I fused mCherry into the C-terminus of transferrin receptor (TfR) which is a type-2 transmembrane protein (2292bp). So, mCherry supposed to express out side of the cell. During the confocal microscopy observation I could see the red signal only inside the cell as granules., stress granules or aggresomes..(not known)?
What could be the reason for this cytoplasmic mis-localization?
Can misfolded protein give its fluorescent signal? Isn't it necessary for the fluorescent protein to get mature to give its signal?
Please help me to find out what has happened to my chimera protein? Your suggestions are greatly appreciated.
Thanks a lot
It is possible that mCherry is folded properly but your TfR is not.
Hi Ranawakage,
it is possible that your mcherry tag disrupt a sequence at the C-terminus of your TfR. These disruptions normally result in the protein of interest being mislocalized, as reported for tubulin and small GTPase (see reports in following PMID). There are several ways to rescue the localization : Tag on N-terminus or internally / used another smaller tag that could be couple to fluorescence, such as the Suntag from Robert Vale.
Hope you find the best combination for your study.
PMID: 18162163
PMID: 24633327
PMID: 25307933
Thanks a lot Debajyoti and Vincent for the comments.. @ Vincent for the references. I found them useful for my work. As you suggested I should go for inserting a linker in between them. I wish your advices if I fail again..:) Thanks again
I am happy to share my success in generating Dimer2 fused Transferrin receptor chimera protein with proper cellular localization...!! In this construct I fused Dimer2 in the N-terminal of CD71 (intracellularly). If you interested please check the attached image..:)
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