The figure below illustrates the structure of an IgG antibody and an exemplary nanoDSF unfolding profile of an IgG. IgG molecules consist of four polypeptide chains: two heavy chains (blue) and two light chains (red). The ‘arms’ of the Y-shaped molecule contain the variable antigen binding sites and are therefore commonly referred to as the Fab region (fragment, antigen-binding). The ‘foot’ of the Y is responsible for the antibody’s immunological properties and is called Fc region (fragment, crystallizable). The Fc region can be subdivided into the C_H2 and C_H3 domains (domains 2 and 3 of the constant parts of the heavy chains, respectively). In thermal unfolding experiments, three separate peaks are often visible, representing the three unfolding events of C_H2 region, Fab region, and C_H3 region. Typically, the C_H2 region unfolds first (at the lowest temperature), followed by Fab and C_H3.

Left panel: Molecular structure of an IgG. Heavy chains are colored blue, light chains are colored red. The three regions that are often seen to unfold separately are the Fab region and the C_H2 and C_H3 domains of the Fc region. Right panel: Thermal unfolding profile of an IgG. The top part shows the ratio of integrated fluorescence at 350/330 nm and the bottom part the corresponding first derivative plotted against the temperature. Vertical lines indicate melting onset (first line from the left) and unfolding transition temperatures. The antibody domains corresponding to the three distinctive unfolding events are indicated.

The interpretation of an IgG unfolding profile is not always as straightforward as shown here. Depending on the exact molecular structure of the IgG, not all three regions may show as separate unfolding events. In some cases, two unfolding events may overlap or even occur simultaneously, precluding a clear separation. Some of the factors that influence the thermal stability of the separate antibody domains are: amino acid sequence and class of IgG, glycosylation pattern and other post‑translational modifications, buffer/formulation conditions and excipients. Also, the height of the unfolding peak does not necessarily allow conclusions about the corresponding domain.