This will aid in the selection of appropriate secondary antibodies to minimize potential antibody cross-reactivity, which can lead to confusing results. Ideally, use a combination of antibodies from two distantly related species such as rat and rabbit, avoiding combinations like mouse and rat or goat and sheep. Use primary antibodies from different host species for each target being probed.This will allow determination of the banding pattern of each antibody prior to a multiplex experiment. Verify the detection of each protein target individually before multiplexing with other targets. Select antibodies designated specifically for western blotting or that list western blotting as an application.The selection of appropriate primary antibodies and fluorescently labeled secondary antibodies is critical when designing a fluorescent multiplex western blot experiment. Selection of antibodies for multiplex western blotting Alexa Fluor Plus secondary antibodies were designed to provide high signal-to-noise ratios and lower cross reactivity, reducing the time needed for optimization. Optimization is required to achieve the best signal-to-noise ratio, but the recommended concentration range, regardless of fluorescent conjugate, is typically between 0.4 and 0.1 µg/mL (1:5,000–1:20,000) for imaging on CCD imaging systems. Secondary antibody concentrations are typically higher in fluorescence applications.Avoid using pens on membranes, as many inks fluoresce. Only handle membranes with gloved hands and clean blunt forceps to limit contamination and scratches on the membranes, which can contribute to background fluorescence and artifacts.
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In addition, limit the use of detergents during blocking steps, as common detergents can auto-fluoresce and increase nonspecific background. Particles and contaminants in wash and blocking buffers can settle on membranes and create fluorescent artifacts.
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Multiplexing helps make research more efficient and productive. While the detection limits are not as low as chemiluminescent detection, fluorescent detection has the unique advantage of allowing multiple targets to be assayed on the same blot at the same time without the need to strip and reprobe. However, if the degree of fluorescent labeling is too high, the signal can also be weak due to the inactivation of the detection reagent or quenching of the signal caused by a phenomenon known as Förster resonance energy transfer (FRET). If the degree of fluorescent labeling is too low, the signal will be weak. Similar to enzymatic reactions, fluorescent reagents must be optimized with respect to the signal-to-noise ratio. Overall, the western blotting procedure is similar between chemiluminescent and fluorescent detection methods, with each method offering specific benefits. Chromogenic enzyme-substrate reactions produce colored products that precipitate onto the membrane, while chemiluminescent detection systems generate enzymatic reactions that produce energy released in the form of light. In contrast, chromogenic and chemiluminescent western detection systems produce signals that are products of enzyme-substrate reactions. Transient light emission from a fluorescent molecule (fluorophore) is produced by the excitation and subsequent release of photons as the excited molecule returns back to its normal state. In fluorescent western blot detection systems, signal is captured in the form of light.