different concentrations of small ions will result in a signal offset.
Whereas the interference optical system is very useful at high concentrations where absorbance is no longer linear, it is not as sensitive as the absorbance optical system at low protein concentrations,
where peptide bond absorption can be detected in the far UV.
Here we will focus on the detection by the third optical module
that is available in analytical ultracentrifugation, the fluorescence
detection system. In our study, we used a Beckman Coulter ProteomeLab XL-I equipped with an Aviv Biomedical fluorescence
detection system (FDS), where the sample is excited by light emitted from a laser at a wavelength of 488 nm and the emission is
detected through a 505–565-nm bandpass filter [8, 9]. After the
rotor loaded with the sample containing cells is mounted into the
vacuum chamber, the FDS is installed above the rotor (Fig. 1a).
The mode of operation is similar to that of a confocal microscope
(Fig. 1b): the excitation light is reflected by a dichroic mirror and
focused through a condensing lens into the sample; the same lens is
used as an objective lens for the emitted light that passes subsequently through the dichroic mirror and the band pass filter. The
emission light is then focused on a pinhole and detected by a
photomultiplier tube (PMT). In order to measure the fluorescence
intensity of the sample as a function of the radial position, the whole
FDS unit is moved along the radial axis by a stepping motor; this
type of radial scan can be taken continuously during the whole
sedimentation process (Fig. 2a).
AUC samples by default are loaded in two-channel centerpieces
(Fig. 1c), where the channels are sector-shaped to prevent collision
of the radially sedimenting molecules with the sidewalls, which
would otherwise result in convection and disturbance of the concentration gradients. For absorbance and interference measurements, one sector is typically filled with the sample and the other
one with the respective buffer to allow for measurements relative to
the buffer signal. Fluorescence measurements do not need a reference beam, and thus both sectors can be filled with sample solution.
Therefore, in an 8-hole rotor (Fig. 1a), up to 14 samples can be
analyzed in one run since the eighth hole has to be reserved for the
accommodation of the FDS calibration cell that is required for
radial calibration and determination of the angular position of
each cell channel [8, 9]. Absorbance and interference measurements require that the light passes through the whole path length
of the solution and intensities are detected below the rotor. Fluorescence intensities, however, are detected above the rotor and
therefore excitation is performed best in the region directly below
the upper window of the AUC cell in order to keep the inner-filter
effect as small as possible. Otherwise, this effect, which is a result of
reduced light intensity due to the light absorption [10] or scattering of the sample, would result in a nonlinearity between fluorescence intensity and concentration.
Analysis of Protein-DNA Interactions by AUC
399
Whereas the interference optical system is very useful at high concentrations where absorbance is no longer linear, it is not as sensitive as the absorbance optical system at low protein concentrations,
where peptide bond absorption can be detected in the far UV.
Here we will focus on the detection by the third optical module
that is available in analytical ultracentrifugation, the fluorescence
detection system. In our study, we used a Beckman Coulter ProteomeLab XL-I equipped with an Aviv Biomedical fluorescence
detection system (FDS), where the sample is excited by light emitted from a laser at a wavelength of 488 nm and the emission is
detected through a 505–565-nm bandpass filter [8, 9]. After the
rotor loaded with the sample containing cells is mounted into the
vacuum chamber, the FDS is installed above the rotor (Fig. 1a).
The mode of operation is similar to that of a confocal microscope
(Fig. 1b): the excitation light is reflected by a dichroic mirror and
focused through a condensing lens into the sample; the same lens is
used as an objective lens for the emitted light that passes subsequently through the dichroic mirror and the band pass filter. The
emission light is then focused on a pinhole and detected by a
photomultiplier tube (PMT). In order to measure the fluorescence
intensity of the sample as a function of the radial position, the whole
FDS unit is moved along the radial axis by a stepping motor; this
type of radial scan can be taken continuously during the whole
sedimentation process (Fig. 2a).
AUC samples by default are loaded in two-channel centerpieces
(Fig. 1c), where the channels are sector-shaped to prevent collision
of the radially sedimenting molecules with the sidewalls, which
would otherwise result in convection and disturbance of the concentration gradients. For absorbance and interference measurements, one sector is typically filled with the sample and the other
one with the respective buffer to allow for measurements relative to
the buffer signal. Fluorescence measurements do not need a reference beam, and thus both sectors can be filled with sample solution.
Therefore, in an 8-hole rotor (Fig. 1a), up to 14 samples can be
analyzed in one run since the eighth hole has to be reserved for the
accommodation of the FDS calibration cell that is required for
radial calibration and determination of the angular position of
each cell channel [8, 9]. Absorbance and interference measurements require that the light passes through the whole path length
of the solution and intensities are detected below the rotor. Fluorescence intensities, however, are detected above the rotor and
therefore excitation is performed best in the region directly below
the upper window of the AUC cell in order to keep the inner-filter
effect as small as possible. Otherwise, this effect, which is a result of
reduced light intensity due to the light absorption [10] or scattering of the sample, would result in a nonlinearity between fluorescence intensity and concentration.
Analysis of Protein-DNA Interactions by AUC
399