I am overexpressing an endogeneous protein with c terminal myc tag in entamoeba. Overexpression has been confirmed by real time pcr. But when i am trying to visualize it in confocal microscopy with anti myc antibody i cannot see any staining. Primary antibody added is in ratio 1:100 and secondary antibody ratio is 1:300. Please suggest that what may be the problem.
HI Shilpa
with anti-myc you should see endogenous staining (I assume you are working with intracellular protein and that all steps of your protocol: fixation, permiabilization, blocking) have been checked earlier.
If you don't see endogenous myc, it may be an antibody problem (if the protocol works for other things).
I hope this can help you
I would try confirming the expression by western blot using both myc antibody and antibody for the protein of your interest. The existence of mRNA does not mean that protein was expressed.
Myc tags are not ideal for microscopy because endogenous c-Myc could also be detected and could interfere with the analysis but it usually doesn't.
- Badges
- Science method