Northern analysis was performed as previously described (Gruner et al., 2016 (link)) using P32 labeled DNA probes. Briefly, Glyoxal-DMSO was used to denature RNA and electrophoresis was performed using 1% BPTE agarose gels. Transfer to Nylon membrane was performed using Turboblotter (Whatman). After transfer and washing, blot was crosslinked using a UV Stratalinker 1800 (Stratagene). DNA probes were prepared using Megaprime DNA labeling system (GE Healthcare, RPN1606) labeled with α32-P dCTP (Perkin Elmer). Probing was performed in a hybridization oven overnight at 45–55°C using ULTRAhyb hybridization buffer (ThermoFisher, AM8669), followed by 3–4 washes at 50–60°C and exposure on phosphoscreen. Image acquisition was performed using a FLA7000IP Typhoon Storage Phosphorimager using Typhoon FLA 7000 control software Version 1.3 (GE Healthcare).
Fla7000ip typhoon storage phosphorimager
Manufactured by GE Healthcare
The FLA7000IP Typhoon Storage Phosphorimager is a high-performance imaging system designed for the analysis and quantification of radioisotope-labeled samples. It utilizes storage phosphor technology to capture and digitize images, providing high sensitivity and resolution for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Megaprime dna labeling system, by GE Healthcare (2 mentions)
Ultrahyb hybridization buffer, by Thermo Fisher Scientific (2 mentions)
Typhoon fla 7000 control software version 1, by GE Healthcare (2 mentions)
α 32p dctp, by PerkinElmer (2 mentions)
Turboblotter, by Cytiva (2 mentions)
Amylose beads, by New England Biolabs (2 mentions)
Odyssey software, by LI COR (2 mentions)
Lambda phosphatase, by New England Biolabs (1 mentions)
4 protocols using fla7000ip typhoon storage phosphorimager
1
Northern Blot Analysis of RNA Transcripts
2
Northern Blot Analysis of RNA Transcripts
3
Purification and Kinase Assay of MBP-CK1
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Bacterial expression of MBP-CK1 fusion proteins were performed in terrific broth media at an OD595 of ~1.2 and 0.4 μM isopropyl β-D-1-thiogalactopyranoside. MBP-CK1 fusion proteins were purified on amylose beads (New England Biolabs) in column buffer (20 mM Tris [pH 7.0], 150 mM NaCl, 2 mM EDTA, 1 mM DTT and 0.1% NP40) and eluted with maltose (10 mM). Kinase reactions were performed with 1000 ng kinase, 1000 ng GAPVD1, 10 μM ATP plus 1 µCi γ-[32P]-ATP in kinase buffer (50 mM Tris [pH 7.5], 10 mM MgCl2, and 5 mM DTT) in 20 µl at 30 °C for 45 minutes. Reactions were quenched by adding SDS-PAGE sample buffer and proteins were separated by SDS-PAGE. Phosphorylated proteins were visualized by autoradiography and relative protein quantities were assessed by Coomassie blue staining relative to known standards utilizing Odyssey software (LI-COR Biosciences). Phosphorylated proteins were visualized and quantitated using an FLA7000IP Typhoon Storage Phosphorimager (GE Healthcare Life Sciences).
4
Kinetic Characterization of CK1 Kinases
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MBP-Hhp1 and MBP-Hhp2 fusion proteins were purified on amylose beads (New England Biolabs) in column buffer (20 mM Tris, pH 7.0, 150 mM NaCl, 2 mM EDTA, and 0.1% NP40) and eluted with maltose (10 mM). Kinase reactions were performed with 500 ng kinase, 500 ng casein, 10 μM ATP plus 1 µCi γ-[32P]ATP in kinase buffer (50 mM Tris, pH 7.5, 10 mM MgCl2, and 5 mM dithiothreitol) in 20 µl at 30°C for 30 min. Reactions were quenched by adding SDS–PAGE sample buffer, and proteins were separated by SDS–PAGE. Phosphorylated proteins were visualized by autoradiography, and relative protein quantities were assessed by Coomassie blue staining relative to known standards using Odyssey software (Li-Cor Biosciences). Kinetic assays were performed using recombinant MBP-Hhp1 and MBP-Hhp2 fusion proteins treated for 2 h with lambda phosphatase (New England Biolabs). These kinase reactions were performed with 0.25 μM of kinase, 25 μM casein, 5 μM cold ATP, 2 μCi [γ32P]-ATP in CK1 kinase buffer. Reactions were quenched at different time points by adding SDS sample buffer, and proteins were separated by SDS–PAGE. Phosphorylated proteins were visualized and quantitated using an FLA7000IP Typhoon Storage Phosphorimager (GE Healthcare Life Sciences). Kinetic measurements were calculated using Prism 6 software. Relative protein quantities were assessed by Coomassie blue staining.
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