Biology Reference
In-Depth Information
Table 5
Co-transformations required to determine the interaction between two proteins (A and B)
Bait clone (BD-)
A
B
A
LamC
B
LamC
P53
LamC
Prey clone (AD-)
B
A
T-ant
B
LamC
A
T-ant
T-ant
Growth in selective media
Yes
Yes
No
No
No
No
Yes
No
Positive and negative yeast two-hybrid controls are indicated in bold
LamC
lamin C,
T-ant
SV40 large T-antigen,
P53
murine p53
bait proteins should also be co-transformed with the rescued
prey-vectors.
This method of testing specifi city can be somewhat cumber-
some if a large number of different library plasmids were isolated,
and if these are to be tested for interaction with several different
baits. For this purpose a mating assay to perform the specifi city test
would be more appropriate (further information could be founded
in
www.clontech.com
, Cat. No. PR033493)
Interestingly, the commonly isolated nonspecifi c interactors
which include heat shock proteins, ribosomal proteins, proteasome
subunits, and other proteins, are not isolated in every interactor
hunt, and in fact do not appear to interact with every bait. This
highlights the importance of using several different bait proteins to
test the specifi city of an interactor.
3.2 Analysis of a
Specifi c Interaction
To determine if two proteins (A and B) interact, a complete or
partial cDNA from each must be cloned into both bait and prey
vectors to express them fused either to the BD or the AD of Gal4.
Protein expression, toxicity and autoactivation of bait and prey
proteins must be tested beforehand to confi rm the interaction as
described in Subheading
3.1.1
.
Interaction is tested by co-transforming yeast cells according
protocol Subheading
3.1.1
. It is advisable to test the interaction
using plasmids expressing protein A fused to AD and B to BD, as
well as plasmids expressing protein B fused to AD and A fused to
BD. Specifi city of the interaction must be also confi rmed using bait
or prey vectors expressing non-related proteins. Table
5
summa-
rizes the co-transformations required to test the interaction and
the expected results for a positive interaction (
see
Note 4
).
1. Cells must be plated onto CM plates to select for co-
transformants. Incubate the plates at 30 °C for 3-5 days.
2. Transfer the colonies onto fresh CM plates. If the colonies are
closely spaced it will be necessary to streak purify to single
colonies to separate the different co-transformants. Incubate
the plates at 30 °C for 2 days.
3. Collect the yeast from one streak using a sterile toothpick and
resuspend them in 100
3.2.1
Test the Interaction
l of deionized sterile water placed in
1.5 ml microcentrifuge tubes.
μ
