6. BIAcore liposomes wash solution: 100 mM NaOH. Weight of
0.4 g of NaOH and transfer to a glass beaker. Add about
100 mL of deionized water to the glass beaker. Mix and transfer to a 100 mL graduated cylinder and adjust to 100 mL with
deionized water.
2.4 Chips
1. Sensor chip SA. The streptavidin (SA) sensor chip is used to
immobilize biotinylated molecules.
2. Sensor chip L1. This sensor chip L1 is used to immobilize
reconstituted liposomes.
3 Methods
Carry out all procedures at room temperature unless otherwise
specified.
3.1 Immobilizing
Biotinylated
Compounds
on BIAcore SA
Sensor Chip
For a BIAcore T200 equipment. The buffer solution is filtered
through a 0.22 μm filter and the instrument is cleaned with Desorb
command before docking the sensor chip (see Note 3). Set the
temperature to 25
C.
1. The SA sensor chip is designed to capture biotinylated peptides, immobilizing and using these as ligands (it can also be
used to capture biotinylated lipids although data are less reliable from a biological point of view than with liposomes that
mimic membrane compartments) (see Note 4). SA sensor chip
provides a sensor surface with covalently attached streptavidin.
Since the streptavidin–biotin interaction is very strong, the
capture is essentially irreversible (Fig. 1).
2. SA sensor chip is acclimated to room temperature before being
inserted into the BIAcore instrument (see Note 5).
3. Insert the SA sensor chip and prime with the BIAcore Running
Buffer.
4. The surface of the chip is conditioned with three consecutive
injections of 5 μL of BIAcore Running Buffer at a flow rate of
10 μL/min.
5. Start a new cycle at a flow rate of 10 μL/min and select only the
flow cell that will be used for immobilization of the biotinylated
peptide, previously solubilized in deionized water as described
in Subheading 2.1, (see Note 6).
6. The immobilization level will determine the binding capacity of
the surface and the maximum response (R max ). In general, low
R max is required for kinetic analysis whereas a high R max is
preferable in concentration measurements. The R max value
can be calculated using the following formula:
80
Pascale Zimmermann and Antonio Luis Egea-Jimenez
0.4 g of NaOH and transfer to a glass beaker. Add about
100 mL of deionized water to the glass beaker. Mix and transfer to a 100 mL graduated cylinder and adjust to 100 mL with
deionized water.
2.4 Chips
1. Sensor chip SA. The streptavidin (SA) sensor chip is used to
immobilize biotinylated molecules.
2. Sensor chip L1. This sensor chip L1 is used to immobilize
reconstituted liposomes.
3 Methods
Carry out all procedures at room temperature unless otherwise
specified.
3.1 Immobilizing
Biotinylated
Compounds
on BIAcore SA
Sensor Chip
For a BIAcore T200 equipment. The buffer solution is filtered
through a 0.22 μm filter and the instrument is cleaned with Desorb
command before docking the sensor chip (see Note 3). Set the
temperature to 25
C.
1. The SA sensor chip is designed to capture biotinylated peptides, immobilizing and using these as ligands (it can also be
used to capture biotinylated lipids although data are less reliable from a biological point of view than with liposomes that
mimic membrane compartments) (see Note 4). SA sensor chip
provides a sensor surface with covalently attached streptavidin.
Since the streptavidin–biotin interaction is very strong, the
capture is essentially irreversible (Fig. 1).
2. SA sensor chip is acclimated to room temperature before being
inserted into the BIAcore instrument (see Note 5).
3. Insert the SA sensor chip and prime with the BIAcore Running
Buffer.
4. The surface of the chip is conditioned with three consecutive
injections of 5 μL of BIAcore Running Buffer at a flow rate of
10 μL/min.
5. Start a new cycle at a flow rate of 10 μL/min and select only the
flow cell that will be used for immobilization of the biotinylated
peptide, previously solubilized in deionized water as described
in Subheading 2.1, (see Note 6).
6. The immobilization level will determine the binding capacity of
the surface and the maximum response (R max ). In general, low
R max is required for kinetic analysis whereas a high R max is
preferable in concentration measurements. The R max value
can be calculated using the following formula:
80
Pascale Zimmermann and Antonio Luis Egea-Jimenez