I have to perform an IHC and I have three different antibodies to test. We already have one made in rabbit and another made in mouse, both antibodies react against human. We have to test a third one, but the only option we have is an antibody made in mouse but conjugated with biotin, so I can use streptavidin conjugated secondary antibody to detect it. May this third antibody be detected also by the secondary against mouse?
Riccardo, a secondary against mouse should work as well. The biotinylation sites are random and usually lysines and N-terminal amines, but not modified to exhaustion. So, even if there are lysines in the paratope and some antibodies cannot bind, overall there should not be any problem
Hi Riccardo, I have tested that dye-conjugated goat anti-mouse secondary antibody binds to biotinylated primary mouse antibody and gives a good signal in immunofluorescence with fixed frozen sections. I also found the signal is still obvious when the primary is dye-conjugated instead of biotinylated, but it's a lot dimmer than if using unconjugated antibody, as mentioned here (https://biotium.com/tech-tips/tech-tip-combined-direct-and-indirect-immunofluorescence-using-primary-antibodies-from-the-same-host/#:~:text=Secondary%20antibodies%20will%20still%20recognize,labeled%20and%20unlabeled%20primary%20antibodies.). So depending on how high the affinity to the Fc vs streptavidin to biotin, and whether the biotin or dye interferes with the epitope, you will get an either strong or weak signal.
Hi Riccardo, you can use a secondary anti-mouse- antibody. If you will use a biotinylated secondary anti-mouse it is possible to inhance the reaction while you are using Strptavidin. Sometimes it makes sence to use the 3-Stepmethod (biotinylated secondyry plus streptavidin) because this method is more sensitive the 2-step or direct labeled methods (primary antibody is labeled).
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