1. Dispense compounds into a 384-well PCR plate using a liquid
handling device, e.g., ECHO 555 acoustic dispenser (see Note
15) or a D300 dispenser (Tecan). Dispense compounds at
66.6Â desired final concentration; for example, dispense
150 nL of a 2 mM DMSO stock of compound to achieve a
final screening concentration of 30 μM.
2. Harvest cells to the optimal density (determined in step 7,
Subheading 3.3), ensuring single-cell suspension (see Note
16), and dispense onto compounds in the 384-well PCR
plate using a liquid dispenser, e.g., Multidrop Combi set to
dispense 10 μL/well (see Note 15).
3. Apply a lid to the PCR plate.
4. Ensure mixing of the cells with the compounds by performing
a brief centrifugation at 300 Â g for 10 s (see Note 17).
5. Incubate the plate under tissue culture conditions (37
C, 5%
CO 2 ) to allow time for compound target engagement. Incubations are typically performed for 1–2 h.
6. Remove the plate from the incubator and replace the lid with
Thermosealing tape (see Note 14).
7. Load the plate into the 384-well PCR instrument, e.g., LightCycler 480 II to perform the heat shock. Set a single cycle to hold
at the target temperature as determined by analysis of data from
Subheading 3.4 for 3 min with a ramp rate of 4.8
C/s, followed
by a hold at 20
C for 1 min with a ramp rate of 2.5
C/s.
8. Remove the plate from the 384-well PCR instrument and
remove the Thermosealing tape.
9. Dilute 2 mL of 5Â SureFire Lysis Buffer in 8 mL of dH 2 O to
give 10 mL of 2Â SureFire Lysis Buffer. Lyse the cells by
addition of 2Â Lysis Buffer using a liquid dispenser, e.g.,
Multidrop Combi to dispense 10 μL/well (see Note 15).
Ensure mixing of cells with Lysis Buffer by performing a brief
centrifugation at 300 Â g for 10 s. Wells will now contain 20 μL
of cell lysates in 1Â Lysis Buffer.
10. Progress to step 11 within 1 h or store at 4
C.
11. Thoroughly mix the cell lysate solution using a liquid handling
platform, e.g., Bravo liquid handler to perform 10 repetitive
cycles of aspirating and dispensing 7 μL. Transfer 3 μL of the
20 μL lysate to an AlphaScreen compatible 384-well plate, e.g.,
ProxiPlate-384, using the liquid handler (see Note 18). Aspirating and dispensing from the PCR plate is performed at
1.5 mm from the bottom of the well. Dispensing into the
AlphaScreen 384-well ProxiPlate-384 plate is performed at
2 mm from the bottom of the well.
12. Quantify thermostable target protein following the steps in
Subheading 3.5.
Quantitative Detection of Ligand Binding by DSF and CETSA HT
211
handling device, e.g., ECHO 555 acoustic dispenser (see Note
15) or a D300 dispenser (Tecan). Dispense compounds at
66.6Â desired final concentration; for example, dispense
150 nL of a 2 mM DMSO stock of compound to achieve a
final screening concentration of 30 μM.
2. Harvest cells to the optimal density (determined in step 7,
Subheading 3.3), ensuring single-cell suspension (see Note
16), and dispense onto compounds in the 384-well PCR
plate using a liquid dispenser, e.g., Multidrop Combi set to
dispense 10 μL/well (see Note 15).
3. Apply a lid to the PCR plate.
4. Ensure mixing of the cells with the compounds by performing
a brief centrifugation at 300 Â g for 10 s (see Note 17).
5. Incubate the plate under tissue culture conditions (37
C, 5%
CO 2 ) to allow time for compound target engagement. Incubations are typically performed for 1–2 h.
6. Remove the plate from the incubator and replace the lid with
Thermosealing tape (see Note 14).
7. Load the plate into the 384-well PCR instrument, e.g., LightCycler 480 II to perform the heat shock. Set a single cycle to hold
at the target temperature as determined by analysis of data from
Subheading 3.4 for 3 min with a ramp rate of 4.8
C/s, followed
by a hold at 20
C for 1 min with a ramp rate of 2.5
C/s.
8. Remove the plate from the 384-well PCR instrument and
remove the Thermosealing tape.
9. Dilute 2 mL of 5Â SureFire Lysis Buffer in 8 mL of dH 2 O to
give 10 mL of 2Â SureFire Lysis Buffer. Lyse the cells by
addition of 2Â Lysis Buffer using a liquid dispenser, e.g.,
Multidrop Combi to dispense 10 μL/well (see Note 15).
Ensure mixing of cells with Lysis Buffer by performing a brief
centrifugation at 300 Â g for 10 s. Wells will now contain 20 μL
of cell lysates in 1Â Lysis Buffer.
10. Progress to step 11 within 1 h or store at 4
C.
11. Thoroughly mix the cell lysate solution using a liquid handling
platform, e.g., Bravo liquid handler to perform 10 repetitive
cycles of aspirating and dispensing 7 μL. Transfer 3 μL of the
20 μL lysate to an AlphaScreen compatible 384-well plate, e.g.,
ProxiPlate-384, using the liquid handler (see Note 18). Aspirating and dispensing from the PCR plate is performed at
1.5 mm from the bottom of the well. Dispensing into the
AlphaScreen 384-well ProxiPlate-384 plate is performed at
2 mm from the bottom of the well.
12. Quantify thermostable target protein following the steps in
Subheading 3.5.
Quantitative Detection of Ligand Binding by DSF and CETSA HT
211