3. 7.5–750 μM ATP (see Note 7).
4. 0 or 400 nM dsDNA oligonucleotide (see Note 8).
5. 5 μM BSA.
The assay is run in 20 μl volume reactions in Corning
384 low-volume plates (see Note 9) in a BMG CLARIOstar plate
reader. Stock solutions of the reaction components are prepared at
two or fourfold the final concentration, adding 10 or 5 μL, respectively, to the wells. The enzyme stock (Chd1) contains 20 μM BSA
to stabilize the enzyme at low concentrations.
Calibration
Calibration of the MDCC-PBP signal is best performed at the
beginning of the experiments (see Fig. 3). Since the fluorescence
signal of the calibration run does not change over time, it can be
used to set up the optics in the plate reader protocol. When the
assay is run for the first time, the P i contamination in the ATP stock
solution is ideally also tested.
1. Set the temperature of the plate reader to 25
C.
2. Set up the method in the plate reader software:
(a) Use fluorescence intensity—plate mode.
(b) Choose plate type: Corning 384 low-volume flat bottom.
(c) Excitation wavelength: 430–8 (430 nm with 8 nm slit
width).
(d) Dichroic: automatic (447.5 nm).
(e) Emission wavelength: 465–8 (465 nm with 8 nm slit
width).
(f) Select top optics, settling time 0.2 s, no. of flashes
40, cycle time 10 s, number of cycles 30.
(g) Shaking frequency 500 rpm, mode double orbital, time
15 s before first cycle.
3. Prepare 60 μM MDCC-PBP solution in reaction buffer. Make
enough solution for the calibration and the experiments you
want to run.
4. Prepare 20 μM BSA solution.
5. In 8-strip PCR tubes, prepare a concentration series of P i
solutions (0, 1, 2, 4, 6, 8, 10, 12 μM) from the 10.53 mM P i
standard stock solution (and, optional, 2 mM ATP solution to
test for P i ).
6. In a 384-well plate, pipette 5 μl MDCC-PBP solution into
10 wells and add 5 μl BSA.
7. With a multichannel pipette, add 10 μl P i standard solution
(and ATP test solution) to the wells, mix by pipetting up and
down a couple of times, and place in the plate reader.
300
Simone Kunzelmann
4. 0 or 400 nM dsDNA oligonucleotide (see Note 8).
5. 5 μM BSA.
The assay is run in 20 μl volume reactions in Corning
384 low-volume plates (see Note 9) in a BMG CLARIOstar plate
reader. Stock solutions of the reaction components are prepared at
two or fourfold the final concentration, adding 10 or 5 μL, respectively, to the wells. The enzyme stock (Chd1) contains 20 μM BSA
to stabilize the enzyme at low concentrations.
Calibration
Calibration of the MDCC-PBP signal is best performed at the
beginning of the experiments (see Fig. 3). Since the fluorescence
signal of the calibration run does not change over time, it can be
used to set up the optics in the plate reader protocol. When the
assay is run for the first time, the P i contamination in the ATP stock
solution is ideally also tested.
1. Set the temperature of the plate reader to 25
C.
2. Set up the method in the plate reader software:
(a) Use fluorescence intensity—plate mode.
(b) Choose plate type: Corning 384 low-volume flat bottom.
(c) Excitation wavelength: 430–8 (430 nm with 8 nm slit
width).
(d) Dichroic: automatic (447.5 nm).
(e) Emission wavelength: 465–8 (465 nm with 8 nm slit
width).
(f) Select top optics, settling time 0.2 s, no. of flashes
40, cycle time 10 s, number of cycles 30.
(g) Shaking frequency 500 rpm, mode double orbital, time
15 s before first cycle.
3. Prepare 60 μM MDCC-PBP solution in reaction buffer. Make
enough solution for the calibration and the experiments you
want to run.
4. Prepare 20 μM BSA solution.
5. In 8-strip PCR tubes, prepare a concentration series of P i
solutions (0, 1, 2, 4, 6, 8, 10, 12 μM) from the 10.53 mM P i
standard stock solution (and, optional, 2 mM ATP solution to
test for P i ).
6. In a 384-well plate, pipette 5 μl MDCC-PBP solution into
10 wells and add 5 μl BSA.
7. With a multichannel pipette, add 10 μl P i standard solution
(and ATP test solution) to the wells, mix by pipetting up and
down a couple of times, and place in the plate reader.
300
Simone Kunzelmann