- The units ofβ-galactosidase activity (Miller Units) are defined
as value of A 420 2000/time of the reaction in minutes
[24]. A regular two-hybrid assay should contain the values of
the controls with the empty plasmids and the values of the
interaction between the AMPK subunit and the putative inter-
actor (Fig.4)(seeNote 7). - In order to validate the results of the two-hybrid analysis it is
necessary to check the correct expression of the fusion proteins
(Fig.5)(seeNote 8).
3.5 Screening
of Yeast Two-Hybrid
Libraries for New
Interactors
The yeast two-hybrid system allows the screening of a cDNA library
using a LexA-AMPK subunit as bait. Different cDNA libraries from
different tissues exist in the market and distinct protocols can be
used to perform the screening, but we still carry out the one based
on the co-transformation of the yeast TAT7 or THY-AP4 strains
with a pBTM116-AMPK subunit plasmid and a commercial cDNA
library in a pGAD-based vector (Clontech) [10, 11]. We carry out
the following steps:- Transform the yeast strain (i.e., THY-AP4) with the bait plas-
mid (i.e., pBTM-AMPKα2) and different amounts of the
selected cDNA library (based in pACT2; prey plasmid). Follow
the steps described in Subheading2.3 (steps 1–6). In this way
we will determine the best ratio between bait and prey plasmids
to obtain the highest number of transformants.
Fig. 4Quantitativeβ-galactosidase assay of the interaction between LexA-AMPKα2 and GAD-AMPKβ1. In the
nomenclature (i.e.,α2/F/β1), the first position corresponds to transformants expressing the bait (0, means
empty pBTM116 vector;α2, means LexA-AMPKα2), the second position corresponds to a third protein being
expressed (0, means empty pWS93 vector; F, means pWS93-F) and the third position corresponds to the prey
(0, means empty pGADT7 vector;β1, means pGADT7-AMPKβ1). The strength of the interaction is a direct
correlation of the levels ofβ-galactosidase activity (values are means of at least six transformants per
condition; bars indicate standard deviation). As observed, the expression of protein F improves the interaction
between LexA-AMPKα2 and GADT7-AMPKβ1 (**p<0.01)
Y2H Interaction Analyses 151