Biology Reference
In-Depth Information
BLOCKING SOLUTION:
40
L 5% BSA
m
10
L 0.37% DM
m
950
L PBS
m
15.7.5.3
Procedure
Load 10
L biotinylated antibody solution to each well. (On epMotion with
TS-50 single channel tool, use multidispense, change tip before each aspiration
and water liquid-type settings.)
Inspect wells for incomplete wetting and centrifuge if necessary.
Incubate 15 min at RT.
Aspirate wells.
Load 10
m
L biotin solution to each well.
Incubate 10 min at RT.
Aspirate wells.
Load 10
m
m
L blocking solution to each well.
Incubate 10 min at RT.
15.8
PROTOCOL 6: AUTOMATED MULTICOLOR,
SINGLE-MOLECULE TIRF MICROSCOPY
The block diagram in
Scheme 15.2
shows the components in our customized SMD-
TIRF microscopy workstation based on a Zeiss AxioVert 200M inverted micro-
scope. The system enables automatic time-lapse, multichannel, and multiposition
single-molecule experiments in a 384-well, small-volume, glass-bottom microplate.
The microplate is in contact with a 100
/1.45NA oil immersion TIRF objective
(
Fig. 15.5
). Multiple positions in a single well may be sampled to increase the sta-
tistics of the single-molecule observables. Multiple wells facilitate systematic vari-
ation of biochemical parameters and routine inclusion of proper controls and
standards.
Brightness and photostability are two important properties of fluorescent labels
employed in SMD methods. The chemical environment can influence these proper-
ties. For example, atmospheric oxygen contributes to irreversible, light-induced re-
actions of fluorophores, leading to photobleaching products. We employ a reducing
and oxidizing system (ROXS) in combination with an enzymatic oxygen scavenger
to minimize photobleaching and blinking of our fluorophores (
Vogelsang et al.
,
2008
). The oxygen scavenger generates a gradient of the partial oxygen pressure
(pO
2
) from high values at the buffer-air interface to an oxygen-depleted environment
close to the glass-bottomwhere the laser excitation beam generates an approximately
100 nm deep, 0.2 mm diameter evanescent wave excitation volume (
Fig. 15.5
). An
oxygen-depleted environment dramatically enhances the photostability of cyanine
dyes, such as Alexa 555 and 647 (
Scheme 15.3
).
