Hi all,
I'm confused about some literature on the dot blot and Far-WB, since it is claimed that Far-WB can be used to detect interaction between prey and bait proteins but there is no such claim for dot blotting. The dot blotting looks easier to me than the Far-WB, so why can't this method measure association of prey and bait proteins?
Thanks!
You could use dot blot if you work with purified protein. But if you have a complex mixture of proteins with dot blot you can't see if you have nonspecific binding for example. You will see signal but not knowing where it comes from ... So WB is more informative.
You could use dot blot if you work with purified protein. But if you have a complex mixture of proteins with dot blot you can't see if you have nonspecific binding for example. You will see signal but not knowing where it comes from ... So WB is more informative.
Dot blots work for complex mixtures if you have carefully characterized your antibodies and confirmed that they only detect your protein of interest and nothing else.
Unless you have very limited amounts of bait protein, I would use Far Western blotting, at least initially. The reason is if your bait protein interacts with more then the intended prey protein, or if there is non-specific binding, you won't know using only dot blots.
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