Density Gradient Separations in Vertical Tube, Near Vertical Tube, Fixed Angle, and Swinging Bucket Rotors: A Comparative Study

Over the years, researchers have used density gradient techniques for separating viruses and macromolecular species such as proteins, subcellular organelles, and nucleic acids. Brakke first used these techniques,1 and a publication by Griffith2 provides a thorough discussion of the rate zonal and isopycnic density gradient procedures. Both fixed angle and swinging bucket rotors have been used for these two methods. The vertical tube rotors with the tube angle fixed parallel to the axis of rotation are also being used for density gradient studies in the ultracentrifuge.

This paper compares separations of similar samples obtained with these four rotor designs. Run times for the rate zonal method were calculated to provide the best resolution for each rotor. Additionally, the rotor instruction manuals were used to obtain optimum rotor speeds for isopycnic banding of DNA in cesium chloride (CsCl) gradients.

Experimental

The models L5-75 and L8-80 ultracentrifuges (Beckman Coulter, Fullerton, CA) were used. Rotors used were the SW 55Ti and SW 28 swinging bucket rotors (Beckman Coulter); F65L-6 × 13.5 mL and F50L-8 × 39 mL fixed angle rotors (FIBERLite Centrifuge Inc., Santa Clara, CA); and VTi 65, NVT 65, and VTi 50 vertical tube rotors (Beckman Coulter). After centrifugation, the gradients were monitored as discussed by Griffith.2

Rate zonal separation (sucrose gradients)

  1. Proteins: a) Bovine serum albumin (BSA), fibrinogen, and a mixture of BSA and fibrinogen were placed on sucrose gradients (10–40% w/w) in the F65L-6 × 13.5 mL, VTi 65, and SW 55Ti rotors. The rotors were run at maximum speeds for 3 hr, 3 hr, and 16 hr, respectively. The run temperature was 5 °C. The short-column (half-filled or short tubes) method by Griffith2 was used in the F65L-6 × 13.5 mL rotor, while the VTi 65 and SW 55Ti rotors had completely filled tubes. Sample volumes were the same in each rotor (0.2 mL, 1%). b) The two molecular forms of human mammary estrogen receptors (4S and 8S) were run in the F65L-6 × 13.5 mL and NVT 65 rotors. Sucrose gradients (10–40% w/w) were used and the rotors had similar loads (0.2 mL). Maximum rotor speeds were maintained for 3 hr and 2.5 hr, respectively. The run temperature was 5 °C.

    A separate experiment was done to show the shifting of the 8S molecular form of the receptor to the 4S position when 0.4 M KCl was added to the sample and gradient. After centrifugation, the model 250 liquid scintillation counter (Beckman Coulter) was used to identify the separated components. No studies were done with the swinging bucket rotor because this separation is documented in the literature. Run times for similar gradients in swinging bucket rotors with full tubes would have been 16 hr at 5 °C. Pavlik et al.3–5 used short-column methods for estrogen receptors, and run times for these studies were 3 hr.

  2. Subcellular particles (polysomes from rat liver): Rat liver polysomes prepared by the method of Noll6 were run on a 10–40% w/w sucrose gradient in the VTi 50, F50L-8 × 39 mL, and SW 28 rotors. The short-column method and similar tubes were used in the F50L-8 × 39 mL and SW 28 rotors. The VTi 50 rotor had full tubes. The sample load was placed on the gradients in the three rotors (2 mL, 40 μg/mL). At 5 °C, rotor speeds and run times were as follows: SW 28 rotor—27,000 rpm for 150 min, F50L-8 × 39 mL rotor—50,000 rpm for 40 min, and VTi 50 rotor—50,000 rpm for 35 min.

Isopycnic banding of DNA

  1. Two DNAs—Micrococcus luteus (1.73 g/ mL) and λ bacteriophage (1.71 g/mL)—were mixed in tris–EDTA (ethylenediaminetetraacetic acid) buffer to obtain an absorbance of 2.0 AUFS at 260 nm. The rotors chosen to observe the separation of this mixture were the SW 55Ti, F65L-6 × 13.5 mL, and NVT 65. The density of the buffered DNA mixture was adjusted with CsCl to 1.60, 1.65, and 1.70 g/mL, respectively, and the run times for the rotors were 16 hr. In the NVT 65 and F65L-6 × 13.5 mL rotors, each tube was loaded with 13.5 mL of CsCl solution, whereas 2 mL was loaded in the SW 55Ti rotors and run at 50,000 rpm. The NVT 65 and F65L-6 × 13.5 mL rotors were run at 65,000 rpm, and the temperature for each run was 20 °C.

