Phosphorimaging

Phosphorimaging is a digital imaging method that detects and measures radiation from labeled biological samples, providing a quantitative alternative to film-based autoradiography. A sample is placed against a storage phosphor screen, whose photostimulable material captures energy emitted by radioactive labels; laser scanning then releases this stored energy as light, which a detector converts into a digital image. In biology, phosphorimaging supports Southern, Northern, and Western blot analysis, as well as nucleic acid and protein localization studies. Its broad dynamic range, sensitivity, and digital workflow enable accurate signal comparison and improve the analysis of molecular experiments.

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Research

JoVE EoE - Chromatography Techniques

Thin Layer Chromatography Based Enzyme Activity Assay: A Technique to Determine In Vitro Pyrophosphokinase Activity Using Thin Layer Chromatography

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2025

This video demonstrates a thin-layer chromatography-based enzyme assay to determine pyrophosphokinase activity using radiolabeled nucleotides. The method provides insights into nucleotide synthesis and phosphotransfer reactions.

Thin-Layer Chromatography-Based Separation of (p)ppGpp Nucleotides Isolated from Stress-Induced Bacteria

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2025

Source: Fernández-Coll, L. and Cashel, M. Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli (p)ppGpp Followed by Thin Layer Chromatography. J. Vis. Exp. (2019)This video presents a high-throughput protocol for measuring the levels and dynamics of the bacterial stress-response nucleotides ppGpp and pppGpp using thin-layer chromatography (TLC). The method provides a powerful, rapid, and cost-effective approach to quantifying (p)ppGpp in bacterial cultures...

Oligopeptide Competition Assay for Phosphorylation Site Determination

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Cited by 3 •

2017

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

Assessing Viral Replication Capacity Using a Radiolabeled Reverse Transcriptase Assay

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2026

Source: Claiborne, D. T. et al. A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses. J. Vis. Exp. (2014)This video demonstrates the assessment of viral replication capacity using a radiolabeled reverse transcriptase assay. Culture supernatants containing progeny virions are added to a reverse transcription mixture containing a radiolabeled nucleotide. The incorporated signal reflects viral output and replication...

Gel Electrophoresis and Northern Blotting to Detect tRNA Charging Levels in E. coli

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2025

Source: Stenum, T. S., et al. Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli. J. Vis. Exp. (2017)This video demonstrates the use of gel electrophoresis and northern blotting to detect and compare the charging levels of transfer RNAs in E. coli by separating aminoacylated and deacylated forms.

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