Dear all,
I am overexpressing my protein of interest in HEK293T. The protein is supposed to be soluble with no particular localization signal, which means it is expected to be in the cytoplasm. Fluorescent protein is attached as a fusion tag for easy visualization. Upon visualization with fluorescent microscope (GFP channel) I realized that vast majority of cells have spots with high concentration of the signal (1-3 spots per cell). These seem to localized in the cytoplasm and are always round and much brighter than the rest of the cell. Does anybody know what might this be? I attach a representative image.
Thank you in advance.
hi
maybe as Proteasome . Due to the absence of a specific signal, your protein will accumulate in the proteasome area .
good luck
Hi Dawid,
it might be that your protein undergoes phase-separation condensing in liquid-like structures (see https://www.ncbi.nlm.nih.gov/pubmed/23415227 for more info) because they look very round to me. However the fact that most of the cells have only one big spot could also look like aggresome formation (accumulation of misfiled proteins eventually targeted for autophagy).
Best,
Raffa
It's possible that overexpression of the protein is causing this. Proteins can behave differently and mislocalize when too much is expressed. Perhaps try a weaker promoter.
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