    The DNA experiment in the NVT 65 and F65L-6 × 13.5 mL rotors was repeated to observe separations in 5 hr. The F65L-6 × 13.5 mL rotor was loaded with 5.0 mL CsCl solution to use the short-column method.

  2. Samples of crude lysate from E. coli bacteria containing plasmids pBR 322 were prepared by the method of Katz et al.7 CsCl solutions were adjusted to densities of 1.55 g/mL, and 3 mL of the nucleic acids containing ethidium bromide (EtBr) solution were added to the CsCl density to fluoresce the DNA. The solutions were run in the VTi 65 and NVT 65 rotors at their maximum speeds, and the run time and temperature were 16 hr at 20 °C; the F65L-6 × 13.5 mL was run at 60,000 rpm at the same temperature with filled tubes. After the centrifugation was terminated, long-wave UV light was used to fluoresce the DNA zones in the centrifuge tubes. The lower zone containing the plasmid DNA was removed by piercing the tube walls with a hypodermic needle and syringe. Thin-walled polyallomer tubes manufactured by Seton Scientific Co. (Los Gatos, CA) were used for this purpose. The EtBr was extracted from the plasmids with CsCl saturated n-butanol followed by low-speed centrifugation (6000 rpm for 15 min). The recovered pellet was resuspended in tris–HCl buffer and analyzed for purity by agarose gel electrophoresis.

Results and discussion

Rate zonal separations (sucrose gradients)

Figure 1 - Rate zonal separations of BSA and fibrinogen in the SW 55Ti swinging bucket rotor.

Figure 2 - Rate zonal separations of BSA and fibrinogen in the F65L-6 × 13.5 mL fixed angle rotor.

Figure 3 - Rate zonal separations of BSA and fibrinogen in the VTi 65 rotor.

Figure 4 - Rate zonal separations of the two molecular forms of estrogen receptors from human mammary tumor in the F65L-6 × 13.5 mL fixed angle rotor.

Figure 5 - Rate zonal separations of the two molecular forms of estrogen receptors from human mammary tumor in the VTi 65 rotor.

Figure 6 - Rate zonal separation of polysomes from rat liver.

  1. Proteins: a) Figures 1–3 show that the swinging bucket rotor provided the best separation of the three rotors, but with the longest run times. Shorter run times are possible with the short-column method without a reduction in sample load. Studies by Pavlik et al. have shown that the run times for proteins in swinging bucket rotors have been reduced by 75% when the short column method is used.3 b) Figures 4 and 5 show that both run times were similar in the VTi 65 and F65L-6 × 13.5 mL rotors. However, the separated zones in the vertical tube rotor were much wider than in the F65L-6 × 13.5 mL rotor because the zones were separated in larger gradient volumes than in the fixed angle rotor. This corresponds to the geometric differences in the gradient reorientation process between the F65L-6 × 13.5 mL rotor and NVT 65 rotor. The result is therefore better separation of the zones with the fixed angle rotor. 
  2. Subcellular particles (polysomes): Figure 6 shows the separation of polysomes in the three rotors. Again, the swinging bucket rotor provided the best separation of the three. Although the same sample load was used in the three rotors, the peak heights of the polysomes were taller in the swinging bucket rotor than in the F65L-6 × 13.5 mL rotor. However, there was loss of the polysomes in the vertical tube after the zones moved toward the centrifugal area during separation.

Isopycnic banding (DNA samples)

Figure 7 - Isopycnic separations of DNA from Micrococcus luteus and λ bacteriophage.

Figure 7 shows the separations of the two DNAs in the three rotor types. Comparable separations in the same run times were observed in the near vertical tube and fixed angle rotors. The swinging bucket rotor did not completely separate the two components.

Figure 8 - Isopycnic separations of DNA from Micrococcus luteus and λ bacteriophage.

