Isolation and characterization of ice-binding proteins from higher plants

The characterization of ice-binding proteins from plants can involve many techniques, only a few of which are presented here. Chief among these methods are tests for ice recrystallization inhibition activity. Two distinct procedures are described; neither is normally used for precise quantitative assays. Thermal hysteresis assays are used for quantitative studies but are also useful for ice crystal morphologies, which are important for the understanding of ice-plane binding. Once the sequence of interest is cloned, recombinant expression, necessary to verify ice-binding protein identity can present challenges, and 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 ice-binding proteins. Here we have attempted to assist researchers wishing to isolate and characterize ice-binding proteins from plants with a few methods critical to success.