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purified two-hybrid proteins?

Piero Morandini pm1 at imiucca.csi.unimi.it
Thu Nov 27 10:46:21 EST 1997


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
question.



>From noel.fong at ucdavis.edu

Piero,

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 <v01550107b084ff5f2838@[159.149.7.179]> 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?

Piero Morandini
Biology Department "L. Gorini"
Plant Physiology and Biochemistry Section
University of Milan
Via Celoria 26,  Torre 4C,
Milan 20133    Italy

tel:  +39-2-2660-4394         Email:  pm1 at imiucca.csi.unimi.it
fax:  +39-2-2660-4399






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