13.5.2 Preparation of Samples
Note that it is important to exclude compounds that affect the refractive index of the solutions (e.g.
glycerol). All samples and buffers should be filtered on 0.22-μm filters or centrifuged 10 min at
10,000 Â g.
• Trap the protein in BAPol as described in Chap. 5, Protocol 5.1. The final protein concentration to use for a good signal depends on the protein under study. For example, in the case of
BR, the protein concentration should be ~0.03 gÁL
À1 . Typical quantities required for the
experiments are 0.06–0.1 μg of total protein.
• Prepare the solutions of antibodies (a non-specific antibody and a specific one for negative
and positive controls, respectively), both solutions at two different concentrations (0.055 and
0.01 gÁL
À1 ). Typical quantities required for the experiments are 6–10 μg of total protein. Note
that these quantities are indicative and should be optimized depending on sample purity.
• Prepare the running buffer, HBS-N, which contains 10 mM HEPES pH 7.4, 150 mM NaCl.
Note that the dextran matrix and the APols are both negatively charged, and so the ionic
strength of buffer can be optimized for an optimal signal with minimal repulsion.
• Prepare the surface regeneration solution (1 M NaCl, 50 mM NaOH).
13.5.3 Measurements
The signal observed during an SPR experiment is proportional to the refractive index, which itself is
proportional to the mass of molecules with the same refractive index. All proteins have approximately
the same refractive index, and the empirical conversion factor (1 RU % 1 pgÁmm
À2 ) can be used for
estimating the mass bound to the surface (Stenberg et al. 1991).
• Follow the instructions of the manufacturer for conditioning the sensor surface.
• After obtaining a stable baseline, immobilize the proteins onto the sensor surface, which
becomes the sample flow cell. For this step, start a new cycle using only the second flow cell
and inject the sample. In the case of qualitative analysis, it is recommended to try to saturate
the surface with the MP/BAPol complexes so as to obtain the highest possible response from
antibody binding in the next step. This can be achieved by repeating consecutive injections
until stabilization of the signal (Note: for a flow rate of 10 μLÁmin
À1 , use consecutive 60-s
injections of MP/BAPol complexes at 0.03 gÁL
À1 to saturate the surface). In the case of
kinetic studies, it is usually preferable to limit the density of immobilized ligand to avoid
diffusion-limited data, analyte depletion, and analyte rebinding.
• Prepare the reference flow cell (the first flow cell), which can either be left unmodified or be
modified with either BAPol alone or a different MP trapped in BAPol.
• Stop the manual run after obtaining a stable signal in both cells.
• Measure the binding of the analyte, for instance, that of an antibody raised against the MP
under study. It is recommended to use two different concentrations of antibodies (0.05 gÁL
À1
for the first injection and 0.01 gÁL
À1 for the second one) and one regeneration solution in each
cycle. The solutions will be added according to the following series of the cycles:
– Cycle 1: blank cycle with running buffer
– Cycle 2: cycle with a non-specific antibody to be used for negative control
– Cycle 3: cycle with a specific antibody raised against the MP under study
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13 Amphipol-Mediated Immobilization of Membrane Proteins and Its Applications
Note that it is important to exclude compounds that affect the refractive index of the solutions (e.g.
glycerol). All samples and buffers should be filtered on 0.22-μm filters or centrifuged 10 min at
10,000 Â g.
• Trap the protein in BAPol as described in Chap. 5, Protocol 5.1. The final protein concentration to use for a good signal depends on the protein under study. For example, in the case of
BR, the protein concentration should be ~0.03 gÁL
À1 . Typical quantities required for the
experiments are 0.06–0.1 μg of total protein.
• Prepare the solutions of antibodies (a non-specific antibody and a specific one for negative
and positive controls, respectively), both solutions at two different concentrations (0.055 and
0.01 gÁL
À1 ). Typical quantities required for the experiments are 6–10 μg of total protein. Note
that these quantities are indicative and should be optimized depending on sample purity.
• Prepare the running buffer, HBS-N, which contains 10 mM HEPES pH 7.4, 150 mM NaCl.
Note that the dextran matrix and the APols are both negatively charged, and so the ionic
strength of buffer can be optimized for an optimal signal with minimal repulsion.
• Prepare the surface regeneration solution (1 M NaCl, 50 mM NaOH).
13.5.3 Measurements
The signal observed during an SPR experiment is proportional to the refractive index, which itself is
proportional to the mass of molecules with the same refractive index. All proteins have approximately
the same refractive index, and the empirical conversion factor (1 RU % 1 pgÁmm
À2 ) can be used for
estimating the mass bound to the surface (Stenberg et al. 1991).
• Follow the instructions of the manufacturer for conditioning the sensor surface.
• After obtaining a stable baseline, immobilize the proteins onto the sensor surface, which
becomes the sample flow cell. For this step, start a new cycle using only the second flow cell
and inject the sample. In the case of qualitative analysis, it is recommended to try to saturate
the surface with the MP/BAPol complexes so as to obtain the highest possible response from
antibody binding in the next step. This can be achieved by repeating consecutive injections
until stabilization of the signal (Note: for a flow rate of 10 μLÁmin
À1 , use consecutive 60-s
injections of MP/BAPol complexes at 0.03 gÁL
À1 to saturate the surface). In the case of
kinetic studies, it is usually preferable to limit the density of immobilized ligand to avoid
diffusion-limited data, analyte depletion, and analyte rebinding.
• Prepare the reference flow cell (the first flow cell), which can either be left unmodified or be
modified with either BAPol alone or a different MP trapped in BAPol.
• Stop the manual run after obtaining a stable signal in both cells.
• Measure the binding of the analyte, for instance, that of an antibody raised against the MP
under study. It is recommended to use two different concentrations of antibodies (0.05 gÁL
À1
for the first injection and 0.01 gÁL
À1 for the second one) and one regeneration solution in each
cycle. The solutions will be added according to the following series of the cycles:
– Cycle 1: blank cycle with running buffer
– Cycle 2: cycle with a non-specific antibody to be used for negative control
– Cycle 3: cycle with a specific antibody raised against the MP under study
618
13 Amphipol-Mediated Immobilization of Membrane Proteins and Its Applications