Understanding the lasers and detectors of your imaging system is critical for fluorophore selection. Select Compatible Fluorophores for Your Imaging System.Here are some tips to maximize the efficacy of your fluorescent western blot experiments: However, the limited detection sensitivity of fluorescent western blotting can pose challenges when detecting antigens with low abundance.Īs with any immunoassay, fluorescent western blotting requires careful preparation and optimization to generate accurate, reproducible results. Additionally, the linear relationship between fluorescence and the quantity of antibody bound to the blot allows for more precise quantification of protein targets. In comparison to chemiluminescent methods, fluorescent western blot detection can improve experimental reproducibility due to the increased stability of fluorescent signals. “For instance, you can compare the abundance of a phosphorylated form of your protein of interest to the total amount of protein. You can also perform normalization of band intensity with an internal control in the same blot, without the inconvenience of stripping and re-probing.” “Multiplexing in fluorescent western blotting allows you to quantify relative protein abundance,” adds Silvia Sbacchi, Team Lead, Immunohistochemistry and Western Blot at Abcam. In contrast, fluorescent detection allows researchers to simultaneously measure up to four different targets using modern imaging systems such as Sapphire FL™ Biomolecular Imager from Azure Biosystems. “This can be especially useful when sample material is limited or there is a need to detect proteins that have a similar molecular weight.” Fee explains that with chemiluminescent methods, detecting similar-sized bands requires the blot to be stripped and re-probed. “A main advantage of fluorescent western blot detection is that it allows for multiplexing,” reports Jade Fee, Application Scientist at Azure Biosystems. However, while chemiluminescent detection involves capturing an image of blots using film or a digital imaging system, fluorescent detection requires the use of an imaging instrument capable of measuring fluorescent signals.Īdvantages of Fluorescent Western Blot Detection Detection can either be direct, using labeled primary antibodies, or indirect, using unlabeled primary antibodies and labeled secondary antibodies. Next, the membrane is blocked to prevent non-specific antibody binding and the target of interest is detected. First, the samples are separated by polyacrylamide gel electrophoresis (PAGE) and transferred to a membrane. Here, Abcam and Azure Biosystems comment on the advantages of fluorescent western blotting and share tips for protocol optimization.Ī standard fluorescent western blot protocol mirrors the steps of conventional western blotting. The introduction of modern digital imaging instruments, combined with advances in antibody labeling, have led many labs to transition to fluorescent western blot detection. While it was once common for researchers to use enzyme-labeled antibodies and chemiluminescent substrates to develop western blots to film, there has been a shift in recent years. Recommended volume per well for 1.0 mm mini gelġ0 μL for electrophoresis, 5 μL for blottingĥ μL for electrophoresis, 2.Fluorescent western blot detection can offer many advantages provided protocols are carefully optimized ~160, 120, 80, 60, 50, 40, 30, 20, 15, 10 kDaĮach protein standard contains a 6X His-tag enabling the detection with the InVision His-tag In-gel StainĬolorimetric or methods that detect phosphorylated proteins such as Pro-Q Diamond phosphoprotein gel stainsĬolorimetric or methods that detect glycosylated proteins such as Pro-Q Glycoprotein stain kits Peppermint Stick Phosphoprotein Molecular Weight StandardsĬand圜ane Glycoprotein Molecular Weight Standards Tagged- proteins in the 10 -250 kDa range contain a Strep-tag™ II sequence and can be detected on western blots using Strep-Tactin™ conjugates or an antibody against Strep-tag™ II sequenceĭirect visualization can be achieved through standard staining protocols (e.g., Coomassie, etc.) Tagged-proteins contain an integral Strep-tag™ II sequence and can be detected on western blots using Strep-Tactin™ conjugates or an antibody against Strep-tag™ II sequence PageRuler Unstained Broad Range Protein LadderĪccurate estimation across a broader range Recommended Volume per well for 1.0 mm gel (μL)Ĭolorimetric, NIR fluorescence (700nm channel, blue bands), RGB fluorescence (550 nm channel, orange bands) Visible monitoring of gel separation and transfer efficiency Spectra Multicolor Broad Range Protein Ladderīest multicolor prestained ladder for routine applicationsĤ colors for improved visualization during separation and transferĪnalysis of high molecular weight proteins
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