All solutions should be prepared using ultrapure water
(prepared by purifying deionized water to achieve a sensitivity of
18 MΩ cm at 25
C) and analytical-grade reagents.
1. 1 M HEPES (stock): Weigh 238 g and dissolve in 900 mL of
ultrapure water in a beaker. Add a stir bar and stir until fully
dissolved. Monitor the pH and add a solid pellet of NaOH
until dissolved. Continue to add and dissolve NaOH pellets
one at a time until the pH approaches the desired quantity.
Fine-tune the pH by addition of small aliquots of 100 mM
solution of NaOH. Add ultrapure water to a final volume to
1 L. Sterile filter the solution (see Note 7) and store for up to
3 months in the dark at room temperature prior to use.
2. 4 M sodium chloride (stock): Weigh 234 g and dissolve in 1 L
of ultrapure water. Filter the solution, sterilize (see Note 7),
and store for up to 6 months in the dark at room temperature
prior to use.
3. 1 M magnesium chloride: Manufacturer-supplied as a solution,
and it has a long shelf life when stored at room temperature in
the dark.
4. 1 M dithiothreitol (DTT stock): Weigh 1.54 g and dissolve in
10 mL of ultrapure water. Filter the solution, sterilize (see Note
7), and store in aliquots of 100 μL for up to 6 months in the
dark at À20
C prior to use.
5. Detergents: Manufacturer-supplied and stored according to
their instructions.
6. High-molecular-weight binding partner: proteins (see Note 8),
manufacturer-supplied or purified in-house, are typically stored
in aliquots of 10–20 μL at high concentration, often around
10 μM, at À80
C.
7. Fluorescently labeled low-molecular-weight binding partners:
manufacturer-supplied or labeled in-house (see Note 9).
8. 1 mM fluorescein (stock): Dissolve 1 mg of fluorescein in 3 mL
of ultrapure water.
3 Methods
3.1 Solution
Handling
Care must be taken when daily removing aliquots of each buffer
component to avoid contamination. Working solutions are made as
follows: half the required volume of ultrapure water is added to a
sterile plastic vessel followed by addition of the required volume of
each of the assay components to give the correct final concentration. Subsequently, the required dilution is made by addition of a
further quantity of ultrapure water. This protocol is followed to
prevent buffer components from precipitating if they are added
Ligand Discovery - Fluorescence Polarization
233
(prepared by purifying deionized water to achieve a sensitivity of
18 MΩ cm at 25
C) and analytical-grade reagents.
1. 1 M HEPES (stock): Weigh 238 g and dissolve in 900 mL of
ultrapure water in a beaker. Add a stir bar and stir until fully
dissolved. Monitor the pH and add a solid pellet of NaOH
until dissolved. Continue to add and dissolve NaOH pellets
one at a time until the pH approaches the desired quantity.
Fine-tune the pH by addition of small aliquots of 100 mM
solution of NaOH. Add ultrapure water to a final volume to
1 L. Sterile filter the solution (see Note 7) and store for up to
3 months in the dark at room temperature prior to use.
2. 4 M sodium chloride (stock): Weigh 234 g and dissolve in 1 L
of ultrapure water. Filter the solution, sterilize (see Note 7),
and store for up to 6 months in the dark at room temperature
prior to use.
3. 1 M magnesium chloride: Manufacturer-supplied as a solution,
and it has a long shelf life when stored at room temperature in
the dark.
4. 1 M dithiothreitol (DTT stock): Weigh 1.54 g and dissolve in
10 mL of ultrapure water. Filter the solution, sterilize (see Note
7), and store in aliquots of 100 μL for up to 6 months in the
dark at À20
C prior to use.
5. Detergents: Manufacturer-supplied and stored according to
their instructions.
6. High-molecular-weight binding partner: proteins (see Note 8),
manufacturer-supplied or purified in-house, are typically stored
in aliquots of 10–20 μL at high concentration, often around
10 μM, at À80
C.
7. Fluorescently labeled low-molecular-weight binding partners:
manufacturer-supplied or labeled in-house (see Note 9).
8. 1 mM fluorescein (stock): Dissolve 1 mg of fluorescein in 3 mL
of ultrapure water.
3 Methods
3.1 Solution
Handling
Care must be taken when daily removing aliquots of each buffer
component to avoid contamination. Working solutions are made as
follows: half the required volume of ultrapure water is added to a
sterile plastic vessel followed by addition of the required volume of
each of the assay components to give the correct final concentration. Subsequently, the required dilution is made by addition of a
further quantity of ultrapure water. This protocol is followed to
prevent buffer components from precipitating if they are added
Ligand Discovery - Fluorescence Polarization
233