1. The lipid capturing surface L1 sensor chip is used for the
immobilization of the vesicles (Fig. 4) prepared as explained
under Subheading 3.4. The L1 sensor chip is equilibrated to
room temperature for 15–30 min before being inserted into
the BIAcore instrument.
2. Insert the L1 sensor chip and prime the instrument with the
BIAcore Running Buffer (25 mM HEPES, pH 7.4,
150 mM NaCl).
3. Start by washing the surfaces that will be used for the vesicle
immobilization with 20 μL of BIAcore detergent solution A at
a flow rate of 5 μL/min, followed by 20 μL of BIAcore detergent solution B at a flow rate of 5 μL/min.
4. Start a new cycle running at a flow rate of 10 μL/min and select
only the flow cell that will be used for immobilization of lipids.
The surface will be conditioned with a 10 μL injection of
BIAcore detergent solution A.
5. Switch the flow rate to 5 μL/min and inject the preparation of
vesicles (Fig. 4). The amount of liposomes attached on the L1
chip will depend on the concentration of liposomes injected
over the chip and the charge of the liposomes (see Note 16).
6. Inject the LUVs till a response of 4000 resonance units (RUs)
is reached.
7. Wash the surface with 20 μL of BIAcore liposomes wash solution at a flow rate of 100 μL/min to remove loosely bound
liposomes.
8. Note the total RUs immobilized; the surface is ready to
be used!
9. If necessary, switch to another channel and proceed with
another immobilization.
3.6 Start
an Experiment
in BIAcore
with an L1 Chip
Single-cycle kinetics (SCK) is recommended for the study of PDZ
domain–liposome interactions. This approach is always valuable
when the surface is difficult to regenerate accurately. The analyte
is injected sequentially in a single cycle, with no regeneration step
between injections (see Note 17) (Fig. 5a).
A predefined method with BIAcore T200 is available “command Single cycle kinetics” (5 sample concentrations per cycle). It is
not possible to modify the predefined variables including the number of analyte injections. Yet other parameters such as flow rate or
association time can be adapted to the experiment.
The following settings are recommended for the measurement
of PDZ domain–lipid interactions.
1. The analyte will be injected at a flow rate of 30 μL/min in
BIAcore running buffer.
PDZ-Peptide and PDZ-Lipid Interactions by SPR
83
immobilization of the vesicles (Fig. 4) prepared as explained
under Subheading 3.4. The L1 sensor chip is equilibrated to
room temperature for 15–30 min before being inserted into
the BIAcore instrument.
2. Insert the L1 sensor chip and prime the instrument with the
BIAcore Running Buffer (25 mM HEPES, pH 7.4,
150 mM NaCl).
3. Start by washing the surfaces that will be used for the vesicle
immobilization with 20 μL of BIAcore detergent solution A at
a flow rate of 5 μL/min, followed by 20 μL of BIAcore detergent solution B at a flow rate of 5 μL/min.
4. Start a new cycle running at a flow rate of 10 μL/min and select
only the flow cell that will be used for immobilization of lipids.
The surface will be conditioned with a 10 μL injection of
BIAcore detergent solution A.
5. Switch the flow rate to 5 μL/min and inject the preparation of
vesicles (Fig. 4). The amount of liposomes attached on the L1
chip will depend on the concentration of liposomes injected
over the chip and the charge of the liposomes (see Note 16).
6. Inject the LUVs till a response of 4000 resonance units (RUs)
is reached.
7. Wash the surface with 20 μL of BIAcore liposomes wash solution at a flow rate of 100 μL/min to remove loosely bound
liposomes.
8. Note the total RUs immobilized; the surface is ready to
be used!
9. If necessary, switch to another channel and proceed with
another immobilization.
3.6 Start
an Experiment
in BIAcore
with an L1 Chip
Single-cycle kinetics (SCK) is recommended for the study of PDZ
domain–liposome interactions. This approach is always valuable
when the surface is difficult to regenerate accurately. The analyte
is injected sequentially in a single cycle, with no regeneration step
between injections (see Note 17) (Fig. 5a).
A predefined method with BIAcore T200 is available “command Single cycle kinetics” (5 sample concentrations per cycle). It is
not possible to modify the predefined variables including the number of analyte injections. Yet other parameters such as flow rate or
association time can be adapted to the experiment.
The following settings are recommended for the measurement
of PDZ domain–lipid interactions.
1. The analyte will be injected at a flow rate of 30 μL/min in
BIAcore running buffer.
PDZ-Peptide and PDZ-Lipid Interactions by SPR
83