2. How critical the P i background is for an assay depends largely
on the sensitivity needed. For measurements of several micromolar P i, and using >15 μM PBP, even 2 μM P i contamination
can be tolerated. Keeping the P i concentration under 2 μM is
easily achieved when following the instructions given here. The
use of a P i mop is generally not necessary. For very sensitive
assays where hundreds of nM or even less P i need to be
measured and lower PBP concentrations are used more care
has to be taken in choosing and preparing the buffers and
nucleotide solutions. It might also be necessary to include the
P i mop (see Subheading 2.5) in some of the reagents.
3. Not all PNPase variants accept 7-methylguanosine as a substrate. The given source works well in our experiments.
4. Multi-well plates, in particular those with a nonproteinbinding coating, can also have significant levels of P i contamination. For the assay we present here this is not a problem.
However, if needed one can reduce P i contamination by rinsing
the plates with P i -free water or even pretreat them with P i mop.
5. When running assays at near ambient temperature, it is not
necessary to take special precautions to minimize temperature
equilibration times. Buffers and diluted solutions are just kept
at room temperature (not on ice) before pipetting into the
plates. However, when measuring at higher temperatures,
e.g., 30
C or 37
C, solutions (and plates) have to be preequilibrated before starting the experiments or temperature
will increase during the initial minutes of the reaction. At
higher temperatures in nonsealed plates, evaporation can also
be a problem, particularly when small volumes and long incubation times are used.
6. All experiments described here were performed with an N- and
C-terminally truncated construct of S. cerevisiae Chd1 (residues 118-1274) [29]. The protein was expressed from pET49b
in E. coli and purified by GSH-affinity, Ni-NTA affinity, and size
exclusion chromatography.
7. Since the ATPase K m of Chd1 is relatively high (>100 μM) and
therefore high ATP concentrations are needed, we used purified ATP for the experiments.
8. A 30 bp double-stranded DNA was generated by annealing
two complementary oligonucleotides with the following
sequences: 5
0 -GTA ACC ACG CGT ATA GAA ACG GGA
CTC ATT-3
0 and the reverse complement.
9. The assay also works well with down to 20 μl in Corning
384 plates with square wells (not small volume). If enough
material is available, the assay can be more robust and accurate
with larger volumes (50–200 μl) and/or 96-well plates.
Phosphate Biosensor Assays
313
on the sensitivity needed. For measurements of several micromolar P i, and using >15 μM PBP, even 2 μM P i contamination
can be tolerated. Keeping the P i concentration under 2 μM is
easily achieved when following the instructions given here. The
use of a P i mop is generally not necessary. For very sensitive
assays where hundreds of nM or even less P i need to be
measured and lower PBP concentrations are used more care
has to be taken in choosing and preparing the buffers and
nucleotide solutions. It might also be necessary to include the
P i mop (see Subheading 2.5) in some of the reagents.
3. Not all PNPase variants accept 7-methylguanosine as a substrate. The given source works well in our experiments.
4. Multi-well plates, in particular those with a nonproteinbinding coating, can also have significant levels of P i contamination. For the assay we present here this is not a problem.
However, if needed one can reduce P i contamination by rinsing
the plates with P i -free water or even pretreat them with P i mop.
5. When running assays at near ambient temperature, it is not
necessary to take special precautions to minimize temperature
equilibration times. Buffers and diluted solutions are just kept
at room temperature (not on ice) before pipetting into the
plates. However, when measuring at higher temperatures,
e.g., 30
C or 37
C, solutions (and plates) have to be preequilibrated before starting the experiments or temperature
will increase during the initial minutes of the reaction. At
higher temperatures in nonsealed plates, evaporation can also
be a problem, particularly when small volumes and long incubation times are used.
6. All experiments described here were performed with an N- and
C-terminally truncated construct of S. cerevisiae Chd1 (residues 118-1274) [29]. The protein was expressed from pET49b
in E. coli and purified by GSH-affinity, Ni-NTA affinity, and size
exclusion chromatography.
7. Since the ATPase K m of Chd1 is relatively high (>100 μM) and
therefore high ATP concentrations are needed, we used purified ATP for the experiments.
8. A 30 bp double-stranded DNA was generated by annealing
two complementary oligonucleotides with the following
sequences: 5
0 -GTA ACC ACG CGT ATA GAA ACG GGA
CTC ATT-3
0 and the reverse complement.
9. The assay also works well with down to 20 μl in Corning
384 plates with square wells (not small volume). If enough
material is available, the assay can be more robust and accurate
with larger volumes (50–200 μl) and/or 96-well plates.
Phosphate Biosensor Assays
313