Generally, Tycho is very robust against other sources of fluorescence in the buffer. In most cases, these kinds of things change the total brightness, or they may change the direction of the unfolding profile, or otherwise contribute, but the unfolding signal is still visible. This is one reason why we use the ratio signal to look at protein unfolding: signals from other sources get canceled out, since they don’t show the characteristic shift upon unfolding.

However, we have seen a few rare cases where the fluorescence signal of the other substance is so high, and the brightness of the target protein is so low, that the unfolding signal can be masked. As an example, below is data from protein samples containing FLAG peptide. FLAG peptide is used to elute FLAG tag containing proteins from columns. FLAG peptide contains a tyrosine residue (sequence is shown below). Tyrosine itself is not very bright, but since its 330 nm brightness is a lot higher than its 350 nm brightness, it has a low ratio signal (around 0.2). FLAG peptide is typically used at pretty high concentrations, for example 0.5 mg/ml. If the concentration and/or intrinsic brightness of the target protein is very low, the FLAG tag signal can potentially mask the protein’s unfolding signal, even though the tag itself doesn’t show an unfolding signal.

The example below shows streptavidin (purple) and FLAG peptide (pink), both separately and mixed. The unfolding profile of streptavidin is clearly decreased in amplitude in the presence of FLAG peptide, and its absolute ratio signal is lowered (green). At very low protein concentrations, the protein unfolding signal may not be visible at all anymore and all that is left is the FLAG peptide signal (a “flat” line at around 0.2 ratio). The critical concentration of FLAG peptide of course depends on the concentration and individual brightness of the protein.

Typically, elution from such a column is followed by more purification steps to remove the remaining FLAG peptide from the sample, because it could interfere with other methods too. Subsequent samples containing less or no FLAG peptide will look fine again in Tycho.

Flag peptide sequence: N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C