-I want to detect differences in protein expression via LiCoR near-infrared secondaries.
-I probe with a RABBIT primary antibody, against my favorite protein.
-I add the *wrong* secondary--Goat anti-rabbit conjugated with AlexaFluor 488. Alexa-488 has almost no emission at the higher LiCoR band (680nm).
-10 minutes later, I realize my mistake.
-I do not dump out the old antibody.
-Instead, I just add the *right* secondary at double the concentration of the wrong antibody to the same solution--Goat anti-rabbit conjugated with IR-Dye680.
-My rationale is that I should still see signal because there will be plenty of Rabbit IgG epitopes, so the wrong antibody should not bind to all of them. As such, there will still be some binding sites left for the right antibody. Moreover, since the right antibody is present at double the concentration, it will compete with the wrong antibody via mass action.
-Will it work? Will I see bands specific to my protein?
Answer: Yes, it did work! I see my protein clearly. The background bands are just about as abundant as they are when I do not accidentally add the wrong secondary. I wanted to share this with the community in case anyone else makes this mistake.
yes, it should work! i fully agree Damian D Guerra
i did this several times. but there are some secondaries, like from jackson, which are polyadsorbed against only the heavy chain etc. those have only few epitopes...
would be nice if you could share pics :-p
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