Upon over-expression of my protein through a piggyBAC-cometGFP vector, apart from seeing its cytoplasmic over-expression, I see some specs around the nucleus which I suppose is the localisation in Golgi bodies. When I look for the endogenous levels of the same protein the specs around the nucleus almost disappear. In such conditions, I assume I have two different systems - (1) Endogenous, and (2) Over-expression with increased specs outside the nucleus. Can you guys suggest some way to improve this problem? I am sure this is related to over-expression only but do not know how to improve it.
The photo attached is of the over-expressed protein.
It could be the protein you are expressing is aggregating to form the clumps you observe. Many proteins will do this if transiently overexpressed but proteins that have coiled-coils domain(s) are often more prone to aggregation. Your protein may be a substrate for autophagy but if that is the case then the aggregates you can see will be positive for LC3.
Try a different construct with the tag spaced away from your protein using an unstructured linker sequence. Transfect less DNA and do a time course of expression so that you can optimise for lower expression.
It could be the protein you are expressing is aggregating to form the clumps you observe. Many proteins will do this if transiently overexpressed but proteins that have coiled-coils domain(s) are often more prone to aggregation. Your protein may be a substrate for autophagy but if that is the case then the aggregates you can see will be positive for LC3.
Try a different construct with the tag spaced away from your protein using an unstructured linker sequence. Transfect less DNA and do a time course of expression so that you can optimise for lower expression.
First thing you should try is express the construct without GFP-tag to check if this is a tag-specific phenomenon, as Matthew suggested previously.
Then you can decrease the amount of transfected DNA to see if lower protein-levels still give the same pattern.
If the pattern remans you can try to colocalize this expression with cellular organelle markers, such as lysosomes, GOLGI, nuclear membrane etc. If you can identify one organelle you can investigate the endogenous protein as well, maybe your protein is not only cytosolic.
Good luck and best regards!
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