I have been performing thermal melt analysis of a protein that can form dimers or other higher order structures. Thus, we were not too surprised to see a melting profile that seems to have two species (one melting out at ~53 and the other at 66o C). I have tested the effects of pH on the stability of this protein and noticed something odd (see Figure). pH doesn't seem to have much of an effect on the position of these two melt peaks (i.e., I still see the 53o and 66o C species). However, the shape of the melt curves seemed to be affected. I am new to this assays, but based on my reading get the impression that the absolute value of RFU doesn't matter, and it is peak position that is important. However, I am seeking some advice on the relative peak height changes that I am seeing. I perform the assay in a BioRad CFX Connect with Applied Biosystem Protein Thermal Shift Dye. The melting protocol ramps from 25o C – 95o C with 0.5o C increments and a hold time at each increment of 10 sec. I do incubate the samples for 30 min at room temperature before subjecting them to melting.