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![[Figure 5]](co5097fig5mag.jpg)
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Figure 5
Combined approach of in situ DLS and SEC: a mixture of thyroglobulin (3 mg ml−1), conalbumin (7 mg ml−1) and RNase A (15 mg ml−1) after passing a Superdex 200GL 5/150 column with a constant flow rate of 200 µl min−1. Proteins in solution were instantly separated and individually analysed inside the capillary by in situ DLS. (a) The total scattering intensity (count rate; blue squares) of the solution is plotted against the retention time and volume of the column. After 100 s of DLS data recording the protein sample solution was injected (`I'). The observed void volume of the column corresponds to approximately 1 ml of retention volume. The obtained peaks were assigned to the individual proteins of the mixture, according to their specific elution properties and upon analysis by SDS–PAGE (d). (b) In parallel, the time course of the radius distribution (blue spheres) for the same experiment was recorded by DLS. Hydrodynamic radii of 9.5 nm (550 kDa), 4.2 nm (85 kDa), 2.9 nm (35 kDa) and 2.0 nm (17 kDa) were determined for the peaks 1 to 4 in the respective chromatogram (a). Therefore, the first peak was assigned to dimeric thyroglobulin (MW: 660 kDa), followed by monomeric conalbumin (MW: 75 kDa; peak 2) as well as dimeric (peak 3) and monomeric (peak 4) RNase A (MW of the monomer: 14 kDa). The diameter of the blue spheres represents the relative abundance of the detected particle radii weighted by scattering intensities in arbitrary units. (c) A set of ACFs is displayed comparatively. The average ACF of the putative thyroglobulin dimer peak [peak 1, (a)] determined by in situ DLS is shown in black. Further, the ACFs of thyroglobulin (red), conalbumin (green) and RNase A (magenta) are shown as obtained from a cuvette instrument, as well as the ACF for the mixture, determined by the same cuvette instrument (blue), for comparison and verification of the protein separation. (d) In order to confirm the protein content of the SEC fractions (a), an aliquot of the identified count-rate peak fractions was subjected to SDS–PAGE analysis. The column input consisting of thyroglobulin, conalbumin and RNase A (`input') is shown for comparison of the molecular weights. Thereby, the identification of the proteins from peaks 1 to 4 based on the determined hydrodynamic radii (b) is confirmed by SDS–PAGE. |
![[Figure 5]](co5097fig5.jpg)
 | JOURNAL OF SYNCHROTRON RADIATION |
ISSN: 1600-5775
