Hi Robert,

I don't have an answer yet, but it's an interesting question. Could you help me and others by sharing some data? Also, I only now learned the term TSA from google, but I'm curious why you would combine these two techniques. SYPRO Orange appears to bind to hydrophobic patches of proteins that become available when they thermally unfold, but TSA's look like something you might use in an ELISA from what I found.

Dear Jeremy Pronchik thanks very much for your response. TSA in this case stands for thermal shift assay. And below is a description of the technique from a website.

"TSA is a highly parallelizable, cost-effective technique for studying protein stability and protein binders. It measures the thermal denaturation of a target protein by monitoring the fluorescence intensity of a dye that binds to exposed hydrophobic regions of the protein during heating. As the protein unfolds, the exposed hydrophobic regions increase, leading to an increase in fluorescence signal. Compounds, such as inhibitors or in general modulators, stabilize the three-dimensional conformation of the target protein and as a consequence, the temperature needed to denature the protein is higher"

Ah ok, I at least understand the problem now.

What are the temperatures where you see the fluorescence spike. If you could give a screenshot of the full set of data, it would help.

The high fluorescence at low temperatures for your protein could be due to:

1. Increased protein stability

2. Reduced protein denaturation

3. Enhanced fluorophore binding

4. Decreased quenching

5. Changes in protein conformation

Temperature-dependent fluorescence:

At lower temperatures:

- Protein movements slow down, reducing quenching.

- Fluorophores may bind more tightly, increasing fluorescence.

- Protein structure becomes more rigid, enhancing fluorescence.

Possible explanations:

1. Hydrophobic interactions: Low temperatures strengthen hydrophobic interactions, stabilizing protein structur

e.

2. Hydrogen bonding: Low temperatures enhance hydrogen bonding, stabilizing protein-fuorophore complex.

3. Reduced protein aggregation: Low temperatures minimize aggregation, increasing fluorescent protein population.

Consider:

1. Protein purification and quality

2. Buffer composition and pH

3. Fluorophore properties and concentration

4. Instrument settings and calibration

To confirm:

1. Perform temperature-dependent fluorescence titration.

2. Measure protein stability using circular dichroism (CD) or differential scanning fluorimetry (DSF).

3. Test protein-fuorophore interaction using fluorescence lifetime measurements.