Biology Reference
In-Depth Information
12.1.1.1.1
Tubulin
Brains are obtained from freshly killed sheep from a local slaughterhouse and are
placed on ice immediately upon removal from animals. The gray matter is separated
from the white matter and blended in cold PEM buffer within 1 h. Particulate matter
and cellular debris are removed at 100,000
g
for 1 h at 4
C. Supernatants are col-
lected and microtubule polymerization is induced by adding 125
m
M GTP, 500
m
M
ATP, and 30% glycerol (in PEM buffer) at 37
C for 1 h with gentle agitation.
Microtubules are then pelleted by centrifugation at 100,000
g
for 30 min at
37
C. The microtubule pellet is depolymerized by resuspending in 100 ml PEM
buffer at 4
C and incubating on ice for 30 min. Microsomes, protein aggregates,
and other polymers are removed by centrifugation at 4
C at 100,000
g
for
30 min. The second cycle of microtubule polymerization is performed by
adding1 mM GTP and 30% glycerol for 1 h at 37
C, and microtubules are pelleted
by centrifugation at 100,000
g
for 1 h at 37
C. Microtubule pellets are resus-
pended in cold PEM buffer and stored at
80
C as “non-PC tubulin” (tubulin con-
taining microtubule-associated proteins, MAPs).
MAPs are removed from tubulin using a phosphocellulose column to obtain
purified tubulin (called PC-tubulin). Tubulin-containing MAPs are loaded on a
PEM buffer (100 mM PIPES, 1 mM EDTA and 1 mM MgCl
2
, pH 6.9)-equilibrated
phosphocellulose column (Whatman P-11) at 4
C. PC-tubulin is eluted with 30 ml
PEM, concentrated to 10 mg/ml and stored at
80
C. Purity of the PC-tubulin, as de-
termined by SDS gel electrophoresis, should be
99% on a Coomassie-stained gel.
>
12.1.1.1.2
Gsa
His-Gs
a
or the mutationally-activated His-Gs
a
Q227L
is purified from BL21 (DE3)
Escherichia coli
(Novagen) (
Linder et al., 1990
). The frozen glycerol stock of bacteria
is cultured overnight in 10 ml LBat 37
C. Four milliliters from this culture are diluted
into 1 l of 2YT medium (16 g/l Bacto-Tryptone, 10 g/l Bacto-yeast extract, and 5 g/l
NaCl) and cultured to 0.4-0.6 OD. Protein expression is induced with 30
m
Misopropyl
b
-
D
-1-thiogalactopyranoside for 20 h at 15
C. After induction, the bacterial pellets are
resuspended in TSG buffer, lysed with lysosyme (10
m
g/ml), and sonicated 10 times for
30 s at 2-min intervals (Fisher Scientific Sonic Dismembrator 550). The crude lysate is
then clarified by ultracentrifugation at 100,000
g
for 90 min at 4
C, and the buffer is
adjusted to GAT buffer (see
Section 2
). The lysate is loaded on a Co
3
þ
superflow resin
(Talon, BD Bioscience) equilibrated in GAT buffer and allowed to flow through at
2 ml/min. The column is washed with GAT buffer containing 0 and 20 mM imidazole
and eluted with 30 and 150 mM imidazole. Finally, the Gs
a
QL is concentrated using
a Pierce ICON concentrator and dialyzed into storage buffer. As a rule, Gs
a
protein pu-
rified in this manner is
85%pure on a Coomassie-stained SDS gel. Proteins are further
purified by HPLC using Gel filtration (Superdex 75, Amersham, England) if higher pu-
rity is needed. To test functionality, wild-type Gs
a
is incubated with NaF (10 mM) and
AlCl
3
(50
m
M). Tryptophan fluorescence is determined with excitation at 280 nm and
emissionat 340 nmbefore andafter the additionof fluoride tomonitor the extent towhich
Gs
a
is converted to the activated state. Note that the Q227L variant is the mutationally
activated form of Gs
a
, as the endogenous GTPase is inactivated.
>
