7. Fill the whole stopped-flow system (drive syringes, mixer and
cuvette, stop syringe, following the instrument manual) with
the P i mop solution and incubate for 20 min.
8. Clean out the system thoroughly with P i -free water.
P i Calibration
1. Prepare 1.8 ml of 2Â MDCC-PBP (20 μM) solution.
2. Prepare a concentration series of 2Â P i standard solutions: 1 ml
of 1, 2, 3, and 4 μM P i .
3. Rinse drive syringes, lines, cuvette, and stop syringe with
buffer.
4. Load the two drive syringes (here C and D) with MDCC-PBP
solution and buffer, respectively, and prime the system (three
pushes with both syringes to completely exchange the solutions
in the two lines and the cuvette).
5. Set the excitation wavelength to 436 nm.
6. Set the photomultiplier voltage, so that the signal is about
10–25% of the maximum to allow detection of an increase in
fluorescence in the presence of P i (around 250–300 V in our
stopped-flow instrument) (see Note 17). Test if you are on the
436 nm Hg line by moving the monochromator wavelength in
1 nm steps in both directions and see where the signal is
maximal. Use this wavelength and readjust photomultiplier
voltage.
7. Record at least three traces at 0 P i , acquiring data for 10 s.
8. Exchange the buffer in syringe D for 1 μM P i solution and
prime: In order to save MDCC-PBP, instead of priming by
pushing both syringes, the PBP syringe (C) is blocked off
(closed valve) and 150 μl of P i solution from the syringe
(D) is pushed through the cuvette manually (in flush mode).
9. Record at least three traces, acquiring data for 10 s. Repeat
steps 8 and 9 for all other P i solutions.
To analyze data:
10. Calculate the average fluorescence signal over time for all P i
concentrations leaving out the initial time points where the
signal is not stable (similar to the steady state experiments in
Fig. 3a).
11. Plot the averaged fluorescence (F) versus P i concentration to
generate the standard curve. Fit a standard curve by linear
regression (see Fig. 3b). The slope gives the fluorescence units
per μM P i (ΔF/Δ [P i ]).
12. P i calibration data are not strictly needed for the analysis of
single-turnover data, but are necessary for the interpretation
of multi-turnover experiments (see Note 18).
Phosphate Biosensor Assays
309
cuvette, stop syringe, following the instrument manual) with
the P i mop solution and incubate for 20 min.
8. Clean out the system thoroughly with P i -free water.
P i Calibration
1. Prepare 1.8 ml of 2Â MDCC-PBP (20 μM) solution.
2. Prepare a concentration series of 2Â P i standard solutions: 1 ml
of 1, 2, 3, and 4 μM P i .
3. Rinse drive syringes, lines, cuvette, and stop syringe with
buffer.
4. Load the two drive syringes (here C and D) with MDCC-PBP
solution and buffer, respectively, and prime the system (three
pushes with both syringes to completely exchange the solutions
in the two lines and the cuvette).
5. Set the excitation wavelength to 436 nm.
6. Set the photomultiplier voltage, so that the signal is about
10–25% of the maximum to allow detection of an increase in
fluorescence in the presence of P i (around 250–300 V in our
stopped-flow instrument) (see Note 17). Test if you are on the
436 nm Hg line by moving the monochromator wavelength in
1 nm steps in both directions and see where the signal is
maximal. Use this wavelength and readjust photomultiplier
voltage.
7. Record at least three traces at 0 P i , acquiring data for 10 s.
8. Exchange the buffer in syringe D for 1 μM P i solution and
prime: In order to save MDCC-PBP, instead of priming by
pushing both syringes, the PBP syringe (C) is blocked off
(closed valve) and 150 μl of P i solution from the syringe
(D) is pushed through the cuvette manually (in flush mode).
9. Record at least three traces, acquiring data for 10 s. Repeat
steps 8 and 9 for all other P i solutions.
To analyze data:
10. Calculate the average fluorescence signal over time for all P i
concentrations leaving out the initial time points where the
signal is not stable (similar to the steady state experiments in
Fig. 3a).
11. Plot the averaged fluorescence (F) versus P i concentration to
generate the standard curve. Fit a standard curve by linear
regression (see Fig. 3b). The slope gives the fluorescence units
per μM P i (ΔF/Δ [P i ]).
12. P i calibration data are not strictly needed for the analysis of
single-turnover data, but are necessary for the interpretation
of multi-turnover experiments (see Note 18).
Phosphate Biosensor Assays
309