPK2 kinase assay Hundred nanograms of DAPK2 was incubated with 750 ng–1.5 μ g substrate in a kinase assay buffer (50 mM HEPES, pH 7.5, 20 mM MgCl2 , 50 μ M ATP, 5 μ Ci [γ - P]ATP or [γ - P]ATP (Perkin-Elmer, Waltham, MA, USA), protease and phosphatase inhibitors, 500 mM EGTA or 0.5 mM CaCl2 , 1 μ M CaM) at 30 °C for 30 min. ..

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  • 99
    PerkinElmer γ 32 p atp
    PXR is phosphorylated in vitro and in cells (A) His-PXR (1 or 2.5 µg) was incubated at 37°C for 30 min with Cdk2 and cyclin E along with [γ- 32 <t>P]-ATP.</t> Samples were resolved on a 4–12% gradient gel, and [γ- 32 P]-ATP incorporation was visualized using a phosphor screen (upper panel), and protein amounts in the samples were detected by SimplyBlue staining of the gel (lower panel). Histone H1 and His-tag were used as a positive and negative substrate control, respectively. The PXR band was indicated with an arrow. (B) Phosphorylation sites identified by using mass spectrometry analysis in His-PXR WT phosphorylated by Cdk2/cyclin E in vitro , and in Flag-PXR WT, Flag-PXR T133A, or Flag-PXR T135A immunoprecipitated from HEK293T cells transiently transfected with corresponding plasmid ( in vivo ). Serine or threonine residues followed by an asterisk (*) indicate phosphorylated residues; UM = unmodified peptide; M = phosphorylated peptide; nd = not detected; nt = not tested. Signal intensities are calculated from area under the curve for the detected precursor ions. (C) Anti-Flag immunoprecipitated samples prepared from HEK293T cells transiently overexpressing either Flag-PXR WT (lanes 1 2) or mutants Flag-PXR T133A (lanes 4 5) or Flag-PXR T135A (lanes 7 8) were resolved on gradient gel and stained using Sypro Ruby stain. (D) Modified peptide sequence TFDTTFS*HFK (asterisk indicating serine phosphorylation), was identified based on assignment of multiple product ions ( b and y ions) in the MS/MS scan of the precursor ion at M/z 665.78. The phosphorylation of serine 167 was confirmed based on the assignment of characteristic “ y-H 3 PO 4 ” ions and other ions (based on a mass loss of 97.9769 Da). (E) Extracted-ion chromatography (XIC) of wild type and mutant PXR sequences showing elution times and signal intensities for the non-modified peptide as well as the singly phosphorylated peptide. Panel (a) and (b) are derived from the immunoprecipitated T133A sample and show the TGAQPLGVQGLTEEQR and T*GAQPLGVQGLTEEQR, respectively. Panel (c) and (d) are derived from the immunoprecipitated T135A sample and show the AGTQPLGVQGLTEEQR and AGT*QPLGVQGLTEEQR, respectively. Panel (e) and (f) are derived from the immunoprecipitated PXR WT sample and show the TGTQPLGVQGLTEEQR and T*GTQPLGVQGLTEEQR/ TGT*QPLGVQGLTEEQR, respectively. Relative abundance (RA) of the signals of the corresponding peptides is noted for each XIC.
    γ 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/PerkinElmer
    Average 99 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    99
    PerkinElmer atp
    The phosphoadaptor subunit <t>Cks1</t> provide processivity for the multiphosphorylation of Sic1 by Cln2-Cdk1 and Clb5-Cdk1. (a) Cln2- and Clb5-Cdk1 complexes were incubated with Sic1ΔC and 32 <t>P-ATP.</t> The reactions also included wild-type Cks1 (wt) or a version with a mutated phosphate-binding site ( mut ; see Supplementary Methods ). Phosphorylated substrates were separated using Phos-Tag SDS-PAGE gels. (b) Reactions were performed in the presence of a phosphopeptide competitor (P) based on the sequence surrounding T45 in Sic1. (c) The phosphorylation of a Sic1ΔC version containing a single Cdk site (Sic1ΔC-T5, with other Cdk consensus sites mutated to alanines) was not affected by Cks1 mut or the phosphopeptide. The standard SDS-PAGE was used. (d) Time courses of Sic1ΔC multiphosphorylation were followed by Phos-Tag SDS-PAGE. (e) The quantified data from (d). The intensities of 32 P-labeled proteins were divided by the number of phosphates as indicated to obtain the levels of different phosphoforms. In the experiments presented in Fig. 1 the enzyme concentrations were chosen to obtain roughly equal substrate labeling.
    Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/atp/product/PerkinElmer
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    atp - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    Image Search Results


