Hi,
I would like to tag two endogenous proteins with a fluorescent tag to calculate their ratio of expression. Preferentially one bright red and one bright green. Which fluorescent proteins would you suggest to allow accurate calculations of ratios? I believe that their emission spectra have to overlap as little as possible. My first thought was to go for dTomato and eGFP.
Dear Nikolaos Tsopoulidis ,
When measuring the ratio of expression between two endogenous proteins, it's important to use fluorescent tags with distinct emission spectra to minimize overlap and ensure accurate measurements. Two commonly used fluorescent tags for measuring protein ratios are eGFP (enhanced green fluorescent protein) and mCherry (a monomeric version of DsRed) as they have distinct excitation and emission spectra, and are relatively bright, which allows for accurate ratio measurements.
eGFP has an excitation peak at 488 nm and an emission peak at 509 nm, while mCherry has an excitation peak at 587 nm and an emission peak at 610 nm. These two proteins have distinct emission spectra, which allows for accurate ratio measurements.
Another option to use is dTomato and eGFP, as dTomato has an excitation peak at 561 nm and an emission peak at 583 nm, which are relatively close to eGFP's excitation and emission peak, but still different enough to allow for accurate ratio measurements.
It's important to note that the specific fluorescence signal of your proteins can vary depending on the specific expression system and conditions, so you may need to test a few different combinations to find the best one for your specific application.
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