Biology Reference
In-Depth Information
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,