Biology Reference
In-Depth Information
Voloshina et al., 1999
)orCydyes(e.g.,Cy3/Cy5)(
Mujumdar, Ernst, Mujumdar,
Lewis, & Waggoner, 1993
), which are commercially available in a wide range of
wavelengths and have been optimized in terms of brightness and photostability. These
dyes are linked to small reactive groups, such as maleimide or succinimidyl esters, that
enable coupling to sulfhydryl or amino groups, resulting in an overall small modifica-
tion of the target receptor (typically
1-2 kDa). However, care must be taken that any
free dye is removed as this can interfere with data interpretation. The labeling effi-
ciency should also be determined, as this will influence the maximum FRET efficiency
possible and should be corrected for. So, although these small dyes have a clear size
advantage over the use of fluorescent FPs, they are experimentally more challenging,
and labeling conditions need to be optimized for each individual case.
<
18.1.2.1
Protocol for site-directed fluorophore labeling of cysteines
Reagents
1.
Purified protein stocks in detergent (0.1% dodecyl maltoside (DDM)/0.01%
cholesteryl hemisuccinate (CHS) (w/v))
2.
Protein buffer: 50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM EDTA, 0.1%
DDM, 0.01% CHS (w/v), 10% glycerol (v/v)
3.
Reducing agent (e.g., dithiothreitol (DTT) or Tris (2-carboxyethyl) phosphine
hydrochloride (TCEP))
4.
Cysteine-reactive fluorophore (e.g., maleimide-Alexa Fluor or Cy dye; typically
as a dimethyl sulphoxide (DMSO) stock for long-term storage or in aqueous
buffer at pH 7 for short-term storage)
5.
PEGm (methoxypolyethylene glycol 5000 maleimide)
18.1.2.1.1
Preparing the receptor for labeling
One should aim to have only one label per monomer to have a homogenous popu-
lation of labeled protein. Thus, a background mutant needs to be constructed in
which native, accessible cysteines are removed by site-directed mutagenesis to en-
sure specific labeling either at one remaining native cysteines or at a newly intro-
duced cysteine. One should rule out the possibility of nonspecific labeling of this
background mutant under the same conditions used for the labeling of the final mu-
tant. Additionally, one should check that the mutant used is still functional, through a
relevant assay such as ligand binding or G protein coupling, or both.
18.1.2.1.2
Reduction of reactive site
The purified receptor is reduced prior to labeling to ensure reactivity of the cysteine
residue, typically with DTT or TCEP, at a concentration in the range of 1-10 mM. It
has been shown that low concentrations (0.1 mM) of the trialkylphosphine reductant
TCEP interfere less withmaleimide labeling than the thiol-based DTT under the same
conditions (
Burmeister Getz, Xiao, Chakrabarty, Cooke, & Selvin, 1999
). Thus,
TCEP can be favorable when labeling with maleimides if reductant removal is not
