Hi, we want to localize a novel protein in cells. We don't have good antibodies to stain endogenous protein due to low expression level and/or low reactivity of the antibody, so we overexpressed the protein and stained it with antibody. The protein colocalizes with lysosomal markers (LysoTracker and LAMP1). The question is, how can we tell whether this is the real localization of the protein, or some artifact caused by protein overexpression? Is there any way to rule out the possibility of artifact? I appreciate your expertise.
There is no simple answer...
1) You can quantify your colocalization and get statistical signification of "R"...
2) Do you have the full sequence of your "new" protein, does it include a lysosomal targeting sequence ? That is worth looking if you think your protein works in the lysosome, and what would be its function....Or do you think it is only degraded there?
3) Did you try to put a tag to your protein and see where it localize after expression within the cell? However be careful tag can be removed and some fluorescent tag do not work within the acidic environment of the lysosome.
You might have to build up the localization of your new protein with different experiments to get final meaningful data.
Good luck!
Is the protein soluble or membrane spanning?
If it is soluble, then it is very unlikely to be an artifact, because overexpression usually causes saturation of specific signal-mediated targeting pathways (ER retention, lysosomal sorting) and results in partial secretion (the default pathway). It does not happen that a secreted protein ends up in the lysosomes due to overexpression.
If it is a membrane spanning protein, then the story is totally different, and it is possible that overexpression saturates retromer-dependent retrieval mechanisms from early and late endosomes, and so you end up with leakage to the lysosomes. Increase the detection-limit by making better antibodies or tag the protein with a more sensitive epitope (hoping that the epitope doesn't alter protein function/targeting).
Good luck indeed.
I Agree with Jürgen, overexertion may push your protein into a specific compartment. You should also be careful as to whether your protein is on the cytosolic or luminal side of the lysosome.
A protease protection assay is useful for determining whether your protein is luminal, cytosolic or membrane spanning. If it is membrane spanning, the you carry out the sassy with N and C-term tags and look to see the disappearance of the tag after protease treatment.
Hope this help.
I appreciate all your comments. To clarify,
1) it is a soluble protein, but it has membrane binding capability when Ca2+ is high. Even under physiological Ca2+, a portion of it binds to the plasma membrane. I would not be surprised if it binds to lysosome membrane. It does not have a signal peptide so it should not be secreted.
2) I do not tag it with fluorescent proteins as we know it interferes with function. Instead, I tagged it with a small epitope tag so I can stain it with either anti epitope tag or the protein's antibody.
3) The protein is stably overexpressed by lentiviral transduction, so it is not crazily overexpressed as in transient transfection with plasmid.
I agree with David's question. It will be really helpful to determine whether the protein is inside or outside of lysosomes. But I will do this experiment slightly differently. I will stain the protein after I selectively permeabilize the plasma membrane with digitonin. If I can still stain it, it is on the outside of lysosome; it not, it is inside. It will help me a lot to know its topology.
In my experience, you can perform some other experiments besides staining to confirm colocolization of a protein, such as Co-IP. If your protein might colocalize with LAMP1, you can detect the direct interaction between these two to see if this is true or artificial.
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