The 5-hr runs in Figure 8 show less separation of the DNA than the 16-hr runs in Figure 7; however, this separation may be acceptable for some experiments. During tube fractionation, the volume of CsCl solution separating the two DNA zones in both rotors for the 16-hr run was measured. The zones in the NVT 65 rotor had a slightly larger volume (0.5 mL) than the F65L-6 × 13.5 mL rotor (0.4 mL). This corresponds to the geometric differences between the reorientation processes in the two rotors. Additionally, the reorientation process is less in the F65L-6 × 13.5 mL rotor than in the NVT 65, and the peak-to-valley ratio is better in the fixed angle rotor than in the near vertical tube rotor. This resulted in better separation of the zones with the fixed angle rotor.

Figure 9 - Purification of pBR 322 plasmids from E. coli by buoyant density gradient centrifugation. Note the RNA contaminant falling off the centrifugal tube wall after the run in the vertical tube (a) and near vertical tube (b) rotors. The contaminant is pelleted in the fixed angle (c) rotor during the run (see text).

Photographs of the fluorescent EtBr–DNA zones separated in the vertical tube, near vertical tube, and fixed angle rotors are shown in Figure 9a–c. The upper zone represents chromosomal DNA, while the lower zone represents plasmid DNA. EtBr also binds to the RNA from the crude lysate. The RNA contaminant is observed pelleted at the bottom of the tube in the F65L-6 ×13.5 mL rotor in Figure 9c. The flocculent contaminant RNA is observed falling from the centrifugal wall of the vertical tube rotor VTi 65 in Figure 9b, and the RNA contaminant is observed falling from the wall of the near vertical tube rotor NVT 65 in Figure 9a.

Conclusion

Rate zonal separation (sucrose gradients)

When proteins are run in sucrose gradients, the swinging bucket rotors provide slightly better separation between components. Since these rotors have the longest pathlength, the run times will be longer than in the vertical tube and fixed angle rotors. During rate zonal separations, particles are separated according to size differences. With multicomponent systems (more than two sample components) or when the sedimentation coefficient differences are very small (less than 4S units), the rotor with the longest pathlength and the highest centrifugal force should be chosen for the best separation between components. If shorter run times are essential, the short-column method2 can be used in swinging bucket rotors. The fixed angle rotor will also provide short run times; however, the separation between components will be better than in the near vertical or vertical tube rotors.

The swinging bucket rotors are therefore recommended for sucrose gradients when the best separation is needed. If shorter run times are essential, the short-column method2 can be used in swinging bucket rotors. The fixed angle will also provide short run times; however, the separation between components will be better than in the near vertical or vertical tube rotors.

Isopycnic banding (cesium chloride gradients)

In this method, samples such as nucleic acids separated according to their density differences in cesium chloride solutions. After the zones have reached their isopycnic position in the tubes, there is no further sedimentation through the gradient. At this point, the zones cease to spread. The best separation therefore depends on the volume of solution that separates the sample zones at the end of the run. The geometry of the rotors is such that during centrifugation, the vertical tube or near vertical tube rotor has the largest volume of solution separating two zones. Flamm et al.8 and Fisher et al.9 demonstrated that swinging bucket rotors have the smallest volume of solution separating the two zones since there is no reorientation of the solution during deceleration of the rotor.

The fixed angle rotor gave similar separations in the same run time as the vertical tube rotor; however, there is a greater than 0.1-mL difference in the volume of CsCl solution separating the two DNA zones as shown in the NVT 65 and the F65L-6 × 13.5 mL rotors. Gradient reorientation in the NVT 65 rotor caused the zones to be larger, thus giving a loss of resolution, as shown in Figures 7 and 8.

When the fixed angle and vertical tube rotors were used to separate plasmid DNA, the run times were similar. With the fixed angle rotor, the contaminating RNA was pelleted at the bottom of the tubes and away from the purified plasmid DNA in a single run. This was reported by Wong et al.10 and Griffith.11 Little12 reported that the RNA contaminant fell off the tube wall of the NVT 65 rotor during centrifugation. The author also mentioned that varying the concentrations of Triton X-100 (Rohm and Haas, Philadelphia, PA) in the sample preparation could prevent the RNA contaminant from adhering to the centrifuge tube walls. However, using Triton X-100 to prevent RNA from adhering to the tube wall would not prevent the plasmid DNA from being contaminated with the RNA. Maniatis et al.13 reported that minor quantities of contaminating RNA greatly reduce the specific activity of 32P-end labeled DNA required for Maxam-Gilbert DNA sequencing.14 This was verified by agarose gel electrophoresis.

