Biology Reference
In-Depth Information
Table 9.2 Buffer X for squid axoplasm experiments (composition and instructions)
ml
stock in
25 ml
buffer X
Grams/
25 ml
stock
M (in
stock)
mM
in X
Chemical
MW
K-Aspartate
171.2
1.0
4.28
350
8.75
Taurine
125.1
Add
by wt.
Add by
wt.
130
0.407 g
Betaine
135.2
1.0
3.38
70
1.75
Glycine
75.07
1.0
1.88
50
1.25
MgCl 2
6H 2 O
203.31
1.0
5.08
12.9
0.323
K 2
EGTA (adjust stock to
pH 7.2 with KOH before
bringing to final volume)
380.4
0.1
0.9510
10
2.5
HEPES (adjust stock to
pH 7.2 with KOH before
bringing to final volume)
238.3
1.0
5.96
20
0.5
CaCl 2 2H 2 O
147.02
1.0
3.68
3
0.075
Glucose
180.2
0.1
0.4505
1
0.25
K 2
ATP (adjust stock to
pH 7.2 with KOH before
bringing to final volume)
583.4
0.2
2.92
(0.1167
g/ml)
1.0
0.25
Combine aliquots of stock reagents as indicated in the table and check pH. Adjust pH to 7.2 with KOH if
needed. Bring to final volume of 25 ml with 20 mM HEPES, pH 7.2 and filter on 45-
m Millipore filter or
equivalent. Store in suitable aliquots at -20 C until ready for use. We routinely prepare aliquots without
ATP, bringing the stock to 20 ml rather than the 25 ml final volume and storing as 0.4 ml aliquots.
Immediately prior to use, an aliquot is thawed and ATP added from a concentrate stock. Experimental
agents can be added at this point and the aliquot brought to a final volume of 0.5 ml.
m
( Sakai & Matsumoto, 1978 ). Dilution of extruded axoplasm by 1000-fold in buffer
X allows determination of stable polymer, soluble polymer, and free tubulin dimer
in axoplasm from a single axon based on the kinetics of extraction for tubulin into
the media ( Morris & Lasek, 1984 ). This approach defines a Kinetic Equilibration
Paradigm (KEP), which provides a unique perspective on microtubule dynamics
in situ .
The KEP method allows one to define discrete pools. The amount of free dimer in
this assay was significantly less than levels reported from in vitro assays of micro-
tubule polymerization, and the level of polymer remaining after 24 h extraction was
15% of the total tubulin ( Morris & Lasek, 1984 ). This is consistent with the observed
stability of microtubules in isolated axoplasm with minimal dilution ( Weiss,
Langford, Seitz-Tutter, & Keller, 1988 ).
For analysis of depolymerization kinetics, axoplasm should be extruded into a
reservoir as described above. The axoplasm will retain its form suspended in the
buffer. At this point, the axoplasm can be picked up with Dumont forceps, taking
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