Hi all, this is a very technical query related to proximity logation assays.
I am trying to locate a viral protein through proximity ligation assay. The vector containing the viral gene is stably integrated in HEK293 cells specifically at a single copy per genome. Hence, the protein level is expected to be suboptimal to pick through western blot.
Under a particular condition, I am trying to detect this protein through PLA since it is highly sensitive technique. I have used two different secondary antibodies from two separate host animals to pick up this particularly rare protein. I get clear signals (dots in satisfactory number) in case of two antibodies together while an absolutely clear background for both the single antibodies. This led me think that indeed the rare protein is present at that condition.
My questions are:-
1. How do I rule out the possibility that these are false negatives?
2. Are there any other supporting experiments that might be asked by the reviwers?
Please give in your valuable suggestions
To answer the first question, ommitting one of the primary antibodies should of cause not give a signal since one of the probes is not able to bind. It will check non specific binding of that probe. What you could do (answer to Q2 as well) is replacing one of the primary antibody with a species matched one of wich you know by single IF/ICC staining is expressed in another cell compartment (read too long distance for PLA signalling). This should not give a reaction while it alows both primaries and both secondaries to bind. Use a well defined antibody which do do create background, because I once pressed the PLA system to give non-specific signals using a dirty controle antibody.
Good luck!
I guess that a negative control with no expression of your viral particle but maintaining primaries antibodies should give you a clue about inespecificity
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