Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

372 Persechini
Fig. 4. Titration with CaM of the acceptor fluorescence of indicators constructed
using L6 (�), L2 (�) or L3 (�) linkers. These data were determined using indicators
based on the EBFP/EGFP FRET pair that is no longer used in the laboratory. Hence,
the acceptor fluorescence emission at 505 nm was monitored, and fluorescence was
excited at 380 nm. We have found that the GFP variants used to construct an indicator
have no effect on its affinity for Ca 2+ –CaM; this is appears to be determined solely by
the linker. Data were fit to Eq. 1 (�,�) or Eq. 2 (�), and the derived apparent K d
values are given in the figure.
levels of Ca 2+ (approx 5 µM) should be sufficient to saturate the Ca 2+ -binding
sites on CaM when it is bound to indicator, we add 0.1 mM CaCl 2 to ensure that
the free-Ca 2+ concentration is not limiting. CaM is adsorbed to surfaces, especially
plastics, and this can result in significant losses from dilute solutions. We
add 0.1 mg/mL BSA to all buffers to prevent this.
3. 5–10 mM BAPTA may be added at the end of an experiment to verify that the
interaction between CaM and the indicator is wholly Ca 2+ -dependent. A high
CaM concentration can also be added to investigate the possibility of a lowaffinity
Ca 2+ -independent interaction with the indicator. We have seen no evidence
of such an interaction with any CaM indicator at CaM concentrations
as high as 10 µM, which is similar to estimates of the total CaM concentration
in fibroblasts (19).
4. If the bound and free concentrations of Ca 2+ –CaM are approximately the same
(“Michaelis-Menten” conditions), then data for the fractional change in fluorescence
produced by each addition of Ca 2+ –CaM can be fit to Eq. 1.