Instruments available: Stratagene MX3005P real time PCR
Equipment location: New Biochemistry 00-059
Equipment coordinator: Dr David Staunton
Equipment charge: £25/run
Sample requirement: approx. 1ug of protein per well
Thermofluor or Differential Scanning Fluorimetry
This technique allows the determination of protein unfolding in a high throughput format for screening ligands or buffer conditions in terms of protein stability. A fluorophore with a low-fluorescence quantum yield in water (e.g. SYPRO Orange) is used as an indicator. Partitioning of the dye into the protein unfolded state results in a significant increase in fluorescence and this can be used to follow protein unfolding in a real time PCR instrument that is programmed for incremental temperature increases rather than PCR cycles. Fluorescence increases as the proportion of unfolded protein increases with increasing temperature and a Tm derived from the midpoint transition or differential plot. Comparison of these plots under different buffer conditions can identify thermal shifts where the Tm has increased due to greater stability or ligand binding. The 96-well format and the small amount of protein per well (approximately 1 µg) make this ideal for high throughput screening.
Ligand screening. New proteins can be screened for ligands by Tm shift. Thermofluor can be used with screens such as Silver Bullet to identify ligands for crystallisation trials.
Condition screening. Buffer conditions such as pH and salts can be explored for recombinant proteins with Thermofluor to improve their solution behaviour.
Stratagene MX3005P real time PCR
The MX3005P takes 96 well RT-PCR plates and can be programmed to follow fluorescence over a wide temperature ramp. A typical temperature ramp rates range from 0.1-10 °C/min but generally 1 °C/min. The fluorescence in each well is measured at 1 °C intervals, over a temperature range spanning the typical protein unfolding temperatures of 25-95 °C. Even though the instrument is equipped with a heated lid condensation on the cover film becomes problematic over 90 °C and so the technique is not suitable for proteins with high Tm (>70 °C).
We use Thermo Scientific AB-0700/W 0.2ml Non-skirted Low Profile 96-well PCR plate and AB-1170 Absolute QPCR Seal sheets.
Specific protein and dye concentrations and sample volumes need to be defined by experimental assay development but a starting point would be;
· 25ul reaction volume per well
· 1:3,000 dilution of SYPRO Orange commercial stock (e.g. Sigma S5692) as a final concentration
· 1ug protein per well
Briefly centrifuge (~1000 × g, 1 min) the sealed assay plate to mix and ensure the sample is all at the bottom of the well before loading in the instrument.
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