2018) (see Chap. 12, § 12.3.4.3). Incorporation of MPs into bioelectronic devices is a very active field
(see e.g. Mahyad et al. 2015; Park et al. 2015; Kumar et al. 2016, and references therein), in which
APols, in particular, tagged APols, have probably a part to play (cf. Pérez-Mitta et al. 2016).
13.5 Protocol 13.1. Amphipol-Mediated Immobilization of Membrane
Proteins for Surface Plasmon Resonance Experiments
Surface plasmon resonance (SPR) (see Rich and Myszka 2005) is a label-free technique based on the
propagation of an evanescent wave along a gold-coated surface. It permits the detection of changes of
refractive index in the vicinity of the surface that result from the binding of partners. This approach
requires the immobilization of one of the interacting partners onto the surface. Various affinity tags can
be used for protein immobilization, including polyhistidine tags, which bind to Ni:NTA motifs, affinity
tags recognized by antibodies, and biotin or streptags binding to streptavidin. Functionalized APols
fulfill several criteria for a specific immobilization of functional MPs onto surfaces: (i) they stabilize
MPs, providing a longer lifetime to the isolated protein, (ii) they form with MPs permanent complexes,
which do not dissociate in the absence of free APols; biosensors can thus be used in surfactant-free
buffers, which simplifies their implementation and considerably limits the risk of missing weak or
moderate interactions with ligands, and, finally, (iii) the complexes formed between MPs and
functionalized APols will bind onto the solid surface of the biosensor without the need for any genetic
or chemical modification of the protein (Charvolin et al. 2009).
The protocol described here is for MPs trapped in biotinylated A8-35 (BAPol), but it can be
adapted to MPs trapped in other functionalized APols, such as HistAPol, ImidAPol, or OligAPol. If the
MP possesses a tag, its trapping with unfunctionalized APol, e.g. plain A8-35, will improve its
stability, and the immobilization of the complexes can be carried out following the same approach.
This protocol is adapted for Biacore instruments (GE Healthcare), which are the most widely
distributed commercial instruments. Specific conditions are indicated in the case of a Biacore X100
instrument. The signal can be optimized by adjusting experimental conditions (Karlsson and Fält
1997). For more details about data analysis, see Rich and Myszka (2005). In the following, comments
and tips are in italics and marked with a pointing hand sign ().
13.5.1 Experimental Setup
MP/BAPols complexes (called ligands in Biacore terminology) are immobilized onto the Biacore
sensor chip before addition of the interacting molecules, for instance, antibodies (called analyte in
Biacore terminology), to reveal specific interactions.
Choose the adequate sensor chips. The binding capacity of most Biacore chips is improved by
pre-coupling of a dextran matrix. CM5 chips, which are the most widely used, exist already
premodified for coupling of various proteins. If the immobilization is carried out via BAPols, the SA
sensor chip (CM5 chips premodified with streptavidin) is used. Note that alternatives to SA sensor
chips are the use of a standard CM5 chip, followed by aminocoupling of streptavidin, or the SA
Capture chip, which is regenerable. In the case of charge repulsion between the sample to be
immobilized and the surface, it is possible to improve the detection signal by using commercial
chips with shorter dextran matrices. Note that two flow cells are required for the measurements: the
sample flow cell, where the total signal will result from specific and non-specific binding onto surfaces,
and the reference flow cell, used to evaluate the non-specific binding of proteins onto surfaces.
13.5 Protocol 13.1. Amphipol-Mediated Immobilization of Membrane Proteins for. . .
617
(see e.g. Mahyad et al. 2015; Park et al. 2015; Kumar et al. 2016, and references therein), in which
APols, in particular, tagged APols, have probably a part to play (cf. Pérez-Mitta et al. 2016).
13.5 Protocol 13.1. Amphipol-Mediated Immobilization of Membrane
Proteins for Surface Plasmon Resonance Experiments
Surface plasmon resonance (SPR) (see Rich and Myszka 2005) is a label-free technique based on the
propagation of an evanescent wave along a gold-coated surface. It permits the detection of changes of
refractive index in the vicinity of the surface that result from the binding of partners. This approach
requires the immobilization of one of the interacting partners onto the surface. Various affinity tags can
be used for protein immobilization, including polyhistidine tags, which bind to Ni:NTA motifs, affinity
tags recognized by antibodies, and biotin or streptags binding to streptavidin. Functionalized APols
fulfill several criteria for a specific immobilization of functional MPs onto surfaces: (i) they stabilize
MPs, providing a longer lifetime to the isolated protein, (ii) they form with MPs permanent complexes,
which do not dissociate in the absence of free APols; biosensors can thus be used in surfactant-free
buffers, which simplifies their implementation and considerably limits the risk of missing weak or
moderate interactions with ligands, and, finally, (iii) the complexes formed between MPs and
functionalized APols will bind onto the solid surface of the biosensor without the need for any genetic
or chemical modification of the protein (Charvolin et al. 2009).
The protocol described here is for MPs trapped in biotinylated A8-35 (BAPol), but it can be
adapted to MPs trapped in other functionalized APols, such as HistAPol, ImidAPol, or OligAPol. If the
MP possesses a tag, its trapping with unfunctionalized APol, e.g. plain A8-35, will improve its
stability, and the immobilization of the complexes can be carried out following the same approach.
This protocol is adapted for Biacore instruments (GE Healthcare), which are the most widely
distributed commercial instruments. Specific conditions are indicated in the case of a Biacore X100
instrument. The signal can be optimized by adjusting experimental conditions (Karlsson and Fält
1997). For more details about data analysis, see Rich and Myszka (2005). In the following, comments
and tips are in italics and marked with a pointing hand sign ().
13.5.1 Experimental Setup
MP/BAPols complexes (called ligands in Biacore terminology) are immobilized onto the Biacore
sensor chip before addition of the interacting molecules, for instance, antibodies (called analyte in
Biacore terminology), to reveal specific interactions.
Choose the adequate sensor chips. The binding capacity of most Biacore chips is improved by
pre-coupling of a dextran matrix. CM5 chips, which are the most widely used, exist already
premodified for coupling of various proteins. If the immobilization is carried out via BAPols, the SA
sensor chip (CM5 chips premodified with streptavidin) is used. Note that alternatives to SA sensor
chips are the use of a standard CM5 chip, followed by aminocoupling of streptavidin, or the SA
Capture chip, which is regenerable. In the case of charge repulsion between the sample to be
immobilized and the surface, it is possible to improve the detection signal by using commercial
chips with shorter dextran matrices. Note that two flow cells are required for the measurements: the
sample flow cell, where the total signal will result from specific and non-specific binding onto surfaces,
and the reference flow cell, used to evaluate the non-specific binding of proteins onto surfaces.
13.5 Protocol 13.1. Amphipol-Mediated Immobilization of Membrane Proteins for. . .
617