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structural similarities with Gs a ( Chen, Yu, Skiba, Hamm, & Rasenick, 2003; Wang
et al., 1990 ), it serves as a good control to determine which regions on Gs a bind
specifically to tubulin. Peptides are synthesized onto a cellulose membrane with a
proprietary PEG spacer (AIMS Scientific Products, Braunschweig, Germany) via
the C-terminal amino acid in sequential spots by the use of a SPOT synthesis kit
(SIGMA genosys, St. Louis, MO, USA) ( Frank, 2002 ). Peptides from Gs a and
Gt a are divided into overlapping peptides (15 amino acids in length with 10 amino
acid overlap between sequential peptide, 70 total spots).
The peptide array membranes are blocked with TBS-containing 0.1% Tween-20
(TBS-T) with 2.5% milk for 1 h, washed with TBS-T, and incubated overnight at
4 Cwith 150 nMtubulin inTBS-Tbuffer. Next, themembranes arewashed three times
with RIPA buffer and incubated with anti a -tubulin antibody (Sigma, St. Louis, MO,
USA), followed by the horseradish peroxidase conjugated secondary antibody (1 h each
at room temperature in the RIPA buffer containing 1% milk), and developed with en-
hanced chemiluminescence (ECL) Western blotting detection reagents (Amersham
Biosciences). Following the tubulin binding determination, themembranes are stripped
by using a stripping buffer and then reprobed with ECL to verify that residual protein-
antibody complexes are removed before reuse. Also, controls are done to verify that
there is no nonspecific binding of the primary or secondary antibody to the membranes
and that no residual tubulin remains bound to the peptides following the stripping pro-
cedure. These peptides identified frompeptide array are then determined for their affin-
ities to immobilized tubulin using the SPR technique described above.
12.1.2 Functional consequence of tubulin-Gs
interaction
Gs a binding to tubulin has several functional consequences including activation of
tubulin GTPase, which can be evaluated using a tubulin GTPase assay, and transfer
of GTP between tubulin and Gs a , which can be determined by a transactivation assay
( Popova et al., 1997; Yan et al., 2001 ). Only the former will be discussed in this chapter.
The nucleotide state on tubulin is an important factor that determines microtubule
dynamics. Both the a - and b -subunits of tubulin bind GTP, but only the GTP on the
b -subunit can be hydrolyzed to GDP and exchanged for a new GTP (E-site). This
hydrolysis occurs due to tubulin's intrinsic GTPase activity, which is normally
activated during the process of polymerization upon addition of another tubulin
dimer ( Carlier & Pantaloni, 1983 ). A flowchart demonstrating the tubulin GTPase
assay protocols is shown in Fig. 12.3 .
a
12.1.2.1 Steady-state tubulin GTPase assay
PC-tubulin is allowed to bind GTP ( Roychowdhury & Rasenick, 1994 ). The samples
are then incubated with Gs a
Q227L (which lacks intrinsic GTPase activity) or ovalbu-
min at 30 C for 30 min and treated with 1% SDS at room temperature for 15 min.
Nucleotide analysis is done by thin-layer chromatography on polyethyleneimine-
cellulose plates. Two microliters of a 10 mM solution of GTP and GDP is spotted
1.5 cm apart on a polyethyleneimine-cellulose thin-layer plate, followed by 2 m l
of each samples. The spots containing GTP or GDP are visualized with a UV lamp,
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