fluorescence emission monitored using a 320 nm cutoff (longpass) filter, which ensures that scattered excitation light does
not reach the detector. However, the exact choice of excitation
wavelength and emission filter will be dictated by the fluorescent probe in the experimental system, for example, in the case
of extrinsic probes that are excited and emit at longer wavelengths. It can also be very useful to check the effects of using
different emission filters. For example, if there are two or more
Trps involved in the binding, the changes in fluorescence signal
upon binding might counteract each other (one increasing and
one decreasing in fluorescence upon binding). Therefore it is
sometimes better to use a bandpass (interference) filter, which
transmits light centered around 330 nm with defined upper
and lower limits (Æ25 nm), or use a long-pass filter transmitting above 360 nm. These options can be determined empirically or with reference to equilibrium measurements (see Note
5). If an intermediate accumulates during the binding, its
fluorescence might also be better captured with these alternative emission filters, resulting in larger amplitudes for one or
both kinetic phases.
4. Set the acquisition (“push”) volume using the threaded backstop adjuster that is located under the stop syringe on Applied
Photophysics SX instruments. Binding experiments are usually
performed with a 1:1 mixing using 2500 μL sample syringes,
allowing rather small volumes (e.g., 100 + 100 μL). In folding
studies, it is common to use asymmetric mixing and larger drive
volumes should then be used to ensure proper mixing (e.g.,
30 + 300 μL). The volume must not affect the observed
kinetics and this should be checked systematically during initial
experiments. To do this, test the reaction at different push
volumes. As long as a decrease in volume does not affect the
rate and amplitude of the kinetic transient the lower volume
can be safely used.
5. Check the integrity of the fluidic system carefully before
making stopped-flow measurements as any leaks will generate
a level of continued flow in the observation cell that may appear
as an extra kinetic phase. Begin every experimental session by a
1–2 s acquisition using “pressure hold” on the sample syringes.
(Typically, a pressure of 3 bar is used in symmetrical mixing
experiments.) Monitor the sample syringes carefully; they
should stand completely still while the pressure is held on and
slightly bounce back when the pressure is released. If there is a
leakage, every connection from the stop syringe all the way
back to the sample syringes must be checked to locate the
leak. If the leak is in one of the valves then it is likely that it
must be replaced with a new one. Leaking tubing connections
can usually be retightened. After prolonged use or work at low
112
Elin Karlsson and Per Jemth
not reach the detector. However, the exact choice of excitation
wavelength and emission filter will be dictated by the fluorescent probe in the experimental system, for example, in the case
of extrinsic probes that are excited and emit at longer wavelengths. It can also be very useful to check the effects of using
different emission filters. For example, if there are two or more
Trps involved in the binding, the changes in fluorescence signal
upon binding might counteract each other (one increasing and
one decreasing in fluorescence upon binding). Therefore it is
sometimes better to use a bandpass (interference) filter, which
transmits light centered around 330 nm with defined upper
and lower limits (Æ25 nm), or use a long-pass filter transmitting above 360 nm. These options can be determined empirically or with reference to equilibrium measurements (see Note
5). If an intermediate accumulates during the binding, its
fluorescence might also be better captured with these alternative emission filters, resulting in larger amplitudes for one or
both kinetic phases.
4. Set the acquisition (“push”) volume using the threaded backstop adjuster that is located under the stop syringe on Applied
Photophysics SX instruments. Binding experiments are usually
performed with a 1:1 mixing using 2500 μL sample syringes,
allowing rather small volumes (e.g., 100 + 100 μL). In folding
studies, it is common to use asymmetric mixing and larger drive
volumes should then be used to ensure proper mixing (e.g.,
30 + 300 μL). The volume must not affect the observed
kinetics and this should be checked systematically during initial
experiments. To do this, test the reaction at different push
volumes. As long as a decrease in volume does not affect the
rate and amplitude of the kinetic transient the lower volume
can be safely used.
5. Check the integrity of the fluidic system carefully before
making stopped-flow measurements as any leaks will generate
a level of continued flow in the observation cell that may appear
as an extra kinetic phase. Begin every experimental session by a
1–2 s acquisition using “pressure hold” on the sample syringes.
(Typically, a pressure of 3 bar is used in symmetrical mixing
experiments.) Monitor the sample syringes carefully; they
should stand completely still while the pressure is held on and
slightly bounce back when the pressure is released. If there is a
leakage, every connection from the stop syringe all the way
back to the sample syringes must be checked to locate the
leak. If the leak is in one of the valves then it is likely that it
must be replaced with a new one. Leaking tubing connections
can usually be retightened. After prolonged use or work at low
112
Elin Karlsson and Per Jemth