I have a protein normally localized on cell membrane (in HEK cells), with a transmembrane domain.
How can I target this protein into ER membrane and make it to accumulate there?
For soluble luminal proteins, appending to the C-terminus of your protein the KDEL (or HDEL) motif is usually enough. But since you have a membrane protein, you could try to engineer the di-Lysine motif KKXX (X any aminoacid), which represents one of the ER retention motif for membrane proteins. If you search these terms, you'll find all the relevant literature examples of application and explanations. Good luck!
Francesco Ferraro , thank you for your answer!
How do you test for protein localization in ER? It appears that ER is everywhere in the cell...
How can I tell if my protein is localized only in ER or both in ER and on outer cell membrane?
Use immunofluorescence microscopy with an ER marker. Calreticulin is one commonly used (fix the cells and probe with an anti-calreticulin antibody). Otherwise, you can used dyes such as ER-Tracker (https://www.thermofisher.com/order/catalog/product/E34250). I never used this, but I have seen papers that do and seems to work well. If you want to test if your protein is also at the plasma membrane, you can fix the cells but not permeabilise them and then use an antibody against your protein. If you get a signal, then the protein reaches the PM. If not, then you protein is retained inside the cell, because you antibody had no access to it (no staining). I suggest that if you are not familiar with how protein localized to cellular compartment look at the confocal/IF microscope you should ask the help of someone who works in membrane traffic and has experience. Regina Kuliawat, at Albert Eistein works on membrane traffic. She might be able to lend a hand. Good luck
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