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. AzureRed protein stain is compatible with downstream Western blotting. It can also provide fluorescent staining of total protein in your gel post-electrophoresis, for relative quantitative comparisons. AzureRed can be imaged with an Azure Imager, or another CCD- or laser-based fluorescent imaging system. 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.
After fluorescent proteins are detected, the fluorescence can be imaged with the Azure 400, Azure 600, new Azure Sapphire FL Biomolecular Imager, or any other digital imager. Learn more about how to get the best results by filling out this form for more information.
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Keep reading if you’re having problems using ponceau! Ponceau S is a fast, reversible protein stain often used to confirm that protein samples have successfully