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optimal. TCEPhas also been shown tobe a stronger and faster reductant at pH
8( Han
&Han, 1994 ).TCEP ismore stable thanDTT in the presence ofmetal ions such as Fe 3 þ
and Ni 2 þ , which can be an important consideration when labeling protein after IMAC
chromatography, while chelating agents such as EGTA adversely affect TCEP stabil-
ity but increase DTT stability ( Burmeister Getz et al., 1999 ).
The receptor is incubated with the reductant for 1-3 h at room temperature or at
4 Cdepending on the stability of the protein. Then, reductantmust be removed, which
can be efficiently done by size-exclusion chromatography (e.g., using a 5 ml Sephadex
G-25 prepacked HiTrap Desalting column, GE Healthcare, or two or more of these in
tandem to increase capacity and resolution) using degassed protein buffer. Peak frac-
tions are collected and protein concentration is determined by A 280 .
<
18.1.2.1.3 Labeling and optimization thereof
Subsequently, the protein is incubated with up to 10 times molar excess of dye over
protein at 4 C for 1-3 h or overnight, depending on the observed labeling efficiency.
Maleimide groups favorably react with cysteines at a pH below 7.5. At higher pH,
labeling of lysines or the N-terminal amide group may occur ( Brewer & Riehm,
1967; Smyth, Blumenfeld, & Konigsberg, 1964 ). After labeling, free dye is removed
by a second round of size-exclusion chromatography (for larger dyes, the use of lon-
ger columns is advisable for optimal separation).
The exact labeling procedure (dye excess, labeling time, temperature, etc.)
needs to be optimized for each case. Labeling tests using PEGm are a convenient
method for optimizing maleimide labeling as they require small amounts of protein
(
1 m g) and allow straightforward visualization of labeling by mobility shift
assay, without the need to remove unreacted reagent ( Lu & Deutsch, 2001 ).
Reduced protein is incubated with PEGm; analogously to labeling with maleimide
fluorophores, reaction with one or more cysteineswillleadtoanincreaseinthe
molecular mass that can be observed as a band shift via SDS-PAGE or Western
blot analysis.
<
18.1.2.1.4 Determining labeling efficiency
The easiest method to establish the labeling efficiency is spectroscopic analysis of
the ratio the A 280 to the absorption at the fluorophore's maximum ( A Fluor ):
½
receptor
label
A Fluor = e Fluor
E labelling ¼
¼
½
ð
A 280
CF
A Fluor
Þ= e Receptor
where
e Receptor
is the extinction coefficient of the GPCR at 280 nm, and CF is a correction factor to
account for the contribution of the fluorophore to the absorption spectrum at 280 nm.
For example, the contribution of AlexaFluor488 at 280 nm is 11% of its absorption
at its maximum at 493 nm; hence, CF Alexa488 ¼ 0.11. These correction factors are
available from literature or can be determined experimentally from the excitation
spectrum of free dye.
e Fluor is the extinction coefficient of the fluorophore at its maximum,
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