S100 proteins are distinct dimeric EF-hand Ca-binding proteins that can bind Zn, Mn, and other transition metals with high affinity at two sites in the dimer interface. Certain S100 proteins, including S100A7, S100A12, S100A8, and S100A9, play key roles in the innate immune response to pathogens. These proteins function via a "nutritional immunity" mechanism by depleting essential transition metals in the infection that are required for the invading organism to grow and thrive. They also act as damage-associated molecular pattern ligands, which activate pattern recognition receptors (e.g., Toll-like receptor 4, RAGE) that mediate inflammation. Here we present protocols for these S100 proteins for high-level production of recombinant protein, measurement of binding affinities using isothermal titration calorimetry, and an assay of antimicrobial activity.