11. Add SYPRO
® Orange at the optimal concentration determined in step 8 and 100 nL DMSO to every well on the plate.
12. Seal the plate with optical sealing film and centrifuge the plate
at 300 Â g for 1 min.
13. Place the plate into the precooled real-time PCR instrument
and ramp the temperature from 20 to 95
C at 1
C/min while
measuring the fluorescence using filters compatible with
SYPRO
® Orange as in step 7.
14. Determine the melting temperature of the protein for each well
(see Note 9) and then calculate the mean melting temperature
and its standard deviation. For a robust DSF screening assay,
the standard deviation should be 0.3
C. If the standard
deviation of the mean melting temperature is significantly
greater than this, try different buffers and/or cofactors. If the
melting temperature is <35
C, it may help to keep the plate
chilled as much as possible during handling and, if possible,
start the temperature ramp from a lower temperature.
3.2 Screening
of Compounds by DSF
1. Prepare the screening plate using liquid handling, e.g., an
ECHO 555 acoustic dispenser to add 100 nL of DMSO to
Columns 1 and 23 and 100 nL of 10 mM positive control
(in DMSO) to Columns 2 and 24 of a 384-well PCR plate. Fill
the remaining wells of the plate with 100 nL of 10 mM test
compounds in DMSO (up to 320 compounds). If a positive
control is unavailable, just add 100 nL DMSO to use these
wells as extra DMSO controls.
2. Seal the plate and centrifuge at 300 Â g for 1 min.
3. Prepare 4 mL of protein in DSF buffer at the concentration
determined in step 8 (Subheading 3.1) and add 10 μL to all wells.
4. Add the volume of 5000Â SYPRO
® Orange determined in
step 8 in Subheading 3.1 to all wells on the plate using a liquid
dispenser, e.g., D300 (Tecan).
5. Seal the plate and centrifuge at 300 Â g for 1 min.
6. Place the plate into the real-time PCR instrument and ramp the
temperature from 20 to 95
C at 1
C/min while measuring
the fluorescence using filters compatible with SYPRO
®
Orange.
7. Determine the melting temperature of the protein in each swell
(see Note 9).
8. Calculate the thermal shift (ΔT m ) of the test compounds by
subtracting the mean melting temperature of the DMSO only
wells from the melting temperature of the protein in the presence of the test compound. A hit is typically defined as a
compound inducing a ΔT m ! 1
C or !3Â the standard
deviation of the DMSO only melting temperature, whichever
is greater.
Quantitative Detection of Ligand Binding by DSF and CETSA HT
207
® Orange at the optimal concentration determined in step 8 and 100 nL DMSO to every well on the plate.
12. Seal the plate with optical sealing film and centrifuge the plate
at 300 Â g for 1 min.
13. Place the plate into the precooled real-time PCR instrument
and ramp the temperature from 20 to 95
C at 1
C/min while
measuring the fluorescence using filters compatible with
SYPRO
® Orange as in step 7.
14. Determine the melting temperature of the protein for each well
(see Note 9) and then calculate the mean melting temperature
and its standard deviation. For a robust DSF screening assay,
the standard deviation should be 0.3
C. If the standard
deviation of the mean melting temperature is significantly
greater than this, try different buffers and/or cofactors. If the
melting temperature is <35
C, it may help to keep the plate
chilled as much as possible during handling and, if possible,
start the temperature ramp from a lower temperature.
3.2 Screening
of Compounds by DSF
1. Prepare the screening plate using liquid handling, e.g., an
ECHO 555 acoustic dispenser to add 100 nL of DMSO to
Columns 1 and 23 and 100 nL of 10 mM positive control
(in DMSO) to Columns 2 and 24 of a 384-well PCR plate. Fill
the remaining wells of the plate with 100 nL of 10 mM test
compounds in DMSO (up to 320 compounds). If a positive
control is unavailable, just add 100 nL DMSO to use these
wells as extra DMSO controls.
2. Seal the plate and centrifuge at 300 Â g for 1 min.
3. Prepare 4 mL of protein in DSF buffer at the concentration
determined in step 8 (Subheading 3.1) and add 10 μL to all wells.
4. Add the volume of 5000Â SYPRO
® Orange determined in
step 8 in Subheading 3.1 to all wells on the plate using a liquid
dispenser, e.g., D300 (Tecan).
5. Seal the plate and centrifuge at 300 Â g for 1 min.
6. Place the plate into the real-time PCR instrument and ramp the
temperature from 20 to 95
C at 1
C/min while measuring
the fluorescence using filters compatible with SYPRO
®
Orange.
7. Determine the melting temperature of the protein in each swell
(see Note 9).
8. Calculate the thermal shift (ΔT m ) of the test compounds by
subtracting the mean melting temperature of the DMSO only
wells from the melting temperature of the protein in the presence of the test compound. A hit is typically defined as a
compound inducing a ΔT m ! 1
C or !3Â the standard
deviation of the DMSO only melting temperature, whichever
is greater.
Quantitative Detection of Ligand Binding by DSF and CETSA HT
207