The C2 domain is a membrane-targeting domain present in numerous signaling proteins that carry out a wide variety of biochemical functions (1-3). In many of these proteins, Ca2+triggers docking of the C2 domain to phospholipid vesicles. Several methods have been utilized to measure the Ca2+-triggered docking of C2 domain proteins, or their isolated C2 domains, to natural or artificial phospholipid membranes in vitro. These include sedimentation of proteins bound to natural membranes (4), synthetic vesicles (5) or sucroseloaded vesicles (6); copurification of radiolabeled vesicles with affinity-tagged proteins bound to agarose beads (7); isolation by size-exclusion chromatography of protein-vesicle complexes (8); monitoring intrinsic fluorescence changes in the C2 domain in response to membrane docking (9); and fluorescence resonance energy transfer (FRET) between protein and vesicles (10-12). Of these approaches, only fluorescence techniques allow measurement of protein docking to membranes in real time. This feature enables equilibrium experiments that: (1) monitor membrane docking in response to incremental changes in Ca2+concentration; (2) test the reversibility of the docking process; and (3) assess the Ca2+-independent component of membrane docking, all in a single sample. Moreover, FRET may be used to directly monitor the kinetics of membrane docking and release in stopped-flow fluorescence measurements (12) (see Note 1). Thus, FRET can measure both the equilibrium and rate constants for membrane association. [ABSTRACT FROM AUTHOR]