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Hi George,
I have observed transport of mRFP foci at high expression levels (just cytoplasmic protein--no tag) but not EGFP or mCherry. In general if your expression is high enough to observe puncta you are probably seeing recycling vesicles and these definitely undergo transport. If you do TIRF microscopy you will see lots of these on the basement membrane moving in a nicely directed fashion. Temperature and general cell stress can also be a contributing factor.
Jay
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Subject: Transport of cytosolic fluorescent proteins?
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Hi all,
My colleague created a mRuby2 fusion protein and observed movement of small puncta, which represent transport of our fusion protein. However, in a control experiment, we also observed transport of small puncta containing the cytoplasmic mRuby2. We have also observed transport of cytoplasmic EGFP and mCherry. We are in the process of testing whether this represents transport within lysosomes or autophagosomes.
However, I wanted to ask an audience with broad experiences. Has anyone else seen what appears to be active transport of cytoplasmic fluorescent proteins?
We want to have confidence that our fusion protein is not being transported due to a tendency for fluorescent proteins to be transported.
Thanks,
George
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