Protocols for Assessing the Nuclease Activity of MIF.

Macrophage migration inhibitory factor (MIF) exhibits nuclease activity critical in DNA damage pathways linked to neurodegenerative diseases and cancer. Here, we describe dual approaches for measuring this nuclease activity, combining biochemical and cellular analysis. Recombinant MIF is subjected to enzymatic assays with L1 DNA substrates, analyzing cleavage patterns via agarose gel electrophoresis and time-dependent kinetics over 5-30 min. SH-SY5Y cells treated with the DNA-damaging agent MNNG, with or without the MIF nuclease inhibitor PAANIB-1, undergo pulse-field gel electrophoresis to assess DNA fragmentation following cellular stress. These assays can be used to test MIF nuclease activity and screen MIF nuclease activity inhibitors.