7-MEG is dissolved in water at 20 mM and stored at À20
C.
PNPase is dissolved in water at 1000 U/ml, shock-frozen in small
aliquots, and stored at À80
C. Repeated freeze–thaw cycles reduce
the activity of PNPase and should be avoided.
For cleaning instruments and cuvettes, a solution of 200 μM
7-MEG and 1 U/ml PNPase in P i -free buffer is prepared. The P i
mop can also be used to pretreat nucleotide and protein solutions
prior to kinetics experiments, but in this case one has to carefully
choose the PNPase concentration so that the removal of P i by the
mop is much slower than the P i release from the system under
study. This is normally only done for stopped-flow experiments.
2.6 Instrumentation
For steady state kinetic assays:
1. Fluorescence plate reader equipped with monochromators for
excitation and emission light or suitable filter set for MDCC or
tetramethylrhodamine fluorescence, ideally with temperature
control (here we use a CLARIOstar reader from BMG
Labtech).
2. 96- or 384-well plates for fluorescence (black plates), here we
use 384-well plates, low volume, NBS (3820, Corning) (see
Note 4).
For pre-steady state kinetics:
1. Stopped-flow instrument, setup for fluorescence detection. We
use a HiTech SF61 DX2 stopped-flow instrument equipped
with a Xe/Hg lamp (TgK Scientific, UK).
2. Long-pass filter suitable for MDCC or tetramethylrhodamine
fluorescence detection (Schott GG 455 for MDCC-PBP or
Schott OG570 for Rho-PBP, both from UQG Optics).
2.7 Data Fitting
Software
1. For steady state experiments, MARS software (BMG Labtech)
is used to determine initial rates by linear regression (alternatively this can be done using Microsoft Excel, GraphPad Prism,
or other software).
2. Prism 7 (GraphPad) is used to run nonlinear least-square fits
and to prepare all plots.
3. For pre-steady state experiments, Kinetic Studio (HiTech) or
Prism 7 (GraphPad) is used for nonlinear least-square fitting
using exponentials.
4. KinTek Explorer (KinTek Corporation) software [24] is used
for all simulations.
294
Simone Kunzelmann
C.
PNPase is dissolved in water at 1000 U/ml, shock-frozen in small
aliquots, and stored at À80
C. Repeated freeze–thaw cycles reduce
the activity of PNPase and should be avoided.
For cleaning instruments and cuvettes, a solution of 200 μM
7-MEG and 1 U/ml PNPase in P i -free buffer is prepared. The P i
mop can also be used to pretreat nucleotide and protein solutions
prior to kinetics experiments, but in this case one has to carefully
choose the PNPase concentration so that the removal of P i by the
mop is much slower than the P i release from the system under
study. This is normally only done for stopped-flow experiments.
2.6 Instrumentation
For steady state kinetic assays:
1. Fluorescence plate reader equipped with monochromators for
excitation and emission light or suitable filter set for MDCC or
tetramethylrhodamine fluorescence, ideally with temperature
control (here we use a CLARIOstar reader from BMG
Labtech).
2. 96- or 384-well plates for fluorescence (black plates), here we
use 384-well plates, low volume, NBS (3820, Corning) (see
Note 4).
For pre-steady state kinetics:
1. Stopped-flow instrument, setup for fluorescence detection. We
use a HiTech SF61 DX2 stopped-flow instrument equipped
with a Xe/Hg lamp (TgK Scientific, UK).
2. Long-pass filter suitable for MDCC or tetramethylrhodamine
fluorescence detection (Schott GG 455 for MDCC-PBP or
Schott OG570 for Rho-PBP, both from UQG Optics).
2.7 Data Fitting
Software
1. For steady state experiments, MARS software (BMG Labtech)
is used to determine initial rates by linear regression (alternatively this can be done using Microsoft Excel, GraphPad Prism,
or other software).
2. Prism 7 (GraphPad) is used to run nonlinear least-square fits
and to prepare all plots.
3. For pre-steady state experiments, Kinetic Studio (HiTech) or
Prism 7 (GraphPad) is used for nonlinear least-square fitting
using exponentials.
4. KinTek Explorer (KinTek Corporation) software [24] is used
for all simulations.
294
Simone Kunzelmann