Analytical ultracentrifugation procedure

In this section, we indicate the general factors that an experimentalist should consider while setting up a cation-mediated equilibrium RNA folding experiment, provide a stepwise protocol for sample preparation and outline the data collection procedure using the absorption optics of the analytical ultracentrifuge. 

G4. Gidez, L. I, Miller, G. J., Burstein, M., and Eder, H. A., Analyses of plasma high density lipoprotein subclasses by a precipitation procedure Correlation with preparative and analytical ultracentrifugation. In Report of the High Density Lipoprotein Methodology Workshop (K. Lippel, ed.), pp. 328-340. U.S. Department of Health, Education and Welfare. NIH publication No. 79-1661, Bethesda, Md., 1979. 

Centrifugation data from measurements with an analytical ultracentrifuge are useful for characterizing glycoenzymes. Pure gly-coenzymes yield symmetrical patterns when examined by this procedure. Sedimentation measurements can be made at several concentrations of the enzyme, and a number of physical constants characteristic of the molecule can be calculated from these data. On the basis of such measurements and calculations, it was determined that chloroperoxidase, which contains carbohydrate, is homogeneous with respect to molecular size,21 and it was concluded that the carbohydrate component in the preparation of chloroperoxidase studied is chemically linked to the protein. 

Here we describe studies of the interaction of interleukin-6 (IL-6) with a soluble form of its cell surface receptor (sIL-6R). A procedure utilising a competition approach is presented which allows the determination of the equilibrium constant in solution thus avoiding any potential problems associated with deviation in kinetic characteristics upon surface immobilisation. In addition, binding characteristics of stable monomeric and dimeric forms of IL-6 are presented to demonstrate both the drastic influence of solute multivalency on kinetic and equilibrium properties and the importance of auxiliary techniques such as analytical ultracentrifugation for the interpretation of SPR data. 

Analytical Ultracentrifugation. Sedimentation coefficients were determined with an ultracentrifuge (Beckman L8-70M) fitted with a schlieren analytical attachment. Photographic images of schlieren (refractive index gradient) profiles were analyzed with a profile projector (Nikon, model VI0) using standard procedures (13). 

Although it is possible to read schheren films with automatic scanning devices (13) that produce data in a form suitable for direct use on a computer, we have elected to use a simple manual procedure in preparing analytical ultracentrifuge data for computer analysis. This not only reduces cost in the developmental phase of the computer solution, but also allows greater control over such reading problems as accurate sens-... 

At present no simple routine method is available for separation, identification and quantitation of lipoproteins. Accurate procedures are based on preparative or analytical ultracentrifugation, reviews of which were given by de Lalla and Gofman (1954), Jahnke and Scholtan (1960), Furman et al. (1961), Pezold... 

Fig. 6. Analytical ultracentrifugal film record demonstrating the various lipoprotein species commonly present in human serum. The lipoprotein concentrate for this run was obtained by Preparative Procedure T3rpe 1. Solution density 1.063 g./ml. at 26°C. Rotor speed is 52,640 r. p. m.

Brown rice, wheat and bean. Several analytical procedures have been developed for rice grain. In the case of rice straw, finely cut samples are added to water and allowed to stand for 2 h, then extracted with acetone. Unpolished rice grain samples are milled with an ultracentrifuge mill and sieved through a 42-mesh screen prior to extraction. 

It would be of considerable interest to extend the technique just presented to problems involving nonlinear equations because there are many situations in ultracentrifugation where nonideality is a dominant feature. Furthermore, it is known (4, 14) that even for two-component systems with nonideality the theory for estimating the sedimentation constant based on a diffusion-free (c = 0) approximation can lead to systematic error. Therefore, the development of an approximate procedure for nonlinear equations would be useful for further progress in analytical separation methods. 

A quantitative method has been developed to separate free and graft copolymers in an ABS sample. The ABS powder is dispersed in MEK and then introduced into the cells of a preparative ultracentrifuge. After the reproducibility of the procedure was ascertained, the method was used to determine the grafting parameters of samples polymerized under specific conditions. This analytical technique is well suited to demonstrate how the grafting efficiency or grafting density is influenced by various polymerization conditions such as mercaptan content, monomer flow rate, emulsifier content, or polybutadiene content. The effects of other variables such as temperature, the initiator system, and characteristics of the polybutadiene latex can also be demonstrated. 

The turbidity due to lipemia may be reduced by ultracentrifugation or precipitation techniques, but these procedures may alter apparent analyte concentrations (Thompson and Kunze 1984). Synthetic lipid emulsions are often used to test for effects caused by lipemia, but these emulsions do not always mimic the physicochemical effects of hyperlipemia caused by perturbations of lipid metabolism (Bornhorst, Roberts, and Roberts 2004). 

It is now apparent from electrophoresis and ultracentrifuge measurements, correlated with separation by neutral salts (167, 208, 209, 210, 211), that the latter are incapable by any known means of accurately fractionating protein mixtures without complicating the operations to a point at which they are of little use as practical analytical procedures. Thus, Dole (212) found that the albumin-globulin ratio measured electrophoretically was about 2/3 of the ratio fotmd by chemical fractionation, an observation which was also checked by Pillemer and Hutchinson (196) by use of a methanol fractionation which was checked against electrophoresis. 

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