Chemistry Reference
In-Depth Information
(ii) Fluorescent antibodies are available for a broad range of epitopes on proteins, but
they are often larger than the protein of interest, may crosslink the target proteins
and therefore may interfere with its mobility and functionality. In addition to
antibodies raised against epitopes speci
c for the particular protein [6], there are
antibodies which have been raised against generally used sequence-tags, for
instance those used for protein puri
cation such as strep-, myc-,
ag- or
polyhistidine-tags [7, 8] or even anti-GFP antibodies which can be used to
double-label GFP fusion proteins with additional or more suitable
uoro-
phores. Once it has been established that these antibodies do not interfere
with the function of the target, they may constitute handy labels in situations
where signal strength is an issue, because they can carry multiple
uorophores
(up to 10). In a comparable approach, target proteins have been selectively
labeled with
uorescent streptavidin either at strep-tags or at biotinylated protein
sites [7
-
9]. In this case, the size of the probe and its potential crosslinking
capability may also change the functionality of the target proteins.
(iii) Green
fluorescent protein (GFP) and its variants [10, 11] can be attached
genetically at different positions to the target protein in a 1:1 stoichiometry,
but it suffers from poor photostability, the tendency to form oligomers and
spectral overlap with other cellular luminescent compounds leading to
substantial auto
uorescence. Also, for the study of proteins in the plasma
membrane it is preferable to label only the properly translocated fraction,
especially in cases where this fraction is small. Although AFPs are not the
best choice for SPT in terms of their photophysical properties, their bene
cial
biochemical properties are responsible for their impact in intracellular FCS
applications [12].
(iv) Direct covalent labeling of particular amino acid side chains bearing
-COOH, -OH, -SH and -NH2 groups by activated chromophores are an
ideal method for imaging puri
ed single proteins but usually lack
selectivity in the case of live cells, resulting in a large
uorescence
background [13]. Nevertheless, single voltage-gated ion channels, labeled by
this approach, have been investigated in living frog oocytes thereby detecting
structural changes of the channel during voltage-gating [14
-
16]. For technical
details we refer to the comprehensive collection of examples, mostly on
ensemble measurements, in The Handbook
-
A Guide to Fluorescence and
Labeling Technologies from Molecular Probes/Invitrogen (http://probes.
invitrogen.com/handbook/).
A novel covalent labeling of speci
c tetracystein sequences by biarsenical-
bearing chromophores was developed by Tsien et al. [17, 18]. Although yet only
applied to ensemble imaging of proteins in living cells, they offer interesting
potential for single-molecule microscopy: the reactive yet non-
uorescent chro-
mophorepermeatesthecellularmembraneandbecomes
fluorescent after reacting
with the tetracystein sequencemotive on a particular target protein inside of a living
cell.
