probes will require appropriate concentrations of B to form the
complex and the different excitation wavelength and emission
range.
6. In the SX series stopped-flow instrument from Applied Photophysics, the tubing going from the left sample syringe to the
observation cell is longer than the tubing from the right
syringe. It is therefore better to have the non-varied protein
in the left syringe and the protein whose concentration will be
changed in the right syringe, since this will save on required
volumes.
7. In some cases, it is advisable to degas solutions before the
experiment; for example, if experiments are performed at
higher than room temperature, there is an increased risk of
formation of air bubbles during mixing. If solutions are
prepared at room temperature and the experiments are performed at lower temperature then there is little risk for bubble
formation. If pressure hold is used during kinetic acquisitions,
this will also suppress outgassing (but see Note 8). In temperature jump experiments, solutions should always be degassed as
the temperature perturbation is always an increase.
8. There is the option of “pressure hold” in stopped-flow instruments. If pressure hold is chosen, the pressure on the drive
rams (pushing the sample syringes) is maintained during the
whole acquisition. If pressure is not held on, then typically it
will be released after 20–30 ms from triggering acquisition.
Close inspection of this time region may reveal artifacts in the
data due to the pressure release in the system. We therefore
routinely use pressure hold for acquisition times up to 500 ms
since the initial 50–100 ms of data is where the majority of
signal change is occurring. However, pressure hold is not
advisable for longer acquisitions (>1 s) as the repeated pressurization over longer time courses may eventually generate a leak
in the fluidic system and premature wear in the valves.
9. Having a measurement of the equilibrium, K d can help anticipate the range of k obs values that may be expected. As a rule of
thumb, if Trp fluorescence is used, preliminary experiments
should be performed with 1 μM A and 10 μM B (final concentrations after mixing). Generally, k on values are in the
range 10
6
–10
8 M
À1 s
À1 . Thus, if the measured K d is 1 μM,
k off will be 1–100 s
À1 since K d ¼ k on /k off . In this case, k obs
will then be 10 μM Â k on + k off (Eq. 2), i.e., between 11 and
1100 s
À1
. If no transient is observed and the fluorescence
level is that measured for the AB complex, then the next
step would be to reduce [B] to 5 μM and/or reduce the
Kinetics of IDP Binding
129
complex and the different excitation wavelength and emission
range.
6. In the SX series stopped-flow instrument from Applied Photophysics, the tubing going from the left sample syringe to the
observation cell is longer than the tubing from the right
syringe. It is therefore better to have the non-varied protein
in the left syringe and the protein whose concentration will be
changed in the right syringe, since this will save on required
volumes.
7. In some cases, it is advisable to degas solutions before the
experiment; for example, if experiments are performed at
higher than room temperature, there is an increased risk of
formation of air bubbles during mixing. If solutions are
prepared at room temperature and the experiments are performed at lower temperature then there is little risk for bubble
formation. If pressure hold is used during kinetic acquisitions,
this will also suppress outgassing (but see Note 8). In temperature jump experiments, solutions should always be degassed as
the temperature perturbation is always an increase.
8. There is the option of “pressure hold” in stopped-flow instruments. If pressure hold is chosen, the pressure on the drive
rams (pushing the sample syringes) is maintained during the
whole acquisition. If pressure is not held on, then typically it
will be released after 20–30 ms from triggering acquisition.
Close inspection of this time region may reveal artifacts in the
data due to the pressure release in the system. We therefore
routinely use pressure hold for acquisition times up to 500 ms
since the initial 50–100 ms of data is where the majority of
signal change is occurring. However, pressure hold is not
advisable for longer acquisitions (>1 s) as the repeated pressurization over longer time courses may eventually generate a leak
in the fluidic system and premature wear in the valves.
9. Having a measurement of the equilibrium, K d can help anticipate the range of k obs values that may be expected. As a rule of
thumb, if Trp fluorescence is used, preliminary experiments
should be performed with 1 μM A and 10 μM B (final concentrations after mixing). Generally, k on values are in the
range 10
6
–10
8 M
À1 s
À1 . Thus, if the measured K d is 1 μM,
k off will be 1–100 s
À1 since K d ¼ k on /k off . In this case, k obs
will then be 10 μM Â k on + k off (Eq. 2), i.e., between 11 and
1100 s
À1
. If no transient is observed and the fluorescence
level is that measured for the AB complex, then the next
step would be to reduce [B] to 5 μM and/or reduce the
Kinetics of IDP Binding
129