I am assuming that you used a ~280 nm excitation filter to excite the tryptophan and tyrosine residues in your protein, but what emission filter setting did you use? Be aware that excitation at 280 nm will also react with a lot of other molecules other than Trp/Tyr and giving off fluorescent emissions at many other wavelengths than Trp/Tyr residues do. Therefore, you need to use a filter which cuts the recorded signal to read that at ~340 nm to only capture the Trp/Try fluorescence. I had the misfortune once to try reading intrinsic Trp/Tyr fluoresence of protein products with platereader and the data seemed completely nonsensical every time I tried it and I eventually gave up. It wasn't until months later when I had to open up the filter wheels (which nobody had opened in years) that I discovered that somebody had removed the 340 nm emission filter at some point (but not changed the settings in the software to reflect this), so where I thought I had only capturing the Trp/Tyr fluorescence signal, I was actually capturing all fluorescence signals.

What QPCR machine exactly did you use, as looking at current ones offered by Biorad none of them can use an excitation frequency of below 450 nm. Of course, they may have offered previous machines that did, but I can't see a reason why they would sell a QPCR machine that could go to such a low wavelength when no common fluorphores are excited by it (plus there would be many problems with other molecules like those in plastics being excited).

How have you processed the data? For data like this a standard way to make it easier to visualise is to take the first derivative of the fluorescence, as that can make it easier to see a definite peak.

Samuel Davis Ive actually already gave up on the analysis and switched to differential scanning calorimetry instead. like you, I only recently discovered that the excitation frequency didnt matched tryptophan's lol. Ive used FRET filter btw. I havent process the data just yet but Im confused. Why do we have to take the 1st derivative of the fluorescence?

Btw thank you so much for your answer!!

Fatin Safiudin No problem at all. DSC will certainly give your answer, although you have to be very careful with calorimetry methods as they are extremely sensitive to changes, you need to get used to doing the loading procedure correctly each time and they eat through protein like no tomorrow (I have a lot of experience using isothermal titration calorimetry to quantify protein-ligand binding events).

Taking the first derivative makes it a lot more obvious where you are getting changes in the fluorescence over the temperature ramp, which makes it much easier to fit the points at which the protein is unfolding. I've attached a comparison showing the raw fluorescence (as a ratio of the emitted fluorescence at 340 nm:350 nm) and the same data as the first derivative. This was for human GABA transporter 1 that I had purified a few years ago (unfortunately this is the best picture I can easily find to hand at the moment showing what I mean) and I measured the unfolding using a Nanotemper Tycho instrument, where you pick up a tiny amount of the protein in a glass capillary and it measures the intrinsic fluorescence emitted at 340 and 350 nm while applying a rapid temperature ramp. You get the analysed data in about 5 minutes. The machine itself is unfortunately quite expensive, but they are very useful for rapidly working out how stable a protein is and you can do up to 8 capillaries at once with the basic Nanotemper Tycho instrument (more expensive instruments do various other things, can read plates etc).

If you were using Sypro Orange dye with your protein then you would have likely got the data you need with the QPCR machine you have (as I mentioned in your other question). In my case with trying to measure Trp fluorescence, it was extremely annoying as I was reading the specific fluorescence of an attached GFP derivative to a protein that I was purifying and that data made perfect sense (luckily, as that was my primary objective). The complementary Trp fluorescence data though made no sense, even though I was exciting it at the correct wavelength of 280 nm and capturing it at 340 nm. I'd previously done Trp fluorescence in a cuvette format successfully, so I couldn't understand why it was so nonsensical. Of course, the reason was obvious once I found out months later that there was actually no 340 nm emission filter in the path...

Hey Samuel Davis im glad i ran into you! btw my qpcr machine couldnt display the data per wavelength as far as i checked. But I did take the first derivative of the data i ve got (without calculating the ratio first like you did) and wow i did get a nicer peak at 55 °C. However, as i told you before i perform dsc and the melting temperature that I got was 46 °C. I get that dsc is more reliable than intrinsic labelled tsa, but the different is too big. ive used similar concentration and everything so what do you think? btw i didnt have an accurate model of my protein so the trp residues whereabout is a mere theory

Fatin Safiudin I would completely disregard what your QPCR assay shows. Unfortunately, as the minimum wavelength that the machine goes to is nowhere near that needed for Trp/Tyr fluorescence, you are measuring the fluorescence of something completely different over the temperature ramp. This could be literally anything in the buffer or the protein. I would concentrate on getting several (ideally at the very least 3, preferentially 5 or more) sets of the reproducible DSC data. To get reliable data from calorimetry methods, you need to make each experiment as closely identical as possible. For any dilution etc, use the same batches of buffer that the protein was purified into (ideally use dialysis or at the least size exclusion to exchange your protein into the final buffer) and immediately before each DSC run filter your sample through a 0.2 um spin filter. Are you planning on looking at how your protein changes in different buffers or in the presence of a binding partner such as ligand/other protein?

The 330:350 nm ratio (I misremembered it at 340:350 nm) isn't strictly necessary, but by dual measurement of both and then expressing as a ratio in theory allows capture of a wider range of how Trp/Tyr residues are changing and being exposed over the temperature ramp. The Nanotemper instruments do all the measurement and analysis (the more advanced models can also work out things like the deltaG and other parameters) and give the data as in various different ways. While the first derivative is useful for visually showing the data, ideally the raw data should be shown as well. Although you may not know the exact location, it is important to remember that the location of any Trp/Tyr residues doesn't strictly matter with measuring their fluorescence. It is all about how these residues are changing in position as the protein conformation changes once something is changed, like adding a binding partner or changing temperature etc. If you can get some Sypro Orange dye to use with your QPCR machine, you will be able to use the built-in thermal shift assay and that will be useful to compare to the DSC data.