We are evaluating a HTP Fluorescence-based Thermal Shift (FTS) Assay to map ligands to various sensor-type proteins.
The theory behind the assay is based on monitoring protein melting profiles (Figure 1). Correctly folded proteins exhibit unfolding with an increase in temperature, resulting in exposure of hydrophobic regions and eventual protein aggregation. Ligand binding to a target protein can stabilize a protein's native state reflected in the increased melting temperature (Tm) of the bound protein. Measurement of the difference in melting temperatures of unbound and ligand-bound proteins can enable determination of ligand binding affinity constants. The assay involves monitoring changes in the fluorescence signal of SYPRO orange dye as it interacts with a protein undergoing thermal unfolding. The dye’s fluorescence signal is quenched in the aqueous environment of a properly folded protein in solution, but becomes unquenched when exposed to the protein’s hydrophobic core upon unfolding. The midpoint (Tm value) of the protein melting curve will increase in the presence of ligands that bind more tightly to the protein’s native state than the unfolded state.
Assay Method Highlights
- High throughput: FTS Assays are performed in 96-well thin-wall white PCR plates, but can also be implemented in a 384-well format. One plate can be screened in less than 30 minutes.
- General Instrumentation: FTS Assays can be run in any RT-PCR machine. We use the Stratagene Mx4000 or the Roche LightCycler 480 quantitative PCR instrument.
- Protein Independent Detection: FTS Assays use the SYPRO orange dye which has been used for this application in a variety of screening approaches. The wavelengths for excitation and emission are 490 and 575 nm, respectively. In addition, this dye has the advantage of a high signal to noise ratio.
- Requires Low Quantity of Sample: Typical reaction conditions will require a reaction volume of 20-40 μl with a protein concentration of 2-50 μM protein, typically < 50 μg per well, and 20-1000 uM ligand.
- High Density Screen: Pooled ligands are used as an initial step to assign protein binding to a specific ligand group. If a significant thermal shift is detected with a hit pool, the protein is then screened against all individual ligands in the pool to determine the specific binding ligand(s).
- Simple Tm Calculation: Temperature midpoint for the unfolding transition is determined using the melting curve analysis software provided with the instrument. The automatic melting temperature (Tm) calculation software was designed for molecular beacon confirmation but can be applied to determine melting curves for protein unfolding. The SYBR green Experimental method generates the negative first derivative (-R’(t)) plot of the melting curve raw fluorescence data (R), from which the Tm can be determined as the minimum peak.
FTS Assay Plate Set Up
Running FTS Assay on LC480 qPCR machine (Roche)
FTS Assay Data Analysis