Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Monitoring Ca 2+ -Calmodulin Concentration 371
Fig. 3. The effect of Ca 2+ –CaM on the excitation and emission spectra for FIP-
CB SM–38 (K d = 45 nm). (Left panel) A series of emission spectra obtained after
successive additions of pure CaM to a 100 nM indicator solution as described in the
text. The emission of the EYFP acceptor, centered at approx 535 nm, decreases with
each addition until the indicator is saturated. The emission of the ECFP FRET donor,
centered at approx 480 nm, behaves in a reciprocal manner. Indicator fluorescence
was excited at 430 nm. (Right panel) The corrected excitation spectra corresponding
to the CaM-free and CaM-saturated indicator are shown. EYFP acceptor emission was
monitored at 530 nm.
(Monmouth Junction, NJ) QM–1 photon-counting fluorometer. Excitation and
emission spectra are corrected for monochromator artifacts using data supplied
by the manufacturer. A relative correction for wavelength-dependent variations
in illumination intensity is also applied to excitation spectra. Samples are incubated
in a 1 cm × 1 cm fused silica cuvet held at 25°C in a water-jacketed cuvet
holder equipped with a magnetic stirrer. In general, 5-nm slits are used on both
the emission and excitation monochromator input and output light paths. Spectra
for titration of pure FIP-CB SM–35 with Ca 2+ –CaM are shown in Fig. 3, and
binding isotherms for indicators with three different K d values for Ca 2+ –CaM
are presented in Fig. 4 (see Note 1).
1. After determination of background fluorescence and water Raman scattering,
indicator is added to a cuvet containing 2 mL of 25 mM Tris-HCl, 0.1 M KCl,
0.5 mM MgCl 2; pH 7.4 to produce the desired final concentration. If fluorescence
data are to be used for a K d determination, then the lowest acceptable indicator
concentration should be used.
2. Small aliquots (approx 2 µL) of a concentrated CaM solution (approx 10 µM) are
added directly to the cuvet. Spectra are taken after each addition. It is not necessary
to correct for the small changes in total volume. Although contaminating