3.2.2 Experimental
Procedure (See Note 11)
1. Ensure the desired long-pass wavelength filters to remove excitation light are inserted in front of the detectors (see Note 12).
2. Turn on the xenon lamp to warm up for 15 min before
performing the first experiment (see Note 13).
3. Drain the 30% ethanol solution from stopped-flow syringes.
4. Wash syringes, mixer, and cuvette with water three times followed by washing with reaction buffer three times each.
5. Set the excitation monochromator to the desired excitation
wavelength and confirm optimal slit width.
6. Prepare syringe A and B reaction mixtures separately (see Note
14).
7. Incubate reaction mixtures at 37
C for 15 min in a water bath
(see Note 15).
8. Centrifuge reactions at 17,000 Â g for 2 min to remove any
precipitate or particles before loading into the stopped-flow (see
Note 16).
9. Draw reaction mixture A up into a 3 mL disposable syringe
using a needle (avoid frothing).
10. Remove needle, insert the syringe into the respective port, and
press slowly the reaction mixture A into stopped-flow syringe A
(avoid capturing an air bubble here). Make sure the stoppedflow is in the load position before injecting reaction mixture
(see Note 17).
11. Repeat steps 7 and 8 for reaction mixture B.
12. Adjust the syringe drive so that the plunger of each stoppedflow syringe is in contact with the motor drive.
13. Switch the stopped-flow into the fire position.
14. Set the duration and number of measurements to be carried
out in the software.
15. Collect data and adjust duration of measurement as needed (see
Note 18). Data should appear similar to that in Fig. 3a.
3.2.3 Data Analysis
1. Fit each recorded time-dependent signal change (referred to as
a fluorescence trace) with an exponential function. Selecting
the correct equation to fit the data is critical and depends on the
reaction components and proposed kinetic mechanism. For a
simple one-step binding mechanism (Eq. 1), a one-exponential
equation (Eq. 8) is fit to the data using TableCurve 2D (Systat
Software) to determine the apparent rate (k app1 ). Here we used
a two-exponential equation (Eq. 9) after initial
one-exponential fits were not describing the data properly
due to an additional kinetic step detected in our experimental
system. For more details on evaluating which equation should
be used to fit the data, see Note 19. In both exponential
280
Harland E. Brandon and Hans-Joachim Wieden
Procedure (See Note 11)
1. Ensure the desired long-pass wavelength filters to remove excitation light are inserted in front of the detectors (see Note 12).
2. Turn on the xenon lamp to warm up for 15 min before
performing the first experiment (see Note 13).
3. Drain the 30% ethanol solution from stopped-flow syringes.
4. Wash syringes, mixer, and cuvette with water three times followed by washing with reaction buffer three times each.
5. Set the excitation monochromator to the desired excitation
wavelength and confirm optimal slit width.
6. Prepare syringe A and B reaction mixtures separately (see Note
14).
7. Incubate reaction mixtures at 37
C for 15 min in a water bath
(see Note 15).
8. Centrifuge reactions at 17,000 Â g for 2 min to remove any
precipitate or particles before loading into the stopped-flow (see
Note 16).
9. Draw reaction mixture A up into a 3 mL disposable syringe
using a needle (avoid frothing).
10. Remove needle, insert the syringe into the respective port, and
press slowly the reaction mixture A into stopped-flow syringe A
(avoid capturing an air bubble here). Make sure the stoppedflow is in the load position before injecting reaction mixture
(see Note 17).
11. Repeat steps 7 and 8 for reaction mixture B.
12. Adjust the syringe drive so that the plunger of each stoppedflow syringe is in contact with the motor drive.
13. Switch the stopped-flow into the fire position.
14. Set the duration and number of measurements to be carried
out in the software.
15. Collect data and adjust duration of measurement as needed (see
Note 18). Data should appear similar to that in Fig. 3a.
3.2.3 Data Analysis
1. Fit each recorded time-dependent signal change (referred to as
a fluorescence trace) with an exponential function. Selecting
the correct equation to fit the data is critical and depends on the
reaction components and proposed kinetic mechanism. For a
simple one-step binding mechanism (Eq. 1), a one-exponential
equation (Eq. 8) is fit to the data using TableCurve 2D (Systat
Software) to determine the apparent rate (k app1 ). Here we used
a two-exponential equation (Eq. 9) after initial
one-exponential fits were not describing the data properly
due to an additional kinetic step detected in our experimental
system. For more details on evaluating which equation should
be used to fit the data, see Note 19. In both exponential
280
Harland E. Brandon and Hans-Joachim Wieden