Biology Reference
In-Depth Information
FIGURE 9.5
Perfusion of axoplasms. Buffers of interest can be perfused into axoplasm for studies of
microtubule biochemistry or imaging. Ideally, sister axoplasms (two axoplasms from a single
squid) are used for comparison to reduce interanimal variability. For biochemistry, the
axoplasms are perfused without a top coverslip while imaging studies require the top
coverslip. In both cases, buffer volumes are kept small relative to the axoplasm volume to
minimize dilution. Typical axoplasms are 5
m
l in volume, so buffer volumes are kept at
20-25
l. This is in contrast to conventional biochemical approaches where dilutions may be
10
3
-10
5
.
m
modifications of tubulins, distribution of microtubule-associated proteins (MAPs),
etc. In addition, experimental manipulation of microtubule modifications can be
performed to understand how a particular modification may alter microtubule
structure, its dynamics and stability, and its affinity for motor proteins and MAPs
(
Song, 2010
).
For biochemical experiments, two “sister” axoplasms of the same length from
one squid are extruded on glass slides as described above, one serves as a control
while the other is used for experimental manipulations (
Fig. 9.5
). This ensures that
the variability between individual squids is minimized. A circle is drawn around each
of the two freshly extruded axoplasms using a PAP Pen (Zymed) or liquid blocker
(Super Pap) to delineate the perfusion area before being placed in a humid chamber.
Placing the slide on a piece of wet paper towel in the chamber may enhance humidity.
After being incubated at 4
C for 10 min, a control axoplasm is perfused with 30
m
lof
þ
buffer X/2
5 mMATP alone, while the experimental axoplasm is perfused with the
appropriate effector mixed in the same buffer. Both axoplasms are incubated for suf-
ficient amount of time at a temperature determined by specific experimental condi-
tions. Axoplasms are transferred into microcentrifuge tubes where they are
homogenized in 30
m
l of SDS (1%) or other proper buffers by trituration with a
P200 pipette. The samples can be analyzed by SDS-PAGE, followed by immunoblot,
autograph (if radio labeled), or direct gel documentation (if fluorescently labeled).
