Biology Reference
In-Depth Information
13.1.3.1
Epifluorescence and single molecule TIRF imaging methods
13.1.3.1.1
Buffers, reagents, and equipment
13.1.3.1.1.1
Buffers
Severing Buffer II: 10% sucrose, 20 mM HEPES, 100 mM NaCl, 3 mM MgCl
2
,
5 mM DTT, 50
m
M ATP, pH 7.0
PEM-100
Imaging buffer: Severing Buffer II, 20
m
M Taxol, 0.1% Pluronic F-127,
0.5 mg/ml bovine serum albumin (BSA), 10 mMDTT, 40 mM glucose, 40
m
g/ml
glucose oxidase, 16
m
g/ml catalase, 8 mM phosphocreatine, 0.16 mg/ml creatine
phosphokinase with 2 mM ATP
13.1.3.1.1.2
Reagents
Taxol
Pluronic F-127 (Sigma-Aldrich, P2443, St. Louis, MO)
BSA (Fraction V, Fisher Scientific, BP1605, Fair Lawn, NJ)
DL-Dithiothreitol (DTT) (Amresco, 0281, Solon, OH)
)-Glucose (Glucose) (Sigma-Aldrich, G5767, St. Louis, MO)
Glucose oxidase from
Aspergillus niger
(Sigma-Aldrich, G2133, St. Louis, MO)
Catalase from bovine liver (Sigma-Aldrich, C3155, St. Louis, MO)
Phosphocreatine (Sigma-Aldrich, P7936, St. Louis, MO)
Creatine phosphokinase from rabbit muscle (Sigma-Aldrich, C3755,
St. Louis, MO)
ATP magnesium salt (Sigma-Aldrich, A9187, St. Louis, MO)
Adenosine 5
0
-(
b
,
g
-imido)triphosphate lithium salt hydrate (AMPPNP) (Sigma-
Aldrich, A2647, St. Louis, MO)
Hexokinase from
Saccharomyces cerevisiae
(Sigma-Aldrich, H4502,
St. Louis, MO)
Stock of severing enzyme (active)
Mutant severing enzyme (inactive) as a negative control
D
-(
þ
13.1.3.1.1.3
Equipment.
Fluorescent species were visualized using epifluores-
cence and total internal reflection fluorescence (TIRF) on a microscope built around
a Nikon Eclipse Ti microscope, as described previously (
Ross & Dixit, 2010
). Epi-
fluorescence is excited using a Xe-Hg lamp (Nikon). TIRF excitation of GFP mol-
ecules used a 488-nm 125 mW Argon-ion air laser (Melles Griot) or a 50-mW
488 nm solid state (SpectraPhysics/Newport). The system has a high numerical ap-
erture objective (60
magni-
fication is placed before the camera to give 67.5 nm/pixel. Images are acquired with
an electron multiplier CCD Cascade II camera (Photometrics, Tucson, AZ) or an
IXON EM-CDD camera (Andor, South Windsor, CT).
,NA
¼
1.49, Nikon, Melville, NY). An additional 4
13.1.3.1.2
Experimental procedures
1.
Using the flow chamber with microtubules, as described above, image the
microtubules to ensure that they are adhered to the surface of the cover glass and
easily detected.
