Biology Reference
In-Depth Information
Methyl cellulose, 1% solution. Prepared in MRB80 buffer under sterile
conditions. Store at 4
C.
Glucose, 1 M in MRB80 buffer. Prepared under sterile conditions, 0.2
m
m
20
Cas10
l aliquots.
Catalase. Available from Sigma-Aldrich, purified from bovine liver. Catalog
number C9322-1G.
Glucose oxidase. Available from Sigma-Aldrich, purified from
Aspergillus
niger
. Catalog number G7141-10KU.
DTT, 1 M stock.
GMPCCP-stabilized MT seeds (color as required).
Tubulin: 100
filtered. Stored at
m
80
C. Cytoskeleton Inc., 5
M stock, stored at
1 mg,
m
catalog number T240-B.
GTP, 50 mM. Stored at
80
C.
Purified
TIPs as required.
Prepared sample chambers.
Sample tube incubator.
Beckman 5
þ
20 mm polyethylene centrifuge tubes (Beckman, catalog
number 343622)
Vacuum grease or vaseline to seal sample chambers.
8.1.4.1
Preparation of an oxygen scavenger system
In any fluorescent system, dye molecules will undergo photodestruction in a process
known as bleaching, and intensity alterations, which is known as blinking. These
phenomena are the result of the fluorophores entering the so-called dark state
which may be temporary or permanent, and are especially important to consider
if the
in vitro
assay is to be used as a single-molecule experiment. If studying an
association/disassociation process, such as EB binding to the MT, then abrupt
photobleaching can cause a major problem in the interpretation of the results of
dwell time. Oxygen scavenging improves fluorophore stability by inhibiting the
triplet state and hence reduces the effects of bleaching and blinking (
Hubner,
Renn, Renge, & Wild, 2001
). There are various methods available in the literature
for oxygen scavenger systems (
Aitken,Marshall,&Puglisi, 2008;Rasnik,McKinney,&
Ha, 2006; Swoboda et al., 2012
). Inour lab,we routinelyuse theglucoseoxidase/catalase
system. This scavenger system is made in the following manner:
5 mg catalase.
10 mg glucose oxidase.
15.4 mg DTT.
Dissolved in 500
l MRB80 buffer.
m
After components have dissolved, centrifuge the sample for 5 min in a desktop cen-
trifuge at 15,000 rpm to remove any large particulate matter. Usually, we make four
100
m
l aliquots and the remaining sample we pipette into 3
m
l aliquots. Flash freeze
everything in liquid nitrogen and store at
80
C.
