Secondary Antibody Problem - (Apr/03/2012 )
I am new in western blotting. Yesterday I performed western with 2 primary antibody:for 1st you need to use anti-rabbit, 2nd is anti-goat. We are using Odyssey scanner, so fluorescent secondary antibodies will give me red-color bands (anti-rabbit or rat) and green-color bands (anti-goat and anti-mouse).But by mistake I treated my membrane with secondary anti-mouse instead anti-goat! But I saw the green bands! How is that possible? Can goat anti-mouse ab binds to primary ab raised against goat?
Sorry if the question is kinda stupid...
you observe cross-reactivity; profile of IB may be similar or the same with anti-mouse as for secondary anti-goat Ab but signals could be weaker; strip and re-probe with correct Ab´s...
If you blocked with milk, anti-goat antibody might react with that.