    PXR is phosphorylated in vitro and in cells (A) His-PXR (1 or 2.5 µg) was incubated at 37°C for 30 min with Cdk2 and cyclin E along with [γ- 32 P]-ATP. Samples were resolved on a 4–12% gradient gel, and [γ- 32 P]-ATP incorporation was visualized using a phosphor screen (upper panel), and protein amounts in the samples were detected by SimplyBlue staining of the gel (lower panel). Histone H1 and His-tag were used as a positive and negative substrate control, respectively. The PXR band was indicated with an arrow. (B) Phosphorylation sites identified by using mass spectrometry analysis in His-PXR WT phosphorylated by Cdk2/cyclin E in vitro , and in Flag-PXR WT, Flag-PXR T133A, or Flag-PXR T135A immunoprecipitated from HEK293T cells transiently transfected with corresponding plasmid ( in vivo ). Serine or threonine residues followed by an asterisk (*) indicate phosphorylated residues; UM = unmodified peptide; M = phosphorylated peptide; nd = not detected; nt = not tested. Signal intensities are calculated from area under the curve for the detected precursor ions. (C) Anti-Flag immunoprecipitated samples prepared from HEK293T cells transiently overexpressing either Flag-PXR WT (lanes 1 2) or mutants Flag-PXR T133A (lanes 4 5) or Flag-PXR T135A (lanes 7 8) were resolved on gradient gel and stained using Sypro Ruby stain. (D) Modified peptide sequence TFDTTFS*HFK (asterisk indicating serine phosphorylation), was identified based on assignment of multiple product ions ( b and y ions) in the MS/MS scan of the precursor ion at M/z 665.78. The phosphorylation of serine 167 was confirmed based on the assignment of characteristic “ y-H 3 PO 4 ” ions and other ions (based on a mass loss of 97.9769 Da). (E) Extracted-ion chromatography (XIC) of wild type and mutant PXR sequences showing elution times and signal intensities for the non-modified peptide as well as the singly phosphorylated peptide. Panel (a) and (b) are derived from the immunoprecipitated T133A sample and show the TGAQPLGVQGLTEEQR and T*GAQPLGVQGLTEEQR, respectively. Panel (c) and (d) are derived from the immunoprecipitated T135A sample and show the AGTQPLGVQGLTEEQR and AGT*QPLGVQGLTEEQR, respectively. Panel (e) and (f) are derived from the immunoprecipitated PXR WT sample and show the TGTQPLGVQGLTEEQR and T*GTQPLGVQGLTEEQR/ TGT*QPLGVQGLTEEQR, respectively. Relative abundance (RA) of the signals of the corresponding peptides is noted for each XIC.

    Journal: Biochemical pharmacology

    Article Title: Identification and Characterization of Phosphorylation Sites within the Pregnane X Receptor Protein

