Zoonens and Popot (2014)). SMA has been functionalized with cysteamine, whose thiol group has
been used as a reactive moiety for further derivatization, but could also be exploited for immobilization
(Lindhoud et al. 2016). Tagged APols make it possible to immobilize MPs onto beads or chips carrying
appropriate groups, namely (i) avidin, streptavidin (SA), or neutravidin for biotin-tagged APols; (ii) a
complementary nucleic acid for ODN-tagged ones; (iii) immobilized Ni
2+ or Co
2+ ions for
polyhistidine and distributed imidazole tags; and (iv) gold or silver surfaces for thiomorpholine and
sulfhydryl ones.
Amphipol-Mediated Immobilization of Membrane Proteins and Its Applications
(# 2018 by Francis Haraux)
A marked advantage of APols is that, because each protein binds several molecules of it,
multiple functions can be associated to the same protein by the simple device of mixing APols. For
instance, a tagged APol can be mixed with a fluorescent one, so as to easily visualize the immobilization process. Seven fluorescent APols (FAPols) derived from either A8-35 or A8-75 have been
synthesized and characterized to date (see Chap. 4, § 4.4, Table 4.5), whose excitation bands range
from 290 to 651 nm and emission bands from 310 to 668 nm (see Chap. 8, Fig. 8.9). Thiolated SMA
has been used to bind Alexa Fluor 488 (Lindhoud et al. 2016). This broad panel of spectral
characteristics greatly facilitates the choice of adequate experimental conditions, as well as
multiplexing.
Figure 13.1 illustrates schematically the basic principle of using tagged APols for immobilizing
MPs onto solid supports. In § 13.2, we will review data pertaining to the use of biotinylated APols for
MP immobilization, in § 13.3 the current state of development of other tagged APols. In § 13.4, a few
other validated or potential applications of tagged APols will be evoked. Table 13.1 presents an
overview of publications that have resorted to the use of tagged APols. Biotin-mediated MP immobilization has been used in five studies using three different APols (Studies 13.1–13.3, 13.5, and 13.6).
Biotinylated A8-35 (BA8-35) has also been used in an EM study (Study 13.4). The other tags, all of
them grafted onto A8-35, have been the object of a single validation study or are still under examination (Studies 13.7–13.11).
13.1 Introduction
593
been used as a reactive moiety for further derivatization, but could also be exploited for immobilization
(Lindhoud et al. 2016). Tagged APols make it possible to immobilize MPs onto beads or chips carrying
appropriate groups, namely (i) avidin, streptavidin (SA), or neutravidin for biotin-tagged APols; (ii) a
complementary nucleic acid for ODN-tagged ones; (iii) immobilized Ni
2+ or Co
2+ ions for
polyhistidine and distributed imidazole tags; and (iv) gold or silver surfaces for thiomorpholine and
sulfhydryl ones.
Amphipol-Mediated Immobilization of Membrane Proteins and Its Applications
(# 2018 by Francis Haraux)
A marked advantage of APols is that, because each protein binds several molecules of it,
multiple functions can be associated to the same protein by the simple device of mixing APols. For
instance, a tagged APol can be mixed with a fluorescent one, so as to easily visualize the immobilization process. Seven fluorescent APols (FAPols) derived from either A8-35 or A8-75 have been
synthesized and characterized to date (see Chap. 4, § 4.4, Table 4.5), whose excitation bands range
from 290 to 651 nm and emission bands from 310 to 668 nm (see Chap. 8, Fig. 8.9). Thiolated SMA
has been used to bind Alexa Fluor 488 (Lindhoud et al. 2016). This broad panel of spectral
characteristics greatly facilitates the choice of adequate experimental conditions, as well as
multiplexing.
Figure 13.1 illustrates schematically the basic principle of using tagged APols for immobilizing
MPs onto solid supports. In § 13.2, we will review data pertaining to the use of biotinylated APols for
MP immobilization, in § 13.3 the current state of development of other tagged APols. In § 13.4, a few
other validated or potential applications of tagged APols will be evoked. Table 13.1 presents an
overview of publications that have resorted to the use of tagged APols. Biotin-mediated MP immobilization has been used in five studies using three different APols (Studies 13.1–13.3, 13.5, and 13.6).
Biotinylated A8-35 (BA8-35) has also been used in an EM study (Study 13.4). The other tags, all of
them grafted onto A8-35, have been the object of a single validation study or are still under examination (Studies 13.7–13.11).
13.1 Introduction
593