(Fig.13.3A). Responses to protein-specific antibodies were typically 10Â higher or more than those to
non-specific ones (Fig. 13.3B). Comparative antibody-binding experiments were carried out with
proteins trapped with either BA8-35 or plain A8-35. Higher signals were observed with BA8-35trapped proteins, with desorption kinetics compatible with the washing away of antibodies, whereas
the signal observed when using ungrafted A8-35 was much weaker. This confirms that most, if not all,
of the response is due to the binding of antibodies to MPs immobilized via the biotin/SA interaction
rather than via non-specific interactions with the support. Taken together, these experiments establish
(i) that each of the four MPs tested can be immobilized onto SA-coated surfaces via the biotinylated
APol and (ii) that, once bound, each of them can be specifically recognized by antibodies directed
against it.
Fig. 13.2 Immobilization of biotinylated A8-35 (BA8-35) and of membrane protein/BA8-35 complexes
onto streptavidin (SA)-coated chips followed by surface plasmon resonance (SPR). (A) Thirty microliters
of a solution of BA8-35 (black line) or of non-biotinylated A8-35 (gray line) were flushed over an
SA-coated sensor chip (SA chip) at a concentration of 0.3 gÁL
À1 in 100 mM NaCl, 5 mM sodium
phosphate buffer (pH 7.4), 1 mM EDTA (NaPh buffer). SPR signals were recorded at 25
C in a Biacore
2000 instrument. (B) Fifty microliters of a solution of cytochrome bc 1 complex trapped either in BA8-35
(black line) or in non-biotinylated A8-35 (gray line) were flushed over an SA chip at a protein concentration of 2 μM in NaPh buffer. (C) Fifty microliters of solutions of tOmpA (thin black line), bacteriorhodopsin (BR; thin gray line), cytochrome b 6 f (dashed thin black line), or cytochrome bc 1 (thick black line), each
of them trapped in BA8-35, were flushed over various flow cells of two SA chips at a concentration of
0.3 gÁL
À1 BA8-35. The cytochrome b 6 f sensorgram, which was recorded in a different session, has been
normalized by using as a reference the response to cytochrome bc 1 , which was measured each time.
Experimental conditions differed in A, B, and C (in particular, two distinct BA8-35 batches were used,
resulting in different binding capacities), so that sensorgrams shown in different panels cannot be directly
compared (From Charvolin et al. 2009. # 2009 National Academy of Sciences, USA).
596
13 Amphipol-Mediated Immobilization of Membrane Proteins and Its Applications
non-specific ones (Fig. 13.3B). Comparative antibody-binding experiments were carried out with
proteins trapped with either BA8-35 or plain A8-35. Higher signals were observed with BA8-35trapped proteins, with desorption kinetics compatible with the washing away of antibodies, whereas
the signal observed when using ungrafted A8-35 was much weaker. This confirms that most, if not all,
of the response is due to the binding of antibodies to MPs immobilized via the biotin/SA interaction
rather than via non-specific interactions with the support. Taken together, these experiments establish
(i) that each of the four MPs tested can be immobilized onto SA-coated surfaces via the biotinylated
APol and (ii) that, once bound, each of them can be specifically recognized by antibodies directed
against it.
Fig. 13.2 Immobilization of biotinylated A8-35 (BA8-35) and of membrane protein/BA8-35 complexes
onto streptavidin (SA)-coated chips followed by surface plasmon resonance (SPR). (A) Thirty microliters
of a solution of BA8-35 (black line) or of non-biotinylated A8-35 (gray line) were flushed over an
SA-coated sensor chip (SA chip) at a concentration of 0.3 gÁL
À1 in 100 mM NaCl, 5 mM sodium
phosphate buffer (pH 7.4), 1 mM EDTA (NaPh buffer). SPR signals were recorded at 25
C in a Biacore
2000 instrument. (B) Fifty microliters of a solution of cytochrome bc 1 complex trapped either in BA8-35
(black line) or in non-biotinylated A8-35 (gray line) were flushed over an SA chip at a protein concentration of 2 μM in NaPh buffer. (C) Fifty microliters of solutions of tOmpA (thin black line), bacteriorhodopsin (BR; thin gray line), cytochrome b 6 f (dashed thin black line), or cytochrome bc 1 (thick black line), each
of them trapped in BA8-35, were flushed over various flow cells of two SA chips at a concentration of
0.3 gÁL
À1 BA8-35. The cytochrome b 6 f sensorgram, which was recorded in a different session, has been
normalized by using as a reference the response to cytochrome bc 1 , which was measured each time.
Experimental conditions differed in A, B, and C (in particular, two distinct BA8-35 batches were used,
resulting in different binding capacities), so that sensorgrams shown in different panels cannot be directly
compared (From Charvolin et al. 2009. # 2009 National Academy of Sciences, USA).
596
13 Amphipol-Mediated Immobilization of Membrane Proteins and Its Applications