    doi: 10.1016/j.bcp.2013.10.015

    Figure Lengend Snippet: PXR is phosphorylated in vitro and in cells (A) His-PXR (1 or 2.5 µg) was incubated at 37°C for 30 min with Cdk2 and cyclin E along with [γ- 32 P]-ATP. Samples were resolved on a 4–12% gradient gel, and [γ- 32 P]-ATP incorporation was visualized using a phosphor screen (upper panel), and protein amounts in the samples were detected by SimplyBlue staining of the gel (lower panel). Histone H1 and His-tag were used as a positive and negative substrate control, respectively. The PXR band was indicated with an arrow. (B) Phosphorylation sites identified by using mass spectrometry analysis in His-PXR WT phosphorylated by Cdk2/cyclin E in vitro , and in Flag-PXR WT, Flag-PXR T133A, or Flag-PXR T135A immunoprecipitated from HEK293T cells transiently transfected with corresponding plasmid ( in vivo ). Serine or threonine residues followed by an asterisk (*) indicate phosphorylated residues; UM = unmodified peptide; M = phosphorylated peptide; nd = not detected; nt = not tested. Signal intensities are calculated from area under the curve for the detected precursor ions. (C) Anti-Flag immunoprecipitated samples prepared from HEK293T cells transiently overexpressing either Flag-PXR WT (lanes 1 2) or mutants Flag-PXR T133A (lanes 4 5) or Flag-PXR T135A (lanes 7 8) were resolved on gradient gel and stained using Sypro Ruby stain. (D) Modified peptide sequence TFDTTFS*HFK (asterisk indicating serine phosphorylation), was identified based on assignment of multiple product ions ( b and y ions) in the MS/MS scan of the precursor ion at M/z 665.78. The phosphorylation of serine 167 was confirmed based on the assignment of characteristic “ y-H 3 PO 4 ” ions and other ions (based on a mass loss of 97.9769 Da). (E) Extracted-ion chromatography (XIC) of wild type and mutant PXR sequences showing elution times and signal intensities for the non-modified peptide as well as the singly phosphorylated peptide. Panel (a) and (b) are derived from the immunoprecipitated T133A sample and show the TGAQPLGVQGLTEEQR and T*GAQPLGVQGLTEEQR, respectively. Panel (c) and (d) are derived from the immunoprecipitated T135A sample and show the AGTQPLGVQGLTEEQR and AGT*QPLGVQGLTEEQR, respectively. Panel (e) and (f) are derived from the immunoprecipitated PXR WT sample and show the TGTQPLGVQGLTEEQR and T*GTQPLGVQGLTEEQR/ TGT*QPLGVQGLTEEQR, respectively. Relative abundance (RA) of the signals of the corresponding peptides is noted for each XIC.

    Article Snippet: Different amount of His-PXR as indicated (1 µg or 2.5 µg) was incubated in kinase buffer with 20 ng Cdk2/cyclin E (EMD Millipore, Billerica, MA), 5 µCi [γ-32 P]ATP (Perkin-Elmer, Santa Clara, CA), and 5 µM cold ATP.

    Techniques: In Vitro, Incubation, Staining, Mass Spectrometry, Immunoprecipitation, Transfection, Plasmid Preparation, In Vivo, Modification, Sequencing, Ion Chromatography, Mutagenesis, Derivative Assay

    5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).

    Journal: RNA

    Article Title: Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)

    doi: 10.1261/rna.5247704

    Figure Lengend Snippet: 5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).

    Article Snippet: Radiolabeled RNAs were prepared with γ-32 P-ATP (PerkinElmer) and T4 PNK (New England Biolabs) and purified by 20% denaturing PAGE followed by ethanol precipitation.

    Techniques: Electrophoretic Mobility Shift Assay, Blocking Assay, Polyacrylamide Gel Electrophoresis, Radioactivity, Positive Control

