The EGSs were tested by preincubating 5′-end-labeled aac(6’)-Ib mRNA (5 pmol) and the EGS (10 pmol) at 25°C for 30 m in a volume of 3 μl before adding this mixture to a solution containing 2.5 pmol of M1 RNA, 70 pmol of C5 protein, 20 mM HEPES-KOH (pH 8.0), 400 mM ammonium acetate, 10 mM magnesium acetate, and 5% glycerol that had been preincubated at 37°C for 15 min in a final volume of 7 μl (54 (link)). After combining both solutions the mix was incubated at 37°C for the times indicated. The reaction was stopped by the addition of 1 volume phenol-chloroform followed by ethanol precipitation. The pellet was resuspended in 10 μl DEPC-treated water, mixed with 10 μl of 2x gel loading buffer, and analyzed by 5% denaturing TBE-PAGE along with labeled RNA Century Marker or RNA Century Marker-Plus (ThermoFisher) as described before (31 (link)). Fluorescence was detected on a Storm 860 Molecular Imager (Molecular Dynamics) and the signal in each band was quantified using Image J (55 ).
Storm 860 molecular imager
Manufactured by GE Healthcare
Sourced in United Kingdom
The Storm 860 Molecular Imager is a laboratory instrument designed for the detection and analysis of fluorescently labeled biomolecules, such as proteins and nucleic acids, in various applications like Western blotting, Fluorescent Gel Imaging, and Phosphorimaging. It utilizes a laser-based excitation system and a charge-coupled device (CCD) camera to capture high-resolution images of labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant software, by GE Healthcare (5 mentions)
Ecf reagent, by GE Healthcare (3 mentions)
Century plus rna marker, by Thermo Fisher Scientific (2 mentions)
Tlc silica gel 60 f254 plate, by Merck Group (2 mentions)
X ray film, by GE Healthcare (2 mentions)
Tgx gel, by Bio-Rad (2 mentions)
Odyssey blocking buffer, by LI COR (2 mentions)
Image studio, by LI COR (2 mentions)
Odyssey clx, by LI COR (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Imagequant tl software, by GE Healthcare (2 mentions)
Mirneasy kit, by Qiagen (1 mentions)
Phosphorimager screen, by GE Healthcare (1 mentions)
Sdf 1α, by Abcam (1 mentions)
Ecl plus substrate, by Cytiva (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Antibody for β actin, by Merck Group (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
29 protocols using storm 860 molecular imager
1
Catalytic Cleavage of aac(6')-Ib mRNA
2
Evaluating aac(6')-Ib mRNA Cleavage by EGSs
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The EGSs were tested by preincubating 5′-end-labeled aac(6’)-Ib mRNA (5 pmol) and the EGS (10 pmol) at 25°C for 30 min in a volume of 3 µl before adding this mixture to a solution containing 2.5 pmol of M1 RNA, 70 pmol of C5 protein, 20 mM HEPES-KOH (pH 8.0), 400 mM ammonium acetate, 10 mM magnesium acetate, and 5% glycerol that had been preincubated at 37°C for 15 min in a final volume of 7 µl [54 (link)]. After combining both solutions the mix was incubated at 37°C for the times indicated. The reaction was stopped by the addition of 1 volume of phenol-chloroform followed by ethanol precipitation. The pellet was resuspended in 10 µl DEPC-treated water, mixed with 10 µl of 2 x gel loading buffer, and analyzed by 5% denaturing TBE-PAGE along with labeled RNA Century Marker or RNA Century Marker-Plus (ThermoFisher) as described before [31 (link)]. Fluorescence was detected on a Storm 860 Molecular Imager (Molecular Dynamics) and the signal in each band was quantified using Image J [55 ].
3
Radiolabeled Protein Production from PCR
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The TNT Quick Coupled Transcription/Translation kit for PCR-generated DNA (Promega) was used to produce proteins of interest directly from PCR products. L -[35S]-Methionine was included to permit monitoring of the radiolabelled proteins formed following separation by SDS–PAGE and visualized by incubating dried gels for 48 h on phosphorimager screens, which were scanned with a STORM 860 molecular imager (Molecular Dynamics).
