3.1.2 Preparation
of NAG3 Solution
1. Weigh out an appropriate mass of the lyophilized powder of
NAG3 into a microtube using an analytical balance. Make sure
the mass is sufficient for accurate weighing in the balance you
are using (usually at least 1 mg).
2. Raise the powder directly in the required volume of the standard assay buffer to give the required concentration. NAG3 is
soluble up to at least 2 mM and is apparently stable for
>6 months when stored at À80
C at that concentration.
It is important that the solution of NAG3 is prepared accurately
to ensure consistency and accuracy of experimental parameters. In
all experiments, concentration errors for the NAG3 solution have a
significant effect on fitted parameters. These methods for determining protein and ligand concentration have in our hands
provided apparently accurate concentrations, as judged by routine
observation of interaction stoichiometries between 0.95 and 1.05
in ITC experiments (see Note 11).
3.2 Thermal Shift
Assay
3.2.1 Thermal Shift
Assay Measurement
The aim is to measure the midpoint of thermal denaturation for
HEWL in the presence and absence of a saturating concentration of
NAG3.
1. Make 500 μL of 20 μM HEWL in standard assay buffer.
2. Make 500 μL of 2 mM NAG3 solution in standard assay buffer.
3. Add SYPRO Orange dye directly to the protein solution at a
250-fold dilution of the dye stock concentration, i.e., 2 μL dye
solution in 500 μL protein solution. Mix thoroughly with a
pipette. The stock solution is supplied by the manufacturer at a
fixed, but unstated concentration labeled as “5000Â.”
4. Mix samples for differential scanning fluorimetry according to
Table 1. After mixing, the final concentration of HEWL will be
10 μM with 10Â SYPRO Orange in the presence and absence
of 1 mM NAG3 (see Note 12).
5. Place an appropriate volume of the sample into micro PCR
tubes or plates, preferably in triplicate for each condition.
6. Perform a thermal ramp from 25 to 95
C at a ramp rate in the
region of 5
C/min, monitoring the fluorescence intensity of
Table 1
Volumes for preparation of samples for the thermal shift assay
Sample HEWL (μL) (in buffer with dye) Buffer (μL) (no dye) 2 mM NAG3 (μL) (in buffer, no dye)
1
200
200
0
2
200
0
200
Interactions by Multiple Methods
55
of NAG3 Solution
1. Weigh out an appropriate mass of the lyophilized powder of
NAG3 into a microtube using an analytical balance. Make sure
the mass is sufficient for accurate weighing in the balance you
are using (usually at least 1 mg).
2. Raise the powder directly in the required volume of the standard assay buffer to give the required concentration. NAG3 is
soluble up to at least 2 mM and is apparently stable for
>6 months when stored at À80
C at that concentration.
It is important that the solution of NAG3 is prepared accurately
to ensure consistency and accuracy of experimental parameters. In
all experiments, concentration errors for the NAG3 solution have a
significant effect on fitted parameters. These methods for determining protein and ligand concentration have in our hands
provided apparently accurate concentrations, as judged by routine
observation of interaction stoichiometries between 0.95 and 1.05
in ITC experiments (see Note 11).
3.2 Thermal Shift
Assay
3.2.1 Thermal Shift
Assay Measurement
The aim is to measure the midpoint of thermal denaturation for
HEWL in the presence and absence of a saturating concentration of
NAG3.
1. Make 500 μL of 20 μM HEWL in standard assay buffer.
2. Make 500 μL of 2 mM NAG3 solution in standard assay buffer.
3. Add SYPRO Orange dye directly to the protein solution at a
250-fold dilution of the dye stock concentration, i.e., 2 μL dye
solution in 500 μL protein solution. Mix thoroughly with a
pipette. The stock solution is supplied by the manufacturer at a
fixed, but unstated concentration labeled as “5000Â.”
4. Mix samples for differential scanning fluorimetry according to
Table 1. After mixing, the final concentration of HEWL will be
10 μM with 10Â SYPRO Orange in the presence and absence
of 1 mM NAG3 (see Note 12).
5. Place an appropriate volume of the sample into micro PCR
tubes or plates, preferably in triplicate for each condition.
6. Perform a thermal ramp from 25 to 95
C at a ramp rate in the
region of 5
C/min, monitoring the fluorescence intensity of
Table 1
Volumes for preparation of samples for the thermal shift assay
Sample HEWL (μL) (in buffer with dye) Buffer (μL) (no dye) 2 mM NAG3 (μL) (in buffer, no dye)
1
200
200
0
2
200
0
200
Interactions by Multiple Methods
55