    Aly2 interacts with and requires Npr1 to promote Gap1 PM-localization. (A) BJ5459 or BJ5459-Npr1-MYC cells expressing GST (pKK212), GST-Aly1 (pKK212-Aly1), or GST-Aly2 (pKK212-Aly2) were grown in SC-0.25% NH 4 . Protein extracts were split, with half used for GST and half for anti-MYC Ab purifications, and copurification assessed by WB. Samples were run on one gel, but line denotes lane removal. (B) WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1 or -Aly2 were grown in MIN-0.25% NH 4 , washed, and inoculated at equal density into either MIN-0.1% GLN or MIN-0.1% citrulline (CIT). Growth was monitored using OD 600 readings, taken every 30 min with a Tecan Genios microtiter plate reader. (C) Growth of WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1, or -Aly2 on MIN-0.5% NH 4 ± AzC. (D) Prototrophic WT (BY4741) and npr1 Δ (2029) with pCK283 and pRS426, - ALY1 , or - ALY2 were assayed for [ 14 C]citrulline uptake. The mean uptake rate ± SDM for three replicates is shown as % relative to WT. (E and F) Prototrophic npr1 ΔΔ (32029) cells with Gap1-GFP (pCK230), pRS313 and pRS425, -Aly1, or -Aly2 were grown in SC-0.5% NH 4 , washed, and grown for 3 h in MIN-0.5% NH 4 and (E) cell extracts were assessed by WB or (F) Gap1-GFP was visualized using fluorescence microscopy (scale bar, 5 μm). (G) GST-Aly1 (pKK212-Aly1) or -Aly2 (pKK212-Aly2) were purified from extracts of WT (BJ5459) or npr1 Δ (BJ5459- npr1 Δ:: KanMX ) cells grown in SC-0.25% NH 4 and assessed by WB. Similar results were obtained using GFP-Aly1 and -Aly2 extracted from WT (BY4741) or npr1 Δ (2029) cells (data not shown). Phosphorylation of GST-Aly2 was further analyzed using mock (−) or lambda phosphatase treatment (λ-PP). (H) pET and pET-Aly2 were purified from E. coli and incubated with [γ- 32 P]ATP kinase cocktail in the presence (+) or absence (−) of Npr1. Proteins were analyzed by SDS-PAGE and imaged on a Typhoon scanner for 32 P quantification or stained for total protein. pET-Aly2 phosphorylation ± Npr1 is shown (left-hand portion of panel). The mean fold-increase in phospho-signal upon addition of Npr1 kinase (normalized for loading) is plotted from three replicate experiments ± SDM for both pET-Aly2 and the pET tag alone (the latter is not phosphorylated by Npr1) in the right-hand portion of the panel.

    Journal: Molecular Biology of the Cell

    Article Title: ?-Arrestins Aly1 and Aly2 Regulate Intracellular Trafficking in Response to Nutrient Signaling

    doi: 10.1091/mbc.E10-07-0636

    Figure Lengend Snippet: Aly2 interacts with and requires Npr1 to promote Gap1 PM-localization. (A) BJ5459 or BJ5459-Npr1-MYC cells expressing GST (pKK212), GST-Aly1 (pKK212-Aly1), or GST-Aly2 (pKK212-Aly2) were grown in SC-0.25% NH 4 . Protein extracts were split, with half used for GST and half for anti-MYC Ab purifications, and copurification assessed by WB. Samples were run on one gel, but line denotes lane removal. (B) WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1 or -Aly2 were grown in MIN-0.25% NH 4 , washed, and inoculated at equal density into either MIN-0.1% GLN or MIN-0.1% citrulline (CIT). Growth was monitored using OD 600 readings, taken every 30 min with a Tecan Genios microtiter plate reader. (C) Growth of WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1, or -Aly2 on MIN-0.5% NH 4 ± AzC. (D) Prototrophic WT (BY4741) and npr1 Δ (2029) with pCK283 and pRS426, - ALY1 , or - ALY2 were assayed for [ 14 C]citrulline uptake. The mean uptake rate ± SDM for three replicates is shown as % relative to WT. (E and F) Prototrophic npr1 ΔΔ (32029) cells with Gap1-GFP (pCK230), pRS313 and pRS425, -Aly1, or -Aly2 were grown in SC-0.5% NH 4 , washed, and grown for 3 h in MIN-0.5% NH 4 and (E) cell extracts were assessed by WB or (F) Gap1-GFP was visualized using fluorescence microscopy (scale bar, 5 μm). (G) GST-Aly1 (pKK212-Aly1) or -Aly2 (pKK212-Aly2) were purified from extracts of WT (BJ5459) or npr1 Δ (BJ5459- npr1 Δ:: KanMX ) cells grown in SC-0.25% NH 4 and assessed by WB. Similar results were obtained using GFP-Aly1 and -Aly2 extracted from WT (BY4741) or npr1 Δ (2029) cells (data not shown). Phosphorylation of GST-Aly2 was further analyzed using mock (−) or lambda phosphatase treatment (λ-PP). (H) pET and pET-Aly2 were purified from E. coli and incubated with [γ- 32 P]ATP kinase cocktail in the presence (+) or absence (−) of Npr1. Proteins were analyzed by SDS-PAGE and imaged on a Typhoon scanner for 32 P quantification or stained for total protein. pET-Aly2 phosphorylation ± Npr1 is shown (left-hand portion of panel). The mean fold-increase in phospho-signal upon addition of Npr1 kinase (normalized for loading) is plotted from three replicate experiments ± SDM for both pET-Aly2 and the pET tag alone (the latter is not phosphorylated by Npr1) in the right-hand portion of the panel.