Investigators have been using a modified prepurification procedure for plasmid isolation. The procedure adds a phenol extraction followed by ethanol precipitation of the DNA to the standard polyethylene glycol method reported by Katz et al.7 Since all of the contaminating protein and RNA are not removed by the extraction, the residual contaminants often recontaminate the sample by falling from the wall of the tube before the plasmid band can be extracted. For this reason, users of vertical tube rotors may perform a second overnight run to further purify the plasmid DNA. The fixed angle rotors are therefore recommended for plasmid DNA separations, especially when it is essential to remove all contaminants. The higher force fields generated by most fixed angle rotors permit separations to be completed in run times equal to those made in the vertical or near vertical tube rotors. Reduced run times at the higher g-forces for fixed angle rotors can be calculated for short pathlength (cone top) polyallomer tubes using the K-Factor formula for these tubes in the fixed angle rotors. The tubes are manufactured by Seton Scientific Co.

References

  1. Brakke, M.K. Density gradient centrifugation: a new separation technique. J. Am. Chem. Soc.1951, 73, 1847–8.
  2. Griffith, O.M. Techniques of Preparative, Zonal, and Continuous Flow Ultracentrifugation, 3rd ed. Beckman Instruments, Inc., Spinco Div.: Palo Alto, CA, 1979.
  3. Pavlik, E.J.; Rutlege, S. Estrogen-binding properties of cytoplasmic and nuclear estrogen receptors in the presence of Triton X-100. J. Steroid Biochem. 1980, 13, 1433–41.
  4. Griffith, O.M. Rapid density gradient centrifugation using short column techniques. Anal. Biochem. 1978, 90, 435–43.
  5. Goral, J.E.; Wittliff, J.L. Comparison of glucocorticoid binding proteins in normal and neoplastic mammary tissues of the rat. Biochemistry1975, 14, 2944–52. 
  6. Noll, H. Polysomes: analysis and structure and function. In Techniques in Protein Biosynthesis, Vol. 2, pp 101–79. Campbell, P.N.; Sargent, J.R., Eds. Academic Press: New York, NY, 1966.
  7. Katz, L.; Kingsbury, D.T.; Helinski, D.R. Stimulation by cyclic adenosine monophosphate of plasmid deoxyribonucleic acid–protein relaxation complex. J. Bacteriol. 1973, 114, 577–91.
  8. Flamm, W.G.; Bond, H.E.; Burr, H.E. Density gradient centrifugation of DNA in a fixed angle rotor. A higher order of resolution. Biochem. Biophys.Acta1966, 129, 310–7.
  9. Fisher, W.D.; Cline, G.B.; Anderson, N.G. Density gradient centrifugation in angle head rotors. Anal. Biochem. 1964, 9, 477–82.
  10. Wong, T.K.; Nicolau, C.; Hofshneider, P.H. Appearance of β-lactamase activity in animal cells upon lipid mediated gene transfer. Gene1980, 10, 87–94.
  11. Griffith, O.M. Rapid isolation of bacterial plasmid DNA by isopycnic centrifugation in fixed angle rotors. Applications Data DS-591. Beckman Instruments, Inc.: Palo Alto, CA, 1981.
  12. Little, S.E. Plasmid separations in NVTTM near vertical tube rotors. Applications Data DS-770. Beckman Instruments, Inc.: Palo Alto, CA, 1998.
  13. Maniatis, T.; Fritsch, E.F.; Sambrook, J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1982.
  14. Maxam, A.M.; Gilbert, W. Proc. Natl. Acad. Sci. USA 1977, 74, 560–4.

Dr. Griffith is Director of Research, FIBERLite Centrifuge Inc., 422 Aldo Ave., Santa Clara, CA 95954, U.S.A.; tel.: 408-988-1103; 408-988-1196; e-mail: Mgriffith@piramoon.com.

Comments