Particles and contaminants in wash and blocking buffers can settle on membranes and create fluorescent artifacts. Use only high-quality filtered buffers, such as Blocker FL Fluorescent Blocking Buffer (Cat.Ability to save sample with multiplexing.No need to strip and reprobe the blot when looking at multiple targets.Greater quantifiable linear range for quantitative western blotting techniques.Fluorescent western blotting provides accurate, quantitative results, stable signals, and the ability to conserve sample due to multiplexing. In addition to enabling multiplexing, fluorescent western blot detection has several other advantages compared to enzyme-based chemiluminescent substrate detection. With the iBright FL1500 Imaging System, one can perform up to a 4-plex fluorescent western blots with the appropriate experimental setup. For example, one can visualize a protein of interest simultaneously with a loading control protein, or differentiate proteins of similar molecular weights, or evaluate complex biological pathways. 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.

Protein ladder western blot