2. The contact time of the analyte with the surface will be around
120 s.
3. At the end of the experiment, wash the cells with 10 μL of
BIAcore detergent solution A at a flow rate of 10 μL/min and
start a new immobilization of LUVs as described under Subheading 3.5 (see Note 18).
4. To determine an apparent K D value, the same principle as
described in Subheading 3.3 can be applied. The response for
each injection will be extracted and fitted considering the injection at time 0 (Fig. 5b). The maximal signals obtained at
equilibrium (Req-max) are plotted against protein concentrations (Fig. 5c).
5. The data analysis can be performed using the BIAevaluation
software provided with the BIAcore T200 instrument. The
affinity constants (association and dissociation constants) can
be calculated from the kinetic measurements.
6. For the study of tripartite complexes on PDZ domain–lipid
membrane interactions see Note 19.
3.7 Study
of Tripartite
Complexes in BIAcore
with a SA Sensor Chip
MCK can be used for the study of the formation of a tripartite
complex when the ligand is immobilized on the chip.
1. Immobilize the biotinylated peptide or lipid of interest as
indicated under Subheading 3.1.
2. Start a new cycle at a flow rate of 30 μL/min in BIAcore
running buffer and verify the optimal equilibration of the
sensor surface (flat baseline).
3. Incubate and inject a fixed concentration of analyte (PDZ
protein) together with the soluble peptide or lipid at a flow
rate of 30 μL/min in BIAcore running buffer.
4. The association and dissociation times together with the regeneration step are the same as described under Subheading 3.2.
5. The concentration of the soluble peptide or lipid is increased in
each injection/cycle whereas the analyte remains constant.
6. Equilibrium responses are plotted against the logarithm of the
peptide (Fig. 6a) or lipid (Fig. 6b) concentration and the data
fitted to the equation log (agonist) versus response.
The Hill coefficient (n H ) is calculated by adjusting the
curve to the Hill–Langmuir equation.
n H < 1 Negative cooperative binding. The PDZ protein is
bound to the immobilized peptide, the presence of another
ligand in solution competes with this PDZ domain–peptide interaction (Fig. 6a).
84
Pascale Zimmermann and Antonio Luis Egea-Jimenez
120 s.
3. At the end of the experiment, wash the cells with 10 μL of
BIAcore detergent solution A at a flow rate of 10 μL/min and
start a new immobilization of LUVs as described under Subheading 3.5 (see Note 18).
4. To determine an apparent K D value, the same principle as
described in Subheading 3.3 can be applied. The response for
each injection will be extracted and fitted considering the injection at time 0 (Fig. 5b). The maximal signals obtained at
equilibrium (Req-max) are plotted against protein concentrations (Fig. 5c).
5. The data analysis can be performed using the BIAevaluation
software provided with the BIAcore T200 instrument. The
affinity constants (association and dissociation constants) can
be calculated from the kinetic measurements.
6. For the study of tripartite complexes on PDZ domain–lipid
membrane interactions see Note 19.
3.7 Study
of Tripartite
Complexes in BIAcore
with a SA Sensor Chip
MCK can be used for the study of the formation of a tripartite
complex when the ligand is immobilized on the chip.
1. Immobilize the biotinylated peptide or lipid of interest as
indicated under Subheading 3.1.
2. Start a new cycle at a flow rate of 30 μL/min in BIAcore
running buffer and verify the optimal equilibration of the
sensor surface (flat baseline).
3. Incubate and inject a fixed concentration of analyte (PDZ
protein) together with the soluble peptide or lipid at a flow
rate of 30 μL/min in BIAcore running buffer.
4. The association and dissociation times together with the regeneration step are the same as described under Subheading 3.2.
5. The concentration of the soluble peptide or lipid is increased in
each injection/cycle whereas the analyte remains constant.
6. Equilibrium responses are plotted against the logarithm of the
peptide (Fig. 6a) or lipid (Fig. 6b) concentration and the data
fitted to the equation log (agonist) versus response.
The Hill coefficient (n H ) is calculated by adjusting the
curve to the Hill–Langmuir equation.
n H < 1 Negative cooperative binding. The PDZ protein is
bound to the immobilized peptide, the presence of another
ligand in solution competes with this PDZ domain–peptide interaction (Fig. 6a).
84
Pascale Zimmermann and Antonio Luis Egea-Jimenez