4. ImmunoAssay Buffer, 10Â (PerkinElmer).
5. Anti-Mouse IgG Alpha Donor beads (PerkinElmer).
6. Anti-Rabbit IgG (Fc specific) AlphaLISA Acceptor beads
(PerkinElmer).
7. AlphaScreen enabled plate reader, e.g., EnVision
(PerkinElmer).
2.4 Thermal Melt
Analysis in Live Cells
by CETSA
1. Cell culture flasks, e.g., T175 flask (ThermoScientific).
2. PCR strip tubes, e.g., MicroAmp
® 8-Tube Strip, 0.2-mL PCR
tubes (ThermoFisher).
3. Strip caps for PCR strip tubes, e.g., MicroAmp
® 8-Cap Strip,
Clear (ThermoFisher).
4. PCR strip-compatible microcentrifuge, e.g., MiniStar Silverline
(VWR).
5. Multi-block thermal cycler, e.g., SimpliAmp™ Thermal Cycler
(ThermoFisher).
6. Electronic adjustable tip spacing multichannel equalizer
pipette, e.g., E1-ClipTip™ (ThermoFisher).
7. Pipette
Tips,
e.g.,
125-μL
tips
for
ClipTip™
384 (ThermoFisher).
8. Centrifuge capable of spinning microplates, e.g., Heraeus Multifuge 1S centrifuge.
9. A well-validated tool compound known to bind the target
protein in cells (see Note 4).
2.5 Quantification
of Thermostable
Target Protein by
AlphaScreen
1. As in Subheading 2.3.
2. AlphaScreen enabled plate reader, e.g., EnVision
(PerkinElmer).
2.6 Screening
of Compounds
at a Single Optimized
Temperature by
CETSA HT
1. 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).
2. Liquid handling device capable of dispensing nanoliter
volumes, e.g., Echo 555 acoustic dispenser (LabCyte) or
D300 liquid dispenser (Tecan).
3. Liquid dispenser capable of dispensing microliter volumes,
e.g., Multidrop Combi and small cassette (ThermoScientific).
4. 384-well PCR machine, e.g., LightCycler480 II (Roche).
5. Liquid handing platform such as the Bravo liquid handler
(Agilent).
6. Tips, 10 μL, 384 in rack, compatible with the liquid handling.
7. As in Subheading 2.5.
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Joseph Shaw and Christopher Stubbs
5. Anti-Mouse IgG Alpha Donor beads (PerkinElmer).
6. Anti-Rabbit IgG (Fc specific) AlphaLISA Acceptor beads
(PerkinElmer).
7. AlphaScreen enabled plate reader, e.g., EnVision
(PerkinElmer).
2.4 Thermal Melt
Analysis in Live Cells
by CETSA
1. Cell culture flasks, e.g., T175 flask (ThermoScientific).
2. PCR strip tubes, e.g., MicroAmp
® 8-Tube Strip, 0.2-mL PCR
tubes (ThermoFisher).
3. Strip caps for PCR strip tubes, e.g., MicroAmp
® 8-Cap Strip,
Clear (ThermoFisher).
4. PCR strip-compatible microcentrifuge, e.g., MiniStar Silverline
(VWR).
5. Multi-block thermal cycler, e.g., SimpliAmp™ Thermal Cycler
(ThermoFisher).
6. Electronic adjustable tip spacing multichannel equalizer
pipette, e.g., E1-ClipTip™ (ThermoFisher).
7. Pipette
Tips,
e.g.,
125-μL
tips
for
ClipTip™
384 (ThermoFisher).
8. Centrifuge capable of spinning microplates, e.g., Heraeus Multifuge 1S centrifuge.
9. A well-validated tool compound known to bind the target
protein in cells (see Note 4).
2.5 Quantification
of Thermostable
Target Protein by
AlphaScreen
1. As in Subheading 2.3.
2. AlphaScreen enabled plate reader, e.g., EnVision
(PerkinElmer).
2.6 Screening
of Compounds
at a Single Optimized
Temperature by
CETSA HT
1. 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).
2. Liquid handling device capable of dispensing nanoliter
volumes, e.g., Echo 555 acoustic dispenser (LabCyte) or
D300 liquid dispenser (Tecan).
3. Liquid dispenser capable of dispensing microliter volumes,
e.g., Multidrop Combi and small cassette (ThermoScientific).
4. 384-well PCR machine, e.g., LightCycler480 II (Roche).
5. Liquid handing platform such as the Bravo liquid handler
(Agilent).
6. Tips, 10 μL, 384 in rack, compatible with the liquid handling.
7. As in Subheading 2.5.
204
Joseph Shaw and Christopher Stubbs