4
Radiolabeling of Vanilla pod metabolites
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The V. planifolia pods harvested at 3, 4, 5, 6, 7, 8 and 9 months following pollination were cut transversely into 1–2 mm thick discs (approximately 25 mg FW) using a scalpel and further dissected to separate the inner and outer part of the pod. Radiolabeled precursors (0.5 μCi) were administered to samples of the inner and outer part of the pod and incubated (30 °C) in 400 mM Tris–HCl pH 8, 20 mM MgCl2 for 6–48 h.
The 14C-labeled products formed in the experiments with fresh V. planifolia pods were extracted by 25% MeOH and applied to Silica Gel 60 F254 TLC plates (Merck,http// www.merck-chemicals.com ). The plates were developed in ethyl acetate : acetone : dichloromethane : methanol : water (40 : 30 : 20 : 10 : 8, by vol.), dried, exposed (48 h) on phosphor-imaging screens (Molecular Dynamics, http://www.moleculardynamics.com ) and the radiolabeled products visualized using a Storm 860 Molecular Imager (Molecular Dynamics). Identification of the radiolabeled compounds formed was guided by co-application of authentic standards.
The same experimental procedure was followed using isolated intact chloroplasts (10 μl, 0.5 μg Chl μl−1) obtained from 8-month-old V. planifolia pods.
The 14C-labeled products formed in the experiments with fresh V. planifolia pods were extracted by 25% MeOH and applied to Silica Gel 60 F254 TLC plates (Merck,
The same experimental procedure was followed using isolated intact chloroplasts (10 μl, 0.5 μg Chl μl−1) obtained from 8-month-old V. planifolia pods.
5
Northern Blot Analysis of Cas Genes
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For Northern blot analysis, total small RNAs were prepared from the PXO99A wild-type strain, its knockouts (Δ) of individual Cas genes by a miRNeasy kit (Qiagen, Beijing, China). Ten micrograms of total RNA extracted from each sample was separated on 12% PAGE gels and then transferred to a HybondTM-N+ positively charged nylon membrane (Roche Diagnostics). The 5S rRNA probe was used as an internal control with oligoprobes, whereas the Xoo repeats was probed using a mixture of P32-labeled DNA antisense oligoprobes. Radiolabeled signals were observed by a Storm 860 Molecular Imager (Molecular Dynamics, Sunnyvale, CA).
6
Kinase Assay for aPKC Substrates
7
Identification of Radiolabelled Vanilla Compounds
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The 14C-labelled products formed in biosynthetic experiments with fresh vanilla pods as well as in in vitro protein assays were applied to Silica Gel 60 F254 TLC plates (Merck, http://www.merck-chemicals.com ). The plates were developed in ethyl acetate: acetone: dichloromethane: methanol: water (40:30:12:10:8, v/v/v/v/v), dried, exposed (48 h) on phosphorimager screens (Molecular Dynamics, http://www.moleculardynamics.com ) and the radiolabelled compounds formed were visualized using a Storm 860 Molecular Imager (Molecular Dynamics). Identification of the radiolabelled compounds formed was guided by co-application of authentic standards. Unambiguous structural verification of the products formed was obtained using LC–MS including accurate mass determination and comparison of retention times and fragmentation patterns with those of authentic reference compounds57 (link).
8
Western Blot Analysis of Hippocampal Proteins
9
Phosphopeptide and Phosphoamino Acid Analysis
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The 32P-labeled Pah1 transferred to PVDF membrane was digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin for phosphopeptide mapping or hydrolyzed with 6 N HCl at 100 °C for phosphoamino acid analysis (101 (link), 102 (link), 103 (link)). The tryptic digests were analyzed on the cellulose TLC plates first by electrophoresis and then by thin-layer chromatography (101 (link), 102 (link), 103 (link)). The acid hydrolysates were mixed with standard phosphoamino acids and separated by two-dimensional electrophoresis on cellulose TLC plates. Radioactive phosphopeptides and phosphoamino acids were visualized by phosphorimaging analysis using a Storm 860 Molecular Imager (GE Healthcare). Nonradioactive phosphoamino acid standards were visualized by ninhydrin staining.
10
Western Blot Analysis Protocol
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