5. The surface is regenerated through 1 pulse of 30 μL of BIAcore
regeneration solution at a flow rate of 30 μL/min (Fig. 2e).
This step will remove the analyte from the surface whereas the
ligand will stay intact. In order to reuse the chip, it is important
to obtain a baseline similar to the one observed at the beginning of the experiment (Fig. 2a, f) (see Note 12).
3.3 Calculate Steady
State Affinity
in Multicycle Kinetics
Analysis
The analysis of steady-state binding data can be applied to 1:1
binding modes (such as in the case of most PDZ domain–peptide
interactions) and it can be performed with the BIAevaluation software provided with the instrument (command “Kinetics/Affinity”)
(Fig. 3).
3.4 Preparation
of Large Unilamellar
Vesicles (LUVs) by
Extrusion
1. Mix the lipids from the stock solutions at the relevant molar
ratio.
2. Dry the lipid mixture under a stream of nitrogen to avoid lipid
oxidation. The lipid films will be kept under high vacuum for at
least 2 h in order to discard residual organic solvent. If the
dried lipids are not used immediately, seal the tube with parafilm and store it at À20
C for not more than 1 week.
3. Hydrate the lipid films by addition of BIAcore Running Buffer
to generate a suspension containing 1.5 mM of total phospholipids. Vortex thoroughly to emulsify the lipid mixture, and use
also a bath sonicator to help lipid suspension.
4. Subject the lipid suspension to 8 rounds of freezing (liquid N 2 )
and thawing (bath set at 40
C). This step will increase the
efficiency of entrapment of water-soluble compounds.
5. Load the sample into one of the gastight syringes and place the
syringe at one end of the extruder (see Note 13). The liposome
preparation should be extruded through 0.1 μm polycarbonate
membranes (see Note 14). A minimum of eleven passes
through the membrane is recommended for most lipids (see
Note 15).
6. You can store the solution containing the liposomes/large
unilamellar vesicles (LUVs) at 4
C. The stock of LUVs is
usually stable for no more than 3–4 days at 4
C.
7. Do not forget to clean the extruder with BIAcore detergent
solution A after use, then rinse it thoroughly with deionized
water, and dry it before storage.
3.5 Immobilizing
LUVs on BIAcore L1
Sensor Chips
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. Set the temperature to
25
C.
82
Pascale Zimmermann and Antonio Luis Egea-Jimenez
regeneration solution at a flow rate of 30 μL/min (Fig. 2e).
This step will remove the analyte from the surface whereas the
ligand will stay intact. In order to reuse the chip, it is important
to obtain a baseline similar to the one observed at the beginning of the experiment (Fig. 2a, f) (see Note 12).
3.3 Calculate Steady
State Affinity
in Multicycle Kinetics
Analysis
The analysis of steady-state binding data can be applied to 1:1
binding modes (such as in the case of most PDZ domain–peptide
interactions) and it can be performed with the BIAevaluation software provided with the instrument (command “Kinetics/Affinity”)
(Fig. 3).
3.4 Preparation
of Large Unilamellar
Vesicles (LUVs) by
Extrusion
1. Mix the lipids from the stock solutions at the relevant molar
ratio.
2. Dry the lipid mixture under a stream of nitrogen to avoid lipid
oxidation. The lipid films will be kept under high vacuum for at
least 2 h in order to discard residual organic solvent. If the
dried lipids are not used immediately, seal the tube with parafilm and store it at À20
C for not more than 1 week.
3. Hydrate the lipid films by addition of BIAcore Running Buffer
to generate a suspension containing 1.5 mM of total phospholipids. Vortex thoroughly to emulsify the lipid mixture, and use
also a bath sonicator to help lipid suspension.
4. Subject the lipid suspension to 8 rounds of freezing (liquid N 2 )
and thawing (bath set at 40
C). This step will increase the
efficiency of entrapment of water-soluble compounds.
5. Load the sample into one of the gastight syringes and place the
syringe at one end of the extruder (see Note 13). The liposome
preparation should be extruded through 0.1 μm polycarbonate
membranes (see Note 14). A minimum of eleven passes
through the membrane is recommended for most lipids (see
Note 15).
6. You can store the solution containing the liposomes/large
unilamellar vesicles (LUVs) at 4
C. The stock of LUVs is
usually stable for no more than 3–4 days at 4
C.
7. Do not forget to clean the extruder with BIAcore detergent
solution A after use, then rinse it thoroughly with deionized
water, and dry it before storage.
3.5 Immobilizing
LUVs on BIAcore L1
Sensor Chips
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. Set the temperature to
25
C.
82
Pascale Zimmermann and Antonio Luis Egea-Jimenez