The two forms can be distinguished based on column chromatography and NMR. The active form has a higher affinity for Cdc20 than does the other form and blocks anaphase in oocyte extracts. In vivo, sequestration of only the active form thwarts the checkpoint.
The forms interchange in vitro very slowly. A fragment of Mad1, however, accelerates transformation of the inactive into the active form. Some Mad2 is always complexed with Mad1 in cells, so the question now is what tells Mad1 to toss off Mad2 (in its active form) so it can bind to Cdc20. ▪