3.4 Thermal Melt
Analysis in Live Cells
by CETSA
Volumes are provided for testing of one compound as a thermal
melt curve but can be scaled as appropriate.
1. Harvest cells to 4 mL at the optimal cell density as determined
in step 7 in Subheading 3.3. Split the cells into two separate
pools of 2 mL. To one pool of cells, add tool compound (see
Note 4) to a final concentration of 10 μM. To the other pool,
add an equivalent volume of DMSO.
2. Aliquot compound-treated cells into four tubes of each eighttube PCR strip, 20 μL/tube, across 12 PCR strips. Aliquot
DMSO-treated cells into the other four tubes of each eighttube PCR strip, 20 μL/tube, across 12 PCR strips. Apply lids
to the PCR tubes. Each eight-tube PCR strip should contain
four tubes of compound-treated cells and four tubes of
DMSO-treated cells, and there should be 12 full eight-tube
PCR strips (Fig. 3).
3. Briefly clarify the PCR strips using a PCR strip microcentrifuge,
place in a PCR rack, and incubate under tissue culture conditions (37
C, 5% CO 2 ) to allow time for compound target
engagement. Incubations are typically performed for 1–2 h.
4. Using a multi-block thermal cycler, e.g., SimpliAmp PCR
machine, heat shock each PCR strip for 3 min at a certain
temperature followed by 1 min at 20
C (see Note 14). Load
the first three PCR strips into the three separate blocks of the
PCR machine and perform the first three heat shocks at 37
C,
40
C, and 42
C. Remove and store at 4
C, then load the next
three PCR strips, and perform the next three heat shocks at
44
C, 46
C, and 48
C. Repeat to perform heat shocks at
50
C, 52
C, and 54
C and 56
C, 58
C, and 60
C (Fig. 3).
5. Following completion of all heat shocks, ensure that all samples
are incubated at 4
C for a minimum of 5 min.
6. Open the PCR tubes and add 20 μL/tube of 2Â SureFire Lysis
Buffer. Briefly clarify the tubes using a PCR strip microcentrifuge and incubate for 10 min at room temperature.
7. Using a 96-well to 384-well convertible multichannel pipette,
e.g., E1 ClipTip, mix each sample by aspirating 10 μL and
dispensing back into the PCR tube, before aspirating 10 μL
and dispensing 3 μL into an AlphaScreen compatible white,
shallow 384-well microplate, e.g., ProxiPlate-384 Plus.
3.5 Quantification
of Thermostable
Target Protein by
AlphaScreen
1. Dilute 0.5 mL of 10Â ImmunoAssay Buffer in 4.5 mL of
dH 2 O to give 5 mL of 1Â ImmunoAssay Buffer. Add primary
antibodies (mouse and rabbit) to the dilution optimized in step
7 in Subheading 3.3.
2. Under subdued light, add to the primary antibody mix 120 μL
of anti-Mouse IgG AlphaDonor beads to give a concentration
Quantitative Detection of Ligand Binding by DSF and CETSA HT
209
Analysis in Live Cells
by CETSA
Volumes are provided for testing of one compound as a thermal
melt curve but can be scaled as appropriate.
1. Harvest cells to 4 mL at the optimal cell density as determined
in step 7 in Subheading 3.3. Split the cells into two separate
pools of 2 mL. To one pool of cells, add tool compound (see
Note 4) to a final concentration of 10 μM. To the other pool,
add an equivalent volume of DMSO.
2. Aliquot compound-treated cells into four tubes of each eighttube PCR strip, 20 μL/tube, across 12 PCR strips. Aliquot
DMSO-treated cells into the other four tubes of each eighttube PCR strip, 20 μL/tube, across 12 PCR strips. Apply lids
to the PCR tubes. Each eight-tube PCR strip should contain
four tubes of compound-treated cells and four tubes of
DMSO-treated cells, and there should be 12 full eight-tube
PCR strips (Fig. 3).
3. Briefly clarify the PCR strips using a PCR strip microcentrifuge,
place in a PCR rack, and incubate under tissue culture conditions (37
C, 5% CO 2 ) to allow time for compound target
engagement. Incubations are typically performed for 1–2 h.
4. Using a multi-block thermal cycler, e.g., SimpliAmp PCR
machine, heat shock each PCR strip for 3 min at a certain
temperature followed by 1 min at 20
C (see Note 14). Load
the first three PCR strips into the three separate blocks of the
PCR machine and perform the first three heat shocks at 37
C,
40
C, and 42
C. Remove and store at 4
C, then load the next
three PCR strips, and perform the next three heat shocks at
44
C, 46
C, and 48
C. Repeat to perform heat shocks at
50
C, 52
C, and 54
C and 56
C, 58
C, and 60
C (Fig. 3).
5. Following completion of all heat shocks, ensure that all samples
are incubated at 4
C for a minimum of 5 min.
6. Open the PCR tubes and add 20 μL/tube of 2Â SureFire Lysis
Buffer. Briefly clarify the tubes using a PCR strip microcentrifuge and incubate for 10 min at room temperature.
7. Using a 96-well to 384-well convertible multichannel pipette,
e.g., E1 ClipTip, mix each sample by aspirating 10 μL and
dispensing back into the PCR tube, before aspirating 10 μL
and dispensing 3 μL into an AlphaScreen compatible white,
shallow 384-well microplate, e.g., ProxiPlate-384 Plus.
3.5 Quantification
of Thermostable
Target Protein by
AlphaScreen
1. Dilute 0.5 mL of 10Â ImmunoAssay Buffer in 4.5 mL of
dH 2 O to give 5 mL of 1Â ImmunoAssay Buffer. Add primary
antibodies (mouse and rabbit) to the dilution optimized in step
7 in Subheading 3.3.
2. Under subdued light, add to the primary antibody mix 120 μL
of anti-Mouse IgG AlphaDonor beads to give a concentration
Quantitative Detection of Ligand Binding by DSF and CETSA HT
209