Biology Reference
In-Depth Information
(DCX), a protein that binds preferentially to 13-pf microtubules, as a readout for
microtubule architecture in mixed populations.
21.1
MATERIALS
1.
Unlabeled tubulin
2.
Alexa Fluor 546-labeled tubulin, Alexa Fluor 546 (Succinimidyl Ester, Life
Technologies A-20002)
3.
TAMRA-labeled tubulin, TAMRA (Succinimidyl Ester, Life Technologies
C1171)
4.
Axonemes (Supporting protocol 2 in
Waterman-Storer, 2001
)
5.
PIPES (Sigma, P6757)
6.
Paclitaxel (Sigma, T7191)
7.
GTP (Jena Biosciences #NU-1012)
8.
GMPCPP (Jena Biosciences #NU-405S)
9.
BRB80 buffer: 80 mm PIPES pH 6.9 (KOH), 1 mM EGTA, 1 mM MgCl
2
filtered (0.22
m
m), degassed, and stored at
20
C
10.
Silanized cover glass (
Gell et al., 2010
)
11.
Cover glass holders or silanized microscope slides (
Gell et al., 2010
)
12.
Double-sided scotch tape
13.
Whatman filter paper (#1001-090)
14.
Antitetramethylrhodamine antibody (Life Technologies A6397)
15.
Pluronic F-127 (Sigma P2443), stock solution of 1% in BRB80, filtered
(0.22
m
m filter), and stored at 4
C
16. b
-Mercaptoethanol (Sigma, M3148), 10
m
l aliquots stored at 4
C
17.
BSA (Sigma, A3059), 10 mg/ml in BRB80, filtered and stored at 4
C
18.
Glucose (Sigma, G7528), 2 mg/ml in water and stored at
20
C
19.
Glucose oxidase (Sigma, G2133), 2 mg/ml in BRB80, flash-frozen and stored at
20
C
20.
Catalase (Sigma, C9322), 0.8 mg/ml in BRB80, flash-frozen and stored
at
20
C
21.
Imaging buffer: BRB80
þ
10
m
M paclitaxel
þ
0.1 mg/ml
b
-Mercaptoethanol
þ
0.1 mg/ml BSA
þ
1:100 dilution of antifade reagents
(glucose, glucose oxidase, catalase)
22.
Beckman Airfuge
23.
TIRF microscope with an EMCCD camera (e.g., Andor iXon
). Commercial
implementations of TIRF (e.g., Zeiss Laser TIRF III) are largely sufficient
for these experiments
24.
Objective heater with controller 37-2 (Pecon GmbH, Germany #0280.004)
25.
DCX-GFP expression vector (
Bechstedt & Brouhard, 2012
)
26.
BL21(DE3) bacteria
27.
LB medium
28.
IPTG (GoldBio, I2481C)
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