Azure BiosystemsIn-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.
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.
FREE WESTERN BLOT eBOOK
Flexible imaging system able to detect three fluorescent targets
The ultimate Western blot imaging system, capable of in-gel fluorescence, and more
Looking for a full list of applications?
Your common Western blotting questions, answered. Western blotting is a widely used analytical technique that can identify one or more specific proteins in a complex
We’ve all been there… Non-specific bands are a perennially frustrating problem in Western blotting. Here are some of the different reasons you might be getting
Why is the background on my Western blot so high? Why is there low (or no) signal? Using too much secondary antibody can result in
