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fluorescent western blotting uses secondary antibodies that are conjugated to fluorescent labels enabling multiple proteins to be viewed simultaneously it is a powerful technique ideal for multiplexing and quantitative analysis over a large dynamic range in this experiment you will need pipettes a pipette man washing buffer blocking buffer primary and secondary antibodies pipette to prevent non-specific binding of the antibody the membrane must be blocked there are many blocking buffers available typically we use a three percent non-fat milk diluted in tris buffered saline with tween 20 or tbst buffer but check your antibodies data sheet for the optimal solution place the membrane in a tray and cover with blocking buffer place the membrane onto a rocking platform and incubate for one hour at room temperature or overnight at four degrees celsius an advantage of the fluorescent western blot method is the ability to detect two separate target proteins without stripping the membran
