There are also two key scientific observations as part of assay
development:
1. The observation of sufficient AlphaScreen signal from optimized antibody pairs to quantify target protein from the cell
line of interest.
2. The observation of a sufficiently large change in the thermal
stability of the target protein upon binding of the tool compound in cells.
2 Materials
All solutions should be prepared using ultrapure water (deionized
water purified to achieve a resistivity of 18.2 MΩ/cm at 25
C) and
analytical-grade reagents. Reagents should be stored as recommended by their suppliers. SYPRO
® Orange (and other fluorescent
dyes) and AlphaScreen
® reagents must be stored in the dark. Protein reagents should generally be thawed quickly and then kept on
ice until use.
2.1 Optimization
of Conditions
and Screening
of Compounds by DSF
Experiments
1. DSF buffer: target dependent, e.g., 50 mM HEPES-Na
(pH 7.5), 100 mM NaCl (see Note 1).
2. Purified protein target of interest: commercially available or
purified in-house. Typically stored at À80
C at 10–100 μM,
aliquoted to minimize freeze–thaw cycles.
3. Liquid dispenser, e.g., D300 liquid dispenser (Tecan).
4. 5000Â SYPRO
® Orange (see Note 2).
5. DMSO (100% v/v).
6. 384-well PCR plate, e.g., 4titude FrameStar 384 well skirted
PCR plate compatible with real-time PCR amplification and
detection instrument and thermosealing tape (4titude).
7. A real-time PCR amplification and detection instrument, e.g.,
LightCycler480 II (Roche).
2.2 Screening
of Compounds by DSF
1. As in Subheading 2.1.
2. Test compounds as 10–100 mM stocks in DMSO (100% v/v).
2.3 Quantitative
AlphaScreen Assay
to Measure Soluble
Target Protein
for CETSA
1. Source several mouse-derived and rabbit-derived antibodies
directed to the target protein. Antibodies should be chosen
to maximize selective recognition of the desired target (see
Note 3).
2. 5Â SureFire Lysis Buffer (PerkinElmer).
3. AlphaScreen compatible white, shallow 384-well microplate,
e.g., ProxiPlate-384 Plus (PerkinElmer).
Quantitative Detection of Ligand Binding by DSF and CETSA HT
203
development:
1. The observation of sufficient AlphaScreen signal from optimized antibody pairs to quantify target protein from the cell
line of interest.
2. The observation of a sufficiently large change in the thermal
stability of the target protein upon binding of the tool compound in cells.
2 Materials
All solutions should be prepared using ultrapure water (deionized
water purified to achieve a resistivity of 18.2 MΩ/cm at 25
C) and
analytical-grade reagents. Reagents should be stored as recommended by their suppliers. SYPRO
® Orange (and other fluorescent
dyes) and AlphaScreen
® reagents must be stored in the dark. Protein reagents should generally be thawed quickly and then kept on
ice until use.
2.1 Optimization
of Conditions
and Screening
of Compounds by DSF
Experiments
1. DSF buffer: target dependent, e.g., 50 mM HEPES-Na
(pH 7.5), 100 mM NaCl (see Note 1).
2. Purified protein target of interest: commercially available or
purified in-house. Typically stored at À80
C at 10–100 μM,
aliquoted to minimize freeze–thaw cycles.
3. Liquid dispenser, e.g., D300 liquid dispenser (Tecan).
4. 5000Â SYPRO
® Orange (see Note 2).
5. DMSO (100% v/v).
6. 384-well PCR plate, e.g., 4titude FrameStar 384 well skirted
PCR plate compatible with real-time PCR amplification and
detection instrument and thermosealing tape (4titude).
7. A real-time PCR amplification and detection instrument, e.g.,
LightCycler480 II (Roche).
2.2 Screening
of Compounds by DSF
1. As in Subheading 2.1.
2. Test compounds as 10–100 mM stocks in DMSO (100% v/v).
2.3 Quantitative
AlphaScreen Assay
to Measure Soluble
Target Protein
for CETSA
1. Source several mouse-derived and rabbit-derived antibodies
directed to the target protein. Antibodies should be chosen
to maximize selective recognition of the desired target (see
Note 3).
2. 5Â SureFire Lysis Buffer (PerkinElmer).
3. AlphaScreen compatible white, shallow 384-well microplate,
e.g., ProxiPlate-384 Plus (PerkinElmer).
Quantitative Detection of Ligand Binding by DSF and CETSA HT
203