In-gel fluorescence has become popular for validating the generation of fusion proteins, analyzing protein-protein interactions, and electrophoretic mobility shift assays. Fluorescent fusion proteins can easily be visualized without having to transfer to a membrane for traditional Western analysis. In the image below, fluorescent proteins were detected in a native gel and imaged with an Azure Imaging System.
Another application of in-gel fluorescence is staining the total protein in a gel before transfer for Western blotting. The AzureRed protein stain can also provide fluorescent staining of total protein in your gel post-electrophoresis, for relative quantitative comparisons. It can be imaged with CCD- or laser-based fluorescent imaging systems and is compatible with downstream Western blotting. By including known concentration standards and imaging the stained gel, you can get quantitative information about your gel bands.
The image above shows the interaction of Fluorescein-labeled Ubiquitin (FL-Ub) with its binding partners, E1 and E2 Ubiquitin-activating enzymes, over a treatment time course. The upper bands represent ubiquitin in complex with E1 or E2. Fluorescence was detected with the Azure c600 using excitation at 470 nm and emission filter at 497 nm.
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