Detection of Emission: Emitted fluorescence is generally detected
by a photomultiplier after passage through cutoff and/or band-pass
filters selected to pass fluorescence while excluding any scattered
exciting light (see Note 4). It is important that the filters selected
maximize the signal change relative to the total signal.
Lamp Selection: Xenon arc lamps have a relatively smooth emission
spectrum while mercury or xenon/mercury lamps have several
intense emission bands which can be used when doing time-based
acquisition at a single wavelength. Emission from deuterium or
quartz halide lamps is less intense but is also less noisy, and these
lamps can be used in absorbance measurements and for fluorescence excitation in the visible region.
Slit Widths: A large slit width can be used to increase the light
intensity for fluorophores with a large Stokes shift (the wavelength
difference between the excitation and emission maxima). If the
Stokes shift is small then the excitation slit width may need to be
reduced to exclude scattered light from the photomultiplier. Alternatively, the wavelength of the exciting light may be set to a shorter
wavelength than the excitation maximum.
Time Constant: The signal-to-noise ratio (S/N) in rapid kinetic
measurements is proportional to the square root of the instrumental time constant and should be selected to be <10% of the half time
of the fastest process being observed (see Note 3). The S/N ratio
can also be improved by averaging several individual records or,
with some instruments, by collecting data at very high sampling
rates and averaging appropriate blocks of data to give the individual
time points.
In most cases, the data may be collected using linear time scales
(see Note 5). Analysis of kinetic transients by fitting one or more
exponential terms to the curves obtained is often straightforward,
and the software supplied with commercially available equipment is
generally adequate (see Subheading 3.5).
2.3 Samples Used
in This Study
Wild-type and cysteine-containing mutants of Drosophila calmodulin were prepared and purified as described elsewhere [12]. The
Asn111Cys calmodulin mutant was labeled with dansyl maleimide
using standard methods. CaM-dependent protein kinase I
(CamKI) was prepared and purified as described [13].
Peptides corresponding to the target sequences from CaMKI
(CaMKIp: IKKNFAKSKWKQAFNATAVVRHMRK ) and neuromodulin (NMp: ATKWQASFRGHITRKKLKG) were synthesized
in-house with and without an N-terminal dansyl label. The chromophoric calcium chelator Quin 2 was obtained from local
suppliers.
88
Stephen R. Martin and Maria J. Schilstra
by a photomultiplier after passage through cutoff and/or band-pass
filters selected to pass fluorescence while excluding any scattered
exciting light (see Note 4). It is important that the filters selected
maximize the signal change relative to the total signal.
Lamp Selection: Xenon arc lamps have a relatively smooth emission
spectrum while mercury or xenon/mercury lamps have several
intense emission bands which can be used when doing time-based
acquisition at a single wavelength. Emission from deuterium or
quartz halide lamps is less intense but is also less noisy, and these
lamps can be used in absorbance measurements and for fluorescence excitation in the visible region.
Slit Widths: A large slit width can be used to increase the light
intensity for fluorophores with a large Stokes shift (the wavelength
difference between the excitation and emission maxima). If the
Stokes shift is small then the excitation slit width may need to be
reduced to exclude scattered light from the photomultiplier. Alternatively, the wavelength of the exciting light may be set to a shorter
wavelength than the excitation maximum.
Time Constant: The signal-to-noise ratio (S/N) in rapid kinetic
measurements is proportional to the square root of the instrumental time constant and should be selected to be <10% of the half time
of the fastest process being observed (see Note 3). The S/N ratio
can also be improved by averaging several individual records or,
with some instruments, by collecting data at very high sampling
rates and averaging appropriate blocks of data to give the individual
time points.
In most cases, the data may be collected using linear time scales
(see Note 5). Analysis of kinetic transients by fitting one or more
exponential terms to the curves obtained is often straightforward,
and the software supplied with commercially available equipment is
generally adequate (see Subheading 3.5).
2.3 Samples Used
in This Study
Wild-type and cysteine-containing mutants of Drosophila calmodulin were prepared and purified as described elsewhere [12]. The
Asn111Cys calmodulin mutant was labeled with dansyl maleimide
using standard methods. CaM-dependent protein kinase I
(CamKI) was prepared and purified as described [13].
Peptides corresponding to the target sequences from CaMKI
(CaMKIp: IKKNFAKSKWKQAFNATAVVRHMRK ) and neuromodulin (NMp: ATKWQASFRGHITRKKLKG) were synthesized
in-house with and without an N-terminal dansyl label. The chromophoric calcium chelator Quin 2 was obtained from local
suppliers.
88
Stephen R. Martin and Maria J. Schilstra