I have been using FlAsH-EDT2 purchased from Carbosynth to develop a fluorescent probe that is basically a fluorescent protein (mCherry or mTurquoise2) with a tetracysteine motif at the C-terminus (essentially as done by the Tsien lab when they first published on this reagent in Science 1998).
Several months ago, I was able to specifically label a different protein in whole cell lysate. Unfortunately, now I get a large amount of background fluorescence from the FlAsH when labeling whole cell lysates (all protein bands in SDS-PAGE become fluorescent) and in plate reader assays (even in no-protein controls). I typically use 10 uM FlAsH-EDT2 and include 100 uM EDT to suppress background fluorescence and to re-quench any FlAsH that has shed its EDT molecules.
Does anyone have tips on how to reduce background labeling in vitro? Or did the reagent somehow go bad such that now it labels non-specifically?? I should mention that I am pretty confident that FlAsH at least binds my protein of interest because I do see a reduction in the emission of mTurquoise2 in the presence of FlAsH due to FRET in a plate reader.
http://science.sciencemag.org/content/281/5374/269
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