Hi,
I am trying to see if my protein of interest (a membrane protein) interacts with EGFR. I transiently transfected glioblastoma cells - high expressors of EGFR - with my protein and carried out my co-IP. I got a negative result (no protein of interest detectable in the blot of IP with EGFR, successfully immunoprecipitated EGFR and lysate has my protein of interest - all checked with immunoblots). Now I am wondering if this is an actual negative or if I need to treat my cells with EGF in order to get activated EGFR and do my co-IP with those cells. Does that matter?
Would appreciate any advice!
I have no experience with this specific protein but i can tell you that anything that changes possibly the confirmation or charge of your protein might obviously change its binding capability especially when it comes to receptors. I by myself have done CO-IPs under several conditions and have seen different results for the binding. You can even observe binding after activation related to time. So without further knowledge of this specific protein, i would think it is worth a shot. I also work with receptors (albeit in yeast) and it makes a huge difference if they are activated or not.
Also, Co-IPs can never really give a negative results in the sense that they can exclude binding. The binding could be transient or depending on compartimentalization and several other factors. You could try cross-linking or if you would be willing to make the switch to microscopy and if it is feasible with your cells, methods involving fluorescence (FRET, BRET, Split-YFP...). Also membrane enriched fractions could be an idea.
Best of Luck with your Co-IP, i know it can be a pain to get it right.
Regards
You might want to check that your protein of interest is also getting to the cell surface. You say it's in the lysate so it sounds like your transfection has worked, but it's possible that it's getting missfolded and stuck in the ER for some reason and not making it to the cell surface to interact with the EGF receptor. There are a couple of ways you could look at this, including just staining the cells and looking by immunofluorescence, or if your protein is glycosylated you can use specfifc sugar digestions to look at how far it is getting through the secretory pathway.
Michaela and Ian - Thank you very much for your input. Those are all great points. My next experiment will be to carry out ICC with these cells to see if the protein gets stuck somewhere. And the Co-IP with activated receptors will follow depending on what I see. I am considering FRET as well, my lab is not equipped for FRET but hopefully will be able to collaborate and run that experiment.
This is a very interesting problem. When is a negative truly negative? Your plan to perform ICC with the transfected glioblastoma cells is a good one but does not address why you can detect both the EGFR and your protein of interest in the lysate. Assuming that you are using a polyclonal Ab for your protein of interest and EGFR, you should have a positive result in either case unless, they are not interacting. If you are using a monoclonal you might want to switch to a polyclonal since the epitope recognized by the Ab may not be available when the proteins are interacting. Therefore no pull down.
I think your first instinct was correct, you should perform your Co-IP with EGF treated and untreated cells before doing any of the other experiments you are thinking about. If as you hypothesize the EGFR needs to be activated to interact with your protein of interest, the Co-IP experiments would be the best objective to test that hypothesis.
All the best
Fedora - Thank you for your input. I am using a monoclonal for the pull down and polyclonals for detection. And I am checking for respective pull down (say EGFR IP with EGFR detection - it works). Was that the concern you expressed or did I miss what you meant?
It certainly seems like doing my Co-IP with EGF treated/untreated cells may be worthwhile. I think the ICC/enzyme digestions will be useful as well because like Ian mentioned, the protein may not even be getting to the membrane, let alone interact with another membrane protein.
The problem with using the monoclonal for the pull down has to do with access. If the epitope recognized by the monoclonal is not accessible when the proteins interact, you will not be able to have a successful pull down but you will be able to detect the protein when it is not part of the complex.
If you are using total lysates of membrane solubilized and cytoplasmic proteins for your pull down, it really does not matter where the protein is located. To provide a more informative answer, I will need to have some background. Have you obtained any other successful pull down results in any other cells with this bait and prey?
If you are using only solubilized membrane proteins then the question of localization of your proteins to the membrane is valid. However, I understand from you that Western immuno detection of the receptor and interacting proteins are positive for the lysate. This means that the proteins are together in the lysate and are not interacting or if interacting, your antibody is not successful at co-imunoprecipitation.
"Have you obtained any other successful pull down results in any other cells with this bait and prey?" - I haven't. This was the only one cell line I have tried. Do you think it might be worthwhile trying this in another cell line?
And I see your point regarding the monoclonal access. I will consider trying the co-IP with a polyclonal to see if there is a difference.
Use the polyclonal for the Co-IP and move on to the exciting questions of your system.
Good luck
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