Several species of polar microalgae are able to live and thrive in the extreme environment found within sea ice, where ice crystals may reduce the organisms' living space and cause mechanical damage to the cells. Among the strategies adopted by these organisms to cope with the harsh conditions in their environment, ice-binding proteins (IBPs) seem to play a key role and possibly contribute to the success of microalgae in sea ice. Indeed, IBPs from microalgae predominantly belong to the so-called "DUF 3494-IBP" family, which today represents the most widespread IBP family. Since IBPs have the ability to control ice crystal growth, their mechanism of function is of interest for many potential applications. Here, we describe methods for a classical determination of the IBP activity (thermal hysteresis, recrystallization inhibition) and further methods for protein activity characterization (ice pitting assay, determination of the nucleating temperature).
Keywords: Antifreeze; Clifton nanoliter osmometer; Diatoms; Ice recrystallization inhibition (IRI); Ice-binding proteins; Lag time; Nucleation; Pitting assay; Recrystallometer; Sea-ice microalgae; Supercooling; Thermal hysteresis (TH).