Biology Reference
In-Depth Information
FIGURE 12.1
Flowchart illustrating the steps in the in vitro binding assays including protein purification,
binding assays, and identifying regions of interaction.
used to demonstrate that G
a
forms stable complexes with tubulin. Here, we will de-
scribe details for the two latter methods used to determine the association between
Gs
a
and tubulin. Flowcharts illustrating the purification and
in vitro
binding assay
protocols are shown in
Fig. 12.1
.
12.1.1.1
Protein purification
For all
in vitro
binding and functional studies, we use purified tubulin and purified
Gs
a
. The purity of tubulin and the Gs
a
is crucial for determining the binding prop-
erties and function of Gs
a
-tubulin complexes. The methods for purification of these
two proteins have been described by many investigators (
Linder, Ewald, Miller, &
Gilman, 1990; Miller & Wilson, 2010; Shelanski, Gaskin, & Cantor, 1973
). Below,
we briefly describe the methods used in this lab.