    Article Snippet: In Vitro Kinase Assays pET and pET-Aly2 were incubated for 30 min at 30°C in kinase buffer (50 mM Tris-HCl, pH 7.5, 20 mM MgCl2 , 1 mM DTT, 1 μM unlabeled ATP, aprotinin, and leupeptin) with 75 nM [γ-32 P]ATP (Perkin Elmer-Cetus) with or without Npr1 kinase (purified from Y258 yeast cells).

    Techniques: Expressing, Copurification, Western Blot, Fluorescence, Microscopy, Purification, Positron Emission Tomography, Incubation, SDS Page, Staining

    The phosphoadaptor subunit Cks1 provide processivity for the multiphosphorylation of Sic1 by Cln2-Cdk1 and Clb5-Cdk1. (a) Cln2- and Clb5-Cdk1 complexes were incubated with Sic1ΔC and 32 P-ATP. The reactions also included wild-type Cks1 (wt) or a version with a mutated phosphate-binding site ( mut ; see Supplementary Methods ). Phosphorylated substrates were separated using Phos-Tag SDS-PAGE gels. (b) Reactions were performed in the presence of a phosphopeptide competitor (P) based on the sequence surrounding T45 in Sic1. (c) The phosphorylation of a Sic1ΔC version containing a single Cdk site (Sic1ΔC-T5, with other Cdk consensus sites mutated to alanines) was not affected by Cks1 mut or the phosphopeptide. The standard SDS-PAGE was used. (d) Time courses of Sic1ΔC multiphosphorylation were followed by Phos-Tag SDS-PAGE. (e) The quantified data from (d). The intensities of 32 P-labeled proteins were divided by the number of phosphates as indicated to obtain the levels of different phosphoforms. In the experiments presented in Fig. 1 the enzyme concentrations were chosen to obtain roughly equal substrate labeling.

    Journal: Nature

    Article Title: Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase

    doi: 10.1038/nature10560

    Figure Lengend Snippet: The phosphoadaptor subunit Cks1 provide processivity for the multiphosphorylation of Sic1 by Cln2-Cdk1 and Clb5-Cdk1. (a) Cln2- and Clb5-Cdk1 complexes were incubated with Sic1ΔC and 32 P-ATP. The reactions also included wild-type Cks1 (wt) or a version with a mutated phosphate-binding site ( mut ; see Supplementary Methods ). Phosphorylated substrates were separated using Phos-Tag SDS-PAGE gels. (b) Reactions were performed in the presence of a phosphopeptide competitor (P) based on the sequence surrounding T45 in Sic1. (c) The phosphorylation of a Sic1ΔC version containing a single Cdk site (Sic1ΔC-T5, with other Cdk consensus sites mutated to alanines) was not affected by Cks1 mut or the phosphopeptide. The standard SDS-PAGE was used. (d) Time courses of Sic1ΔC multiphosphorylation were followed by Phos-Tag SDS-PAGE. (e) The quantified data from (d). The intensities of 32 P-labeled proteins were divided by the number of phosphates as indicated to obtain the levels of different phosphoforms. In the experiments presented in Fig. 1 the enzyme concentrations were chosen to obtain roughly equal substrate labeling.

    Article Snippet: The general composition of the assay mixture was as follows: 50 mM Hepes pH 7.4, 100 mM NaCl, 0.1% NP-40, 20 mM imidazole, 2% glycerol, 2 mM EGTA, 0.2 mg/ml BSA, 500 nM Cks1 and 500 μM ATP (with added γ-32 P-ATP (Perkin Elmer)).

    Techniques: Incubation, Binding Assay, SDS Page, Sequencing, Labeling