temperatures, the sample syringes can become worn and prone
to leaking and so may need to be replaced. However, before
this step it is worthwhile to remove the syringe plungers and
boil them for a couple of minutes. The cleaning and expansion
of the plunger tip can make them work without leaking and
extend their useable lifespan.
6. Introduce protein A into one of the sample syringes at 1 μM
final concentration after mixing if using Trp fluorescence (see
Note 6) and buffer into the other syringe. Let the solutions
equilibrate to the set temperature (see Note 7). Make 2–3 drive
shots until the fluorescence signal has stabilized, i.e., the tubing
is filled with the respective solutions. The number of drive shots
necessary to fill the tubing will depend on the push volume and
the length of tubing connecting the syringe to the cell. Record
a measurement of protein A mixed with buffer from the other
drive syringe and check if this gives a constant signal and flat
transient. When measuring over longer acquisitions this may
reveal a slow linear decrease in signal that is due to photobleaching. Adjust the detection gain on the instrument photo
multiplier tube (PMT) such that this signal level is in the
middle of the instrument’s dynamic range (e.g., 5 V for a
0–10 V scale).
7. Replace the buffer solution with protein B at 10 μM final
concentration after mixing, and make 2–3 drives such that the
tubing is filled with protein B all the way to the observation
cell. At this point, you may observe whether the fluorescence
signal decreases or increases upon addition of B. If the signal
increases such that it is close to the PMT upper limit adjust the
gain such that the end point signal is not higher than 80% of
detection maximum. If B contains a fluorescent probe, this
must also be taken into account before conducting an experiment with a series of concentrations of B; the highest concentration of B, and thus highest fluorescence, will then dictate
what PMT voltage can be used. In this respect, remember that
the fluorescence may decrease upon binding but the initial
fluorescence of unbound A and B could potentially be even
higher and saturate the detector. In such a case, set the PMT
voltage so that the extrapolated starting fluorescence is around
80% of detection maximum.
8. Make the first acquisitions with an intermediate time base of 1 s
and look for a transient (see Note 8). Then adjust the time base
according to what you observe in the experiment in terms of
time for the transient (see Note 9) and make further adjustments of the fluorescence signal if necessary. As a rule of
thumb, a kinetic transient should be monitored over 5τ for
optimal curve fitting. Even if you observe a transient that is
completed within 1 s, make a new experiment where you
Kinetics of IDP Binding
113
to leaking and so may need to be replaced. However, before
this step it is worthwhile to remove the syringe plungers and
boil them for a couple of minutes. The cleaning and expansion
of the plunger tip can make them work without leaking and
extend their useable lifespan.
6. Introduce protein A into one of the sample syringes at 1 μM
final concentration after mixing if using Trp fluorescence (see
Note 6) and buffer into the other syringe. Let the solutions
equilibrate to the set temperature (see Note 7). Make 2–3 drive
shots until the fluorescence signal has stabilized, i.e., the tubing
is filled with the respective solutions. The number of drive shots
necessary to fill the tubing will depend on the push volume and
the length of tubing connecting the syringe to the cell. Record
a measurement of protein A mixed with buffer from the other
drive syringe and check if this gives a constant signal and flat
transient. When measuring over longer acquisitions this may
reveal a slow linear decrease in signal that is due to photobleaching. Adjust the detection gain on the instrument photo
multiplier tube (PMT) such that this signal level is in the
middle of the instrument’s dynamic range (e.g., 5 V for a
0–10 V scale).
7. Replace the buffer solution with protein B at 10 μM final
concentration after mixing, and make 2–3 drives such that the
tubing is filled with protein B all the way to the observation
cell. At this point, you may observe whether the fluorescence
signal decreases or increases upon addition of B. If the signal
increases such that it is close to the PMT upper limit adjust the
gain such that the end point signal is not higher than 80% of
detection maximum. If B contains a fluorescent probe, this
must also be taken into account before conducting an experiment with a series of concentrations of B; the highest concentration of B, and thus highest fluorescence, will then dictate
what PMT voltage can be used. In this respect, remember that
the fluorescence may decrease upon binding but the initial
fluorescence of unbound A and B could potentially be even
higher and saturate the detector. In such a case, set the PMT
voltage so that the extrapolated starting fluorescence is around
80% of detection maximum.
8. Make the first acquisitions with an intermediate time base of 1 s
and look for a transient (see Note 8). Then adjust the time base
according to what you observe in the experiment in terms of
time for the transient (see Note 9) and make further adjustments of the fluorescence signal if necessary. As a rule of
thumb, a kinetic transient should be monitored over 5τ for
optimal curve fitting. Even if you observe a transient that is
completed within 1 s, make a new experiment where you
Kinetics of IDP Binding
113