Fluorescence-based Protein Brightness Assay

Cells were transfected with Tandem FP constructs containing a T2A linker, mTurquoise2 as reference and a fluorescent protein of interest. Cells expressing two separate FPs in equal amounts were imaged on a widefield fluorescence microscope (Axiovert 200 M; Carl Zeiss GmbH) equipped with a xenon arc lamp with monochromator (Cairn Research, Faversham, Kent, UK), using a 40x objective (oil-immersion Plan-Neo- fluor 40×/1.30; Carl Zeiss GmbH). Orange FPs were excited with 510 nm light and emission was detected with a BP572/25 filter. As reference, mTurquoise2 was excited with 420 nm light and emission was detected with a BP470/30 filter. Clover and mNeonGreen are excited with 500 nm light and emission was detected with a BP535/30 filter. To prevent cross excitation, reference mTurquoise2 was excited with 405 nm light and emission was detected with a BP470/30 filter. After subtraction of background signal, the mean fluorescence intensity of the cells was calculated. The fluorescence intensity of the protein of interest relative to the fluorescence intensity of the reference mTurquoise2 reveals the relative brightness of the protein of interest^{42 (link)}.