In-gel Fluorescence

Fluorescence detection in-gel electrophoresis

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.

Fluorescent protein in native gel.
Fluorescent protein in native gel imaged with an Azure imager

Visualizing proteins in-gel without Western Blotting

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.

Imaging fluorescent proteins in-gel for a gel-shift assay

Fluorescein-labeled-Ubiquitin

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.

Systems to use for the best in-gel fluorescence results

After fluorescent proteins are detected, the fluorescence can be imaged with a digital imager, such as the Azure 400, Azure 600, or Azure Sapphire Biomolecular Imager.

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Azure 400 Visible fluorescent imager

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The ultimate Western blot imaging system, capable of in-gel fluorescence, and more

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