3 Methods
Perform all steps at room temperature unless otherwise indicated.
Ensure appropriate waste disposal throughout and appropriate
safety measures when using automated liquid handling devices.
3.1 Optimization
of Conditions for DSF
Experiments
The concentrations of protein and SYPRO
® Orange dye must be
optimized for the protein of interest in order to maximize reproducibility while minimizing reagent consumption. Figure 2a
depicts the plate layout and exemplar results for the optimization
experiment described below.
1. Prepare protein solutions at 1.25 μM, 2.5 μM, 5 μM, and
10 μM in DSF buffer by adding 300 μL of 10 μM protein in
DSF buffer to a 0.5-mL polypropylene tube. Mix 150 μL of
this solution with 150 μL of DSF buffer in a fresh tube. Take
150 μL of the resulting solution and mix with 150 μL of DSF
buffer in another tube. Repeat once more.
2. To a PCR instrument compatible 384-well microplate, e.g.,
4titude FrameStar 384 well skirted PCR plate, add 10 μL of
1.25 μM protein to wells A1–A12, 2.5 μM protein to wells B1–
B12, 5 μM protein to wells C1–C12, and 10 μM protein to
wells D1–D12 (see Note 5).
3. Briefly centrifuge the plate at 300 Â g to ensure that the
solution is at the bottom of the wells.
4. Add DMSO and SYPRO
® Orange to the PCR plate using a
liquid dispenser, e.g., D300 (Tecan), as shown in Table 1.
5. Seal the plate with the optical sealing film and centrifuge the
plate at 300 Â g for 1 min.
6. Precool the real-time PCR instrument by running a method to
hold the block temperature at 20
C for 5 min with a blank
plate (see Note 6).
7. When the precooling method finishes, immediately insert the
prepared PCR plate and run a method that will hold the
temperature at 20
C for 30 s and then run a temperature
gradient from 20 to 95
C at 1
C/min. Measure the fluorescence throughout the gradient (at least 1 acquisition per 1
C)
using filters compatible with SYPRO
® Orange (λ ex ¼ 470 nm,
λ em ¼ 570 nm, see Note 7).
8. Visually inspect the melt curves to choose the optimal protein/
dye combination for screening, considering reproducibility and
the signal/background ratio (see Fig. 2b for more detail and see
Note 8).
9. Assess across-plate reproducibility of screening conditions for
DSF experiment. If protein is not limiting, prepare 4 mL of
Quantitative Detection of Ligand Binding by DSF and CETSA HT
205
Perform all steps at room temperature unless otherwise indicated.
Ensure appropriate waste disposal throughout and appropriate
safety measures when using automated liquid handling devices.
3.1 Optimization
of Conditions for DSF
Experiments
The concentrations of protein and SYPRO
® Orange dye must be
optimized for the protein of interest in order to maximize reproducibility while minimizing reagent consumption. Figure 2a
depicts the plate layout and exemplar results for the optimization
experiment described below.
1. Prepare protein solutions at 1.25 μM, 2.5 μM, 5 μM, and
10 μM in DSF buffer by adding 300 μL of 10 μM protein in
DSF buffer to a 0.5-mL polypropylene tube. Mix 150 μL of
this solution with 150 μL of DSF buffer in a fresh tube. Take
150 μL of the resulting solution and mix with 150 μL of DSF
buffer in another tube. Repeat once more.
2. To a PCR instrument compatible 384-well microplate, e.g.,
4titude FrameStar 384 well skirted PCR plate, add 10 μL of
1.25 μM protein to wells A1–A12, 2.5 μM protein to wells B1–
B12, 5 μM protein to wells C1–C12, and 10 μM protein to
wells D1–D12 (see Note 5).
3. Briefly centrifuge the plate at 300 Â g to ensure that the
solution is at the bottom of the wells.
4. Add DMSO and SYPRO
® Orange to the PCR plate using a
liquid dispenser, e.g., D300 (Tecan), as shown in Table 1.
5. Seal the plate with the optical sealing film and centrifuge the
plate at 300 Â g for 1 min.
6. Precool the real-time PCR instrument by running a method to
hold the block temperature at 20
C for 5 min with a blank
plate (see Note 6).
7. When the precooling method finishes, immediately insert the
prepared PCR plate and run a method that will hold the
temperature at 20
C for 30 s and then run a temperature
gradient from 20 to 95
C at 1
C/min. Measure the fluorescence throughout the gradient (at least 1 acquisition per 1
C)
using filters compatible with SYPRO
® Orange (λ ex ¼ 470 nm,
λ em ¼ 570 nm, see Note 7).
8. Visually inspect the melt curves to choose the optimal protein/
dye combination for screening, considering reproducibility and
the signal/background ratio (see Fig. 2b for more detail and see
Note 8).
9. Assess across-plate reproducibility of screening conditions for
DSF experiment. If protein is not limiting, prepare 4 mL of
Quantitative Detection of Ligand Binding by DSF and CETSA HT
205