the reaction is much slower than this, unwanted signal drifts could
impair the data quality. If the reaction is too fast, then the initial rate
may be underestimated, and this depends on the sampling rate of
the plate reader.
With a new reaction system, one can vary the enzyme concentration to optimize the assay in advance. Testing the linearity of the
reaction rate with enzyme concentration is also good practice, for
example, to exclude problems with enzyme stability at low enzyme
concentration. However, nonlinearity does not necessarily point to
an artifact and could instead be a physiological feature of the
system, for example, due to the enzyme activity being regulated
by self-assembly [25].
Fitting of Initial Rates
As a rule of thumb, to capture the initial rate prior to substrate
depletion and significant product inhibition, one should only use
measurements made before 10% of the substrate is depleted. In
practice, there are other, often assay-specific, reasons that define the
range over which the product formation is linear with time.
Figure 2a shows example traces of P i formation during ATP hydrolysis by Chd1 with two different ATP concentrations. The signal
appears unstable for the first minute of the reaction, likely due to
the settling of the meniscus in the wells and/or temperature equilibration. Subsequently, the signal reaches a linear steady state phase.
At high substrate concentration, the signal plateaus due to saturation of MDCC-PBP. At low substrate concentration, the signal
deviates from linearity much later in time but at lower fluorescence
level, likely due to substrate depletion. While 10% turnover can be
used as an upper limit to fit initial rates, visual inspection is required
to judge the linear range to be fit (grey brackets in Fig. 2a).
3.1.3 Example Protocol:
Steady State Kinetic
Assay—Chd1 ATPase
Chd1 is an ATP-dependent chromatin remodeler that uses ATP
hydrolysis to move and position nucleosomes along DNA
[26, 27]. The intrinsic ATPase activity of Chd1 is low, but ATP
hydrolysis is strongly stimulated by the binding of double-stranded
DNA (dsDNA) or nucleosomes [28, 29], which relieves an intramolecular autoinhibition [28]. MDCC-PBP is used here to measure the K m and k cat for the basic and dsDNA-stimulated ATPase
of Chd1.
Buffer and Concentrations
The assay is performed in buffer containing 30 mM Tris–HCl
pH 7.5, 50 mM KCl, 5 mM MgCl 2 , and 1 mM TCEP (using
components with minimal P i , see Subheading 2.2) at 25
C (see
Note 5). The buffer is prepared freshly and kept at room temperature throughout the experiments.
The final concentrations of assay components are:
1. 15 μM MDCC-PBP.
2. 100 nM and 2 nM Chd1 (in the absence and presence of DNA,
respectively) (see Note 6).
Phosphate Biosensor Assays
299
impair the data quality. If the reaction is too fast, then the initial rate
may be underestimated, and this depends on the sampling rate of
the plate reader.
With a new reaction system, one can vary the enzyme concentration to optimize the assay in advance. Testing the linearity of the
reaction rate with enzyme concentration is also good practice, for
example, to exclude problems with enzyme stability at low enzyme
concentration. However, nonlinearity does not necessarily point to
an artifact and could instead be a physiological feature of the
system, for example, due to the enzyme activity being regulated
by self-assembly [25].
Fitting of Initial Rates
As a rule of thumb, to capture the initial rate prior to substrate
depletion and significant product inhibition, one should only use
measurements made before 10% of the substrate is depleted. In
practice, there are other, often assay-specific, reasons that define the
range over which the product formation is linear with time.
Figure 2a shows example traces of P i formation during ATP hydrolysis by Chd1 with two different ATP concentrations. The signal
appears unstable for the first minute of the reaction, likely due to
the settling of the meniscus in the wells and/or temperature equilibration. Subsequently, the signal reaches a linear steady state phase.
At high substrate concentration, the signal plateaus due to saturation of MDCC-PBP. At low substrate concentration, the signal
deviates from linearity much later in time but at lower fluorescence
level, likely due to substrate depletion. While 10% turnover can be
used as an upper limit to fit initial rates, visual inspection is required
to judge the linear range to be fit (grey brackets in Fig. 2a).
3.1.3 Example Protocol:
Steady State Kinetic
Assay—Chd1 ATPase
Chd1 is an ATP-dependent chromatin remodeler that uses ATP
hydrolysis to move and position nucleosomes along DNA
[26, 27]. The intrinsic ATPase activity of Chd1 is low, but ATP
hydrolysis is strongly stimulated by the binding of double-stranded
DNA (dsDNA) or nucleosomes [28, 29], which relieves an intramolecular autoinhibition [28]. MDCC-PBP is used here to measure the K m and k cat for the basic and dsDNA-stimulated ATPase
of Chd1.
Buffer and Concentrations
The assay is performed in buffer containing 30 mM Tris–HCl
pH 7.5, 50 mM KCl, 5 mM MgCl 2 , and 1 mM TCEP (using
components with minimal P i , see Subheading 2.2) at 25
C (see
Note 5). The buffer is prepared freshly and kept at room temperature throughout the experiments.
The final concentrations of assay components are:
1. 15 μM MDCC-PBP.
2. 100 nM and 2 nM Chd1 (in the absence and presence of DNA,
respectively) (see Note 6).
Phosphate Biosensor Assays
299