Isolation and Characterization of Ice-Binding Proteins from Higher Plants

The characterization of ice-binding proteins (IBPs) from plants can involve many techniques, a few of which are presented here. Chief among these methods are tests for ice recrystallization inhibition, an activity characteristic of plant IBPs. Two related procedures are described, both of which can be used to demonstrate and quantify ice-binding activity. First, is the traditional "splat" assay, which can easily be set up using common laboratory equipment, and second, is our modification of this method using superhydrophobic coated sapphire for analysis of multiple samples in tandem. Thermal hysteresis is described as another method for quantifying ice-binding activity, during which ice crystal morphology observations can be used to provide clues about ice-plane binding. Once ice-binding activity has been evaluated, it is necessary to verify IBP identity. We detail two methods for enriching IBPs from complex mixtures using ice-affinity purification, the "ice-finger" and "ice-shell" methods, and we highlight their advantages and limitations for the isolation of plant IBPs. Recombinant IBP expression, necessary for detailed ice-binding analysis, can present challenges. Here, a strategy for recovery of soluble, active protein is described. Lastly, verification of function in planta borrows from standard protocols, but with an additional screen applicable to IBPs. Together, these methods, and a few considerations critical to success, can be used to assist researchers wishing to isolate and characterize IBPs from plants.