A fluorescence detection system for the analytical ultracentrifuge and its application to proteins, nucleic acids, and viruses

Abstract

A fluorescence detection system was developed for the analytical ultracentrifuge Spinco model E. Fluorescence is excited by a laser beam which is focussed into the cell and illuminates an area with a dimension of 60 ~tm in radial direction. For scanning the laser beam is moved in radial direction. After passing the cell, the laser beam is quenched by a carbon light trap and a set of optical filters. Fluorescence emission intensity is monitored by a photomultiplier located behind the light trap and the set of filters. The sensitivity of the detection system was tested by applying it to the sedimentation analysis of proteins and nucleic acids. Bovine serum albumin (BSA) was covalently labelled with the fluorescence-dye fluorescein-isothiocyanate (FITC), and its sedimentation coefficient could be determined even if BSA was analyzed in a concentration as low as 10-10 M. Nucleic acids were labelled non-covalently by the intercalating dye ethidium bromide. Only 8 ng RNA were needed for the determination of the sedimentation coefficient. The particular advantages of the fluorescence detection system were exploited for the establishment of a new method for quantitative virus detection. To tobacco mosaic virus (TMV) a monoclonal anti-TMV antibody from mouse was bound, and to this a second, anti-mouse antibody that carried the fluorescence-label HTC was attached. Either by UV-irradiation or by incubation with glutaraldehyde, the first antibody was covalently crosslinked to TMV, and the second antibody to the first. In CsC1 density centrifugation with fluorescence detection as little as 3.2 ng virus/80 IA or 6 x 10 s virus particles/ml were recorded in a well expressed band at the corresponding buoyant density. Tenfold lower concentration would result still in a significant band. The sensitivity compares well with those of the most advanced techniques from immunology. Due to the specific labelling of viruses by antibodies it will be possible to carry out quantitative physical characterization of virus containing samples without purifying the virus. Future applications of the fluorescence detection system and of the virus detection technique are discussed.

A multiwavelength emission detector for analytical ultracentrifugation

“…9 Additionally, integral uorescence intensities of particle and protein ensembles were used for analysis in AUC. [10][11][12] Combining spectral information at several wavelengths 13 led to the development of multiwavelength extinction AUC (MWL-AUC) 14 extending AUC analysis towards complex and oen coupled size-dependent hydrodynamic, thermodynamic and optical particle properties opening up vast new possibilities, including e.g. the identication of different species 4,5 or the determination of two-dimensional size distributions of anisotropic plasmonic particles.…”

Are fluorescence-detected sedimentation velocity data reliable?

“…The noise characteristics and potential sources of systematic errors for these systems have been described [6]. Fluorescence detectors for the analytical ultracentrifuge have also been developed [7-9] and are now commercially available (AU-FDS, AVIV Biomedical). Fluorescence detection greatly enhances AUC sensitivity and selectivity and allows analysis of high affinity interactions as well as labeled molecules present in complex media such as serum or in the presence of high concentration of crowding agents [10,11].…”

NUTS and BOLTS: Applications of fluorescence-detected sedimentation

“…While a custom-built fluorescence detector was described for the Model E analytical ultracentrifuge [6], its use was restricted to applications where low concentration detection was necessary [7]. The AU-FDS is the first commercially available fluorescence detection system for the XL-I AUC, and its confocal design extends the useable concentration range by reducing inner filter effects and minimizing artifacts resulting from steep refractive index gradients [4].…”