paper is used or two solutions are prepared in parallel and the
volume of acid/base needed for the adjustment of one is measured,
then the same volume is added to the other, P i -free, buffer.
Other than inorganic phosphate, most buffer components are
compatible with P i detection by phosphate-binding protein,
including
common
reducing
agents
(DTT,
TCEP,
β-mercaptoethanol). Most nucleotide hydrolyzing enzymes
depend on Mg
2+ (or Mn
2+ ) as a cofactor. Therefore 1 to 10 mM
MgCl 2 is generally included in the buffer for these reactions.
Adding BSA or detergent can help to prevent sticking of proteins to the surface of tubes, plates, and cuvettes and therefore
stabilizes enzymes at very low concentration. We generally use
BSA (5–20 μM) for enzyme solutions at concentrations of 0.5 μM
or lower.
2.3 Nucleotide
Solutions
Nucleotide solutions are generally the biggest source of P i contamination due to the spontaneous hydrolysis of phosphodiester bonds
in water. The stability of nucleoside triphosphates depends on the
nature of the nucleotide. Purine nucleotides (G and A) are less
stable than pyrimidine nucleotides (C and T).
For ATPase reactions, we use adenosine 5
0 -triphosphate
disodium salt hydrate (BioXtra, >99% (HPLC)) from SigmaAldrich. This ATP source normally contains between 0.2 and
0.5% (mol/mol) inorganic phosphate and can be used at up to
500–1000 μM in PBP-based assays. GTP, guanosine
5
0 -triphosphate sodium salt hydrate (>95% by HPLC), can also
be purchased from Sigma-Aldrich, but at lower purity than ATP.
Both nucleotides can be further purified by anion-exchange chromatography if needed for a particular assay [11].
Solutions are made up at 20–100 mM in equimolar Tris–HCL
(pH 8.0–9.0). If necessary, the pH is adjusted with NaOH using
pH paper. All nucleotide solutions are stored at À20
C or À80
C
(purified nucleotides).
2.4 P i Standard
The fluorescence response of MDCC-PBP or Rho-PBP is calibrated with an inorganic phosphate standard. Here we use a Centripur
® phosphate standard solution, 1000 mg/ml PO 4
3À (Merck),
corresponding to 10.53 mM phosphate ions.
2.5 P i Mop
To remove P i contaminations from multi-well plates and the
stopped-flow instrument, a P i mop [21] system is used which
consists of 7-methylguanosine (M0627, Sigma-Aldrich) and purine
nucleoside phosphorylase (N8264, Sigma-Aldrich) (see Note 3).
PNPase (purine nucleoside phosphorylase) catalyzes the phosphorolysis of 7-methylguanosine (7-MEG) to form ribose-1-phosphate and guanine, thereby removing free phosphate from the
solution. With this system, P i concentrations can be lowered to
about 0.1 μM [1].
Phosphate Biosensor Assays
293
volume of acid/base needed for the adjustment of one is measured,
then the same volume is added to the other, P i -free, buffer.
Other than inorganic phosphate, most buffer components are
compatible with P i detection by phosphate-binding protein,
including
common
reducing
agents
(DTT,
TCEP,
β-mercaptoethanol). Most nucleotide hydrolyzing enzymes
depend on Mg
2+ (or Mn
2+ ) as a cofactor. Therefore 1 to 10 mM
MgCl 2 is generally included in the buffer for these reactions.
Adding BSA or detergent can help to prevent sticking of proteins to the surface of tubes, plates, and cuvettes and therefore
stabilizes enzymes at very low concentration. We generally use
BSA (5–20 μM) for enzyme solutions at concentrations of 0.5 μM
or lower.
2.3 Nucleotide
Solutions
Nucleotide solutions are generally the biggest source of P i contamination due to the spontaneous hydrolysis of phosphodiester bonds
in water. The stability of nucleoside triphosphates depends on the
nature of the nucleotide. Purine nucleotides (G and A) are less
stable than pyrimidine nucleotides (C and T).
For ATPase reactions, we use adenosine 5
0 -triphosphate
disodium salt hydrate (BioXtra, >99% (HPLC)) from SigmaAldrich. This ATP source normally contains between 0.2 and
0.5% (mol/mol) inorganic phosphate and can be used at up to
500–1000 μM in PBP-based assays. GTP, guanosine
5
0 -triphosphate sodium salt hydrate (>95% by HPLC), can also
be purchased from Sigma-Aldrich, but at lower purity than ATP.
Both nucleotides can be further purified by anion-exchange chromatography if needed for a particular assay [11].
Solutions are made up at 20–100 mM in equimolar Tris–HCL
(pH 8.0–9.0). If necessary, the pH is adjusted with NaOH using
pH paper. All nucleotide solutions are stored at À20
C or À80
C
(purified nucleotides).
2.4 P i Standard
The fluorescence response of MDCC-PBP or Rho-PBP is calibrated with an inorganic phosphate standard. Here we use a Centripur
® phosphate standard solution, 1000 mg/ml PO 4
3À (Merck),
corresponding to 10.53 mM phosphate ions.
2.5 P i Mop
To remove P i contaminations from multi-well plates and the
stopped-flow instrument, a P i mop [21] system is used which
consists of 7-methylguanosine (M0627, Sigma-Aldrich) and purine
nucleoside phosphorylase (N8264, Sigma-Aldrich) (see Note 3).
PNPase (purine nucleoside phosphorylase) catalyzes the phosphorolysis of 7-methylguanosine (7-MEG) to form ribose-1-phosphate and guanine, thereby removing free phosphate from the
solution. With this system, P i concentrations can be lowered to
about 0.1 μM [1].
Phosphate Biosensor Assays
293