Ultracentrifugation density gradient

Most organelle membranes, such as the tonoplast (6) and the Golgi apparatus (7), can be separated by density gradient ultracentrifugation of plant cell homogenates. However, other effective methods for the isolation of the plasma membrane (8,9) have been described. Moreover, another method that uses an aqueous two-phase system for the isolation of ER is also described (10). Those interested in these details for these methods should consult the original articles. 

Table 3.13 Density gradient ultracentrifugation of sub-cellular organelles Organelle Density (p)...

The site of assembly of the Lp(a) particle, by covalent linkage of apo-B100 to apo(a), is not definitively established. White et al. (W12) proved in baboon hepatocytes that inside the cell two types of apo(a) existed, of which only the larger form was recovered from the culture medium. The lower-molecular-weight form proved to be a precursor with a prolonged residence time in the endoplasmatic reticulum. Density gradient ultracentrifugation and immunoblot analysis showed that the majority of apo(a) was secreted into the medium in a... 

X. Sun, D. Luo, J. Liu, D.G. Evans, Monodisperse chemically modified graphene obtained by density gradient ultracentrifugal rate separation, ACS Nano, 4 (2010) 3381-3389. 

Separation of SWNTs based on chirality and diameter with surfactants has also been evidenced by density-gradient ultracentrifugation [86]. Finally, a recent study has demonstrated the separation of semiconducting nanotubes from metallic ones by chemical interaction of the former with attached amine-terminated silane molecules assembled on a silicon wafer. In a separate experiment, metallic nanotubes were also... 

For the convenience of the reader, we have outlined the method of sequential flotation employed in our laboratory for separating chylomicrons VLDL, LDL, HDLa, HDLs, VHDL, and d> 1.25 bottom (Table 1). This method, the result of years of experience, has been highly reproducible in terms of the normal human population examined in this laboratory. Such a method may not necessarily apply to dyslipoproteinemic states, where modifications may be necessary, depending on the type of abnormality under consideration. It should also be stressed that any lipoprotein isolated is in need of purification this may be achieved by ultracentrifugation based on the assumption that contaminants are in loose association with the main complex. Whenever this purification is not achieved, other methods may be used as outlined below. For a discussion of the application of density gradient ultracentrifugation to the study of plasma lipoproteins, the reader is referred to a recent review (L3). 

Several investigators have presented evidence for low molecular weight forms exhibiting urease activity. Hand (33), in 1939, obtained diffusion data indicating particles of 17,000 daltons or less that retained enzymic activity. More recently, sucrose density gradient ultracentrifugation (34)... 

M., A one-step separation of human serum high density lipoproteins 2 and 3 by rate-zonal density gradient ultracentrifugation in a swinging bucket rotor. J, Lipid Res. 23, 1342-1353 (1982). 

Fig- ( ) Gel electrophoresis patterns of immune serum and affinity purified antibodies [A], Density gradient ultracentrifugation (B), top pattern glucose oxidase, lower pattern mesquite antibody. 

Another method for determining homogeneity and molecular size of antibodies is density gradient ultracentrifugation [33], A sample of anti-... 

Jensen, R. H., and N. Davidson Spectrophotometric, potentiometric, and density gradient ultracentrifugation studies of the binding of silver ion by DNA. Biopolymers 4, 17 (1966). 

Sedimentation-FFF. Retention measurements give the effective particle mass m (buoyant mass). If the particle density is known, the particle mass m, particle volume Vp, and hydrodynamic diameter dH can be calculated [80,81]. Apart from the particle dimensions, the density can be determined as well [82] as the difference in the densities of the solute and the solvent, Ap, is linearly correlated to X. Fractionation can be used in regions where the solvent density is lower than the solute density (pps. The determination of particle density in a single experiment is possible by sedimentation-flotation focusing-FFF [72,73,83] analogous to density gradient ultracentrifugation. 

Figure 3. Potassium bromide density gradient ultracentrifugation of M. sexta hemolymph. The less dense yellow colored lipophorin floats above the layer of more dense ordinary proteins, including the blue insecticyanin. Arrow designates position of lipophorin in the gradient.

Figure 4. Distribution of [1 ]C—DDT in larval M. sexta hemolymph. 19 h after topical application, hemoTymph was subjected to density gradient ultracentrifugation as shown in Figure 3. Following centrifugation the tube was fractionated and the radioactivity in each fraction determined. Most of the labeled pesticide was found in the lipophorin fraction.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (4-15 percent acrylamide gradient slab) of M. sexta adult hemolymph following density gradient ultracentrifugation. Centrifuge tubes were fractionated and aliquots applied to the gel. 

Chung, B.H. (1980) Preparative and quantitative isolation of plasma lipoproteins rapid single discontinuous density gradient ultracentrifugation in a vertical rotor. 

The molecular size and molecular homogeneity of antibodies may be determined by a sucrose density-gradient ultracentrifugation method.31 32 Samples of 0.2 mL of 0.4% solutions of each antibody preparation are placed carefully on top of separate sucrose density-gradient tubes prepared from 5,10,20,30, and 40% sucrose. The tubes are centrifuged in a swinging... 

Density gradient ultracentrifugation revealed that the antibodies in the two sets had the same molecular size of 1.5 X 105 daltons. 

Density gradient ultracentrifugation is used to measure hpoprotein subclasses it is performed in a vertical rotor with measurement of cholesterol continuously in the fractions eluted from the gradient. Mathematical curve resolution derives the component lipoprotein profiles and allows calculation of their cholesterol concentrations. The method can determine concentrations of VLDL, IDL, LDL, Lp(a), and HDL cholesterol. LDL cholesterol subclasses can be expressed separately or combined to give a measurement similar to that provided by the Friedewald equation or beta-quantification. A disadvantage is that the procedure is technically demanding and requires instrumentation not usually available in clinical laboratories. 

Hokanson JE, Krauss RM, et al. Single vertical spin density gradient ultracentrifugation. Methods Enzymol 1986 128 181-209. 

Fig- 2. Percent of total FAEEs in fractions obtained by density gradient ultracentrifugation. Pooled sera from 15 intoxicated emergency-room patients were subjected to density gradient ultracentrifugation and serum fractions were isolated. Lipids were extracted and FAEEs were isolated by thin-layer chromatography and quantitated by gas chromatography. (From Doyle 1994 with permission.)... 

---