One advantage of fluorescence detection is the high sensitivity,
which allows measurements even at concentrations below 1 nM
fluorophore and therefore makes it possible to determine binding
constants of high-affinity interactions that are not accessible with
absorbance or interference detection. Since proteins and nucleic
acids are normally not excitable at 488 nm, they have to be extrinsically labeled or proteins alternatively can be expressed as GFP or
YFP fusions. It should be checked, however, whether introduction
of the label influences the properties of the system. This is most
easily done by checking whether application of mixtures of the
labeled and unlabeled form of the fluorescing molecule produces
different results. Although the requirement for labeling might look
like a disadvantage at first sight, the fact that only the specifically
labeled molecule is detected allows the investigation of the behavior
Fig. 1 Analytical ultracentrifugation with fluorescence detection. (a) View into the rotor chamber of the
analytical ultracentrifuge ProteomeLab XL-I (Beckman Coulter, USA) that is equipped with an An-50 Ti rotor
with AUC cells and the fluorescence detection system (FDS, AVIV Biomedical, USA). The FDS is mounted above
the rotor and contains the light source, detector, and optical devices. (b) Schematic representation of the FDS
according to Nelson et al. [9]; for details, see text. (c) In an assembled AUC cell, the cell housing (1) holds the
two-channel centerpiece (3) sandwiched between two window assemblies (2). The cell assembly is closed at
the top using a screw ring gasket (5) and a screw ring (4). Filling holes at the front side of the assembled AUC
cells are sealed with plug gaskets and housing plugs (6). The housing shown is a special fluorescence cell
housing (Nanolytics Instruments, Germany), where the filling holes are shifted 4.5 mm to the top and a 9 mm
elevation is included at the bottom of the housing, allowing the placement of a 3-mm centerpiece at the top of
the housing without the need of extra spacers
400
Andrea Bogutzki and Ute Curth
which allows measurements even at concentrations below 1 nM
fluorophore and therefore makes it possible to determine binding
constants of high-affinity interactions that are not accessible with
absorbance or interference detection. Since proteins and nucleic
acids are normally not excitable at 488 nm, they have to be extrinsically labeled or proteins alternatively can be expressed as GFP or
YFP fusions. It should be checked, however, whether introduction
of the label influences the properties of the system. This is most
easily done by checking whether application of mixtures of the
labeled and unlabeled form of the fluorescing molecule produces
different results. Although the requirement for labeling might look
like a disadvantage at first sight, the fact that only the specifically
labeled molecule is detected allows the investigation of the behavior
Fig. 1 Analytical ultracentrifugation with fluorescence detection. (a) View into the rotor chamber of the
analytical ultracentrifuge ProteomeLab XL-I (Beckman Coulter, USA) that is equipped with an An-50 Ti rotor
with AUC cells and the fluorescence detection system (FDS, AVIV Biomedical, USA). The FDS is mounted above
the rotor and contains the light source, detector, and optical devices. (b) Schematic representation of the FDS
according to Nelson et al. [9]; for details, see text. (c) In an assembled AUC cell, the cell housing (1) holds the
two-channel centerpiece (3) sandwiched between two window assemblies (2). The cell assembly is closed at
the top using a screw ring gasket (5) and a screw ring (4). Filling holes at the front side of the assembled AUC
cells are sealed with plug gaskets and housing plugs (6). The housing shown is a special fluorescence cell
housing (Nanolytics Instruments, Germany), where the filling holes are shifted 4.5 mm to the top and a 9 mm
elevation is included at the bottom of the housing, allowing the placement of a 3-mm centerpiece at the top of
the housing without the need of extra spacers
400
Andrea Bogutzki and Ute Curth