Dear yeast netters,
A few weeks ago a posted a question on the biochemical confirmation for a
bait/prey interaction. Here is the only answer I got and once again the
>From noel.fong at ucdavis.edu
In many of the articles that I read on interactants identified through a
2H search, I often see confirmation by GST-fusion proteins in vitro, not
by purifying the 2H fusions. For a reference of this approach, try
O'Neill et al JBC 1997, Apr 11, 272(15): 10035-40.
In article <firstname.lastname@example.org> you wrote:
: Dear yeast netters,
: after many false positives, I fished out of a two-hybrid library what seems
: to be a true interactor of my pet protein.
: Now it seems to me time to seek biochemical evidence for the interaction.
: I suspect I will need for this decent amounts of the purified proteins.
:: Question is: what is the feeling/experience of the people about this?
: Should one first generate a clone for expression in coli or try to purify
: it from yeast cells?
: Is it better to keep the fusion protein as it is in the two-hybrid or
: eliminate everything that is not coding for the interactor/bait protein?
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