blu003h250uc  (Revvity)

Journal: iScience

Article Title: Maintenance of a host-specific minority mutation in the West Nile virus NS3

doi: 10.1016/j.isci.2023.107468

Figure Lengend Snippet:

Article Snippet: α 32 P-ATP , Perkin Elmer , Cat# BLU003H250UC.

Techniques: Virus, Mutagenesis, Recombinant, Transfection, Sequencing, Plasmid Preparation, Software, Ubiquitin Proteomics

Journal: iScience

Article Title: Maintenance of a host-specific minority mutation in the West Nile virus NS3

doi: 10.1016/j.isci.2023.107468

Figure Lengend Snippet:

Article Snippet: α 32 P-ATP , Perkin Elmer , Cat# BLU003H250UC.

Techniques: Virus, Mutagenesis, Recombinant, Transfection, Sequencing, Plasmid Preparation, Software, Ubiquitin Proteomics

Journal: Methods in enzymology

Article Title: Measurement of ATP utilization in RNA unwinding and RNA chaperone activities by DEAD-box helicase proteins

doi: 10.1016/bs.mie.2022.04.004

Figure Lengend Snippet: RNA binding and unwinding by DEAD-box helicase proteins. (A), Structural views of ssRNA binding by the helicase core, as shown for Mss116 (Del Campo & Lambowitz, 2009b). The ssRNA (purple) binds to Mss116 (blue) in a crimped conformation, incompatible with a standard duplex geometry. Also shown are a bound ATP analog (orange) and a coordinated Mg2+ ion (green). (B) ATP-dependent RNA unwinding by DEAD-box helicases. In the dominant pathway, highlighted in yellow, the ATP-bound form of the helicase binds dsRNA and produces local unwinding (starting from top left; ATP is indicated by a red triangle). This unwinding reaches completion, resulting in the dissociation of one of the strands, producing the ssRNA-bound form shown in panel A. ATP hydrolysis and release of Pi weaken binding of ssRNA (bound ADP is indicated by a purple circle), resulting in ssRNA dissociation (bottom right). Thus, this pathway results in complete helix unwinding with hydrolysis of one ATP. Less populated pathways produce ATPase activity without complete duplex unwinding (top two rows, counterclockwise) and complete duplex unwinding without ATP hydrolysis (left column, top to bottom).

Article Snippet: For radioactivity-based measurements of ATPase activity [γ-32P]ATP, 10 μCi/μL (3000 Ci/mmol; Perkin-Elmer, BLU003H250UC)Note: This less-concentrated ATP is better suited for ATPase measurements than the product used for oligonucleotide labeling, because of the much lower background contamination level of Pi (the product of ATP hydrolysis).150 mM EDTA (pH 8.0) for quenching ATPase reactions.

Techniques: RNA Binding Assay, Binding Assay, Activity Assay

Journal: Methods in enzymology

Article Title: Measurement of ATP utilization in RNA unwinding and RNA chaperone activities by DEAD-box helicase proteins

doi: 10.1016/bs.mie.2022.04.004

Figure Lengend Snippet: Results of ATP utilization measurement in RNA unwinding by the DEAD-box helicase CYT-19 (Chen et al., 2008). (A) RNA unwinding substrate and labeling strategy. The RNA helix (red) was appended to a flanking DNA helix, which was shown to increase CYT-19 activity (Tijerina, Bhaskaran, & Russell, 2006). In the pulse phase, the RNA helix was formed by adding labeled CCCUCUA5 (labeling indicated by asterisk), and unwinding of this helix was trapped in the chase phase by excess unlabeled CCCUCUA5. Results are shown for RNA unwinding measurements (B) and ATPase measurements (C). For both sets of experiments, CYT-19 was 2 μM and the duplex was 0.5 μM. For ATPase measurements, excess of the substrate strand was present (1 μM). For RNA unwinding measurements, this strand was increased to 5 μM in panel C, which was shown to increase the signal for RNA unwinding without changing the observed rate constant. Conditions for all measurements were 25 °C, 10 mM Mg2+, and 50 μM ATP-Mg2+. These measurements gave an ATP utilization value of 1.1 ± 0.1.

Article Snippet: For radioactivity-based measurements of ATPase activity [γ-32P]ATP, 10 μCi/μL (3000 Ci/mmol; Perkin-Elmer, BLU003H250UC)Note: This less-concentrated ATP is better suited for ATPase measurements than the product used for oligonucleotide labeling, because of the much lower background contamination level of Pi (the product of ATP hydrolysis).150 mM EDTA (pH 8.0) for quenching ATPase reactions.

Techniques: Labeling, Activity Assay

Journal: Methods in enzymology

Article Title: Measurement of ATP utilization in RNA unwinding and RNA chaperone activities by DEAD-box helicase proteins

doi: 10.1016/bs.mie.2022.04.004

Figure Lengend Snippet: Experimental workflows for measurement of ATP utilization during helicase-promoted RNA unwinding. Workflows are shown for ATPase (A) and RNA unwinding (B) rate measurements. Asterisks indicate radioactively labeled components (ATP or RNA).

Article Snippet: For radioactivity-based measurements of ATPase activity [γ-32P]ATP, 10 μCi/μL (3000 Ci/mmol; Perkin-Elmer, BLU003H250UC)Note: This less-concentrated ATP is better suited for ATPase measurements than the product used for oligonucleotide labeling, because of the much lower background contamination level of Pi (the product of ATP hydrolysis).150 mM EDTA (pH 8.0) for quenching ATPase reactions.

Techniques: Labeling

Journal: Methods in enzymology

Article Title: Measurement of ATP utilization in RNA unwinding and RNA chaperone activities by DEAD-box helicase proteins

doi: 10.1016/bs.mie.2022.04.004

Figure Lengend Snippet: Experimental workflows for measurement of ATP utilization during a helicase-promoted RNA folding transition. Workflows are shown for ATPase (A), RNA refolding (B), and combined ATPase and refolding (C) rate measurements. Asterisks indicate radioactively labeled components (ATP and/or ribozyme substrate oligonucleotide). In (C), the concentrations of folding quench components should be adjusted to be 80% lower than those listed in Section 3.3.2 (step 3) to account for the slightly larger volume required to provide sufficient sample for both TLC and gel analysis.

Article Snippet: For radioactivity-based measurements of ATPase activity [γ-32P]ATP, 10 μCi/μL (3000 Ci/mmol; Perkin-Elmer, BLU003H250UC)Note: This less-concentrated ATP is better suited for ATPase measurements than the product used for oligonucleotide labeling, because of the much lower background contamination level of Pi (the product of ATP hydrolysis).150 mM EDTA (pH 8.0) for quenching ATPase reactions.

Techniques: Labeling