I am trying to overexpress 15 different proteins in HEK293T cells by lentiviral transduction. The lentivirus plasmid was acquired from GenScript and I have confirmed that it has the protein and a FLAG tag. I have been trying to overexpress several proteins (both cytoplasmic and membrane) in HEK293Ts (not all 15 proteins in one, but 15 different cell lines), and performed western blots probed against FLAG. So far, I have had expression of 7 different proteins, and they happen to be cytoplasmic, while membrane proteins do not appear in the western blot. I bought an antibody to the specific protein (not FLAG) and it did appear in the western blot. Is it possible that the FLAG tag is buried within the lipid membrane and not reachable by the antibody? If so, wouldn't lyse of the cells with RIPA buffer take care of this?
I also notice that the expression of each protein gives the same levels every time, some high and some low (thick dark bands or light bands on WB). Is this due to the nature of the protein? Or the construct/plasmid? (All plasmids are about 8-11kb)
Simply do an IF against FLAG-membrane proteins. If you can see the signal, the problem is in your lysis protocol, if you don't see the signal, the problem is in your plasmid.
Are you able to see the protein at the right molecular weight in your WB? Otherwise, have you confirmed the localization of your proteins? If you are doing an SDS-PAGE and not a native gel, SDS will solubilize enough the lipid membranes and make them accesible for the antibody to bind during WB, but sometimes it can be your protein that is way too bulky around your FLAG tag to allow the binding of your AB.
For the expression part, i didn't understand if you are getting the same levels every time or no (low high bands?)
Do your constructs have signal sequences for being trafficked to the membrane? Do you find these proteins at least in the cytoplasmic fractions?
(Edit: Membrane protein which are on the way to their destination should always be present in the cytosolic fraction).
Thank you for all your answers!
Alessandro Palma I will try the IFA to see how that goes.
Jorgaq Pata Yes, I always get the same levels of protein, and they do appear in the WB at the correct band size. There are just some plasmids/lentivirus that work best at transducing the cells. I have a couple of proteins that are seen in the WB always as a big dark band, while other (always the same ones) have to play with the exposure to see it.
Wolfgang Schechinger I am not sure. So far I have been able to see all of my proteins (by WB) that are cytoplasmic (according to Uniprot) and not the membrane localized ones (also according to Uniprot)
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