How to detect your target proteins using fluorescence-based reagents

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Fluorescent detection is growing in popularity because it offers time savings and the ability to detect multiple proteins simultaneously. This approach uses antibodies labelled with fluorescent dyes. Visualization of the target protein is achieved by exciting the fluorescent dye using imaging systems equipped with an appropriate light source. No enzymes or substrates are used. When getting started with fluorescent western blotting, some reagents and steps will need to be optimized to help ensure background fluorescence does not interfere with detection of the protein of interest. Consider using fluorescent-friendly sample buffers without bromophenol blue, such as Invitrogen Fluorescent Compatible Sample Buffer. For example, sample buffers containing bromophenol blue will fluoresce and can contribute to increased background. To eliminate a major source of background fluorescence, use membranes with low autofluorescence, such as nitrocellulose and low-fluorescent PVDF membranes. Additionally, particles and contaminants in blocking and wash buffers can settle on membranes and create fluorescent artifacts. Use high-quality, filtered buffers such as Thermo Scientific Blocker FL Fluorescent blocking buffer. Use only detergent-free blocking buffers, as common detergents autofluorescence and will increase background. The selection of appropriate primary antibodies and fluorescently labeled secondary antibodies is critical when designing a fluorescent western blot experiment. For example, when performing two-plex multiplexing experiments, use primary antibodies from different host species to avoid cross-reactivity. Ideally, use a combination of antibodies from two distantly related species such as mouse and rabbit, avoiding combinations like mouse and rat or goat and sheep. In this video, you will learn how to perform a two-plex fluorescent western blot with Invitrogen Alexa Fluor Plus 680 and Alexa Fluor Plus 800. After protein transfer wash the membrane in deionized water with agitation to remove excess transfer buffer. Prepare the blocking buffer. Dilute the Blocker FL Fluorescent Blocking Buffer from 10X to 1X with deionized water. Incubate the membrane with a sufficient volume of blocking buffer for 30-60 minutes at room temperature with agitation. Dilute the two primary antibodies per supplier recommendations in sufficient volume of blocking buffer. Incubate the membrane with gentle agitation with the primary antibodies for 1 hour to overnight. When incubating overnight, place at 4°C. Wash the membrane 6 times for 5 minutes each in wash buffer with agitation. Prepare dilutions of the conjugated secondary antibody of 0.4 to 0.1 µg/mL, or 1:5,000 to 1:20,000, in an appropriate volume of wash buffer. Alternatively, the secondary antibody can be diluted in blocking buffer. Detergent can now be added if needed. Incubate the membrane in diluted secondary antibody for 1 hour at room temperature with agitation, protecting the membrane from light. Wash 6 times for 5 minutes each in wash buffer with agitation, protecting the membrane from light. Blots can be imaged immediately while still wet, or may be dried prior to imaging. Place each blot in a sheet protector or on a clean surface prior to imaging to prevent contamination. Image on the Invitrogen iBright Imaging system using the fluorescence detection mode; select the Alexa Fluor 680 and 800 channels, and click Smart Exposure. Find out more information about fluorescent detection for western blotting at thermofisher.com/5Steps-multiplexwestern

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