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Swinging bucket rotors
Swinging bucket (SW) rotors are held in a horizontal position, relative to the axis of rotation during centrifugation. Due to this position, the path length that the particles need to travel is the equivalent of the full length of the tube (Figure 2). A SW rotor system consists of a rotor body, which attaches to the centrifuge drive. The buckets are secured into the arms of the rotor body using trunnion pins.
PURIFICATION PROTOCOLS
Figure 4. Vertical rotors – at speed, at rest inside the rotor, at rest outside the rotor
Near-vertical tube rotors
Figure 2. SW rotors - at speed, at rest inside the rotor, at rest outside the rotor
Fixed angle rotors
When using fixed angle (FA) rotors, the tubes are fixed between 20 and 45° relative to the axis of rotation. As seen in figure 3, the path length is therefore smaller than the length of the tube, making run times faster. During centrifugation, the gradient must re-orientate relative to the centrifugal force. The cavities in these rotors range in volume from 0.2 ml to 1 L, and speeds from single digits to 1,000,000 x g can be achieved, making them ideal for pelleting and the isopycnic banding of DNA.
Tubes are held at a narrow angle of <10° relative to the axis of rotation (Figure 5), providing a shorter path length and consequently, shorter run times. This allows for components that do not band under separation conditions to pellet at the bottom or float at the top of the tube, away from the band of interest. These rotors are ideal for density gradient separations where short run times are vital.
Choosing the correct density gradient media is also important, especially for the purification of cells, viruses, subcellular membranes and macromolecules. Sucrose is the most commonly used gradient media for rate-zonal centrifugation as well as for the separation of cellular organelles and viruses, whereas alkali metal salts, including CsCl, are most often used for isopycnic centrifugation and the purification of nucleic acids and other macromolecules. Cells and individual organelles are most effectively purified using iodinated media, e.g. nycodenz. Colloidal silica (percoll) and polysaccharides (Ficoll) are effective across the range of cells, viruses and organelles.
INCREASING PELLETING EFFICIENCY
The clearing factor (k-factor) is one of the most important parameters for consideration when selecting a rotor. It is essentially a calculation of the relative pelleting efficiency and can be used to estimate the time required to pellet a particle of a known sedimentation coefficient (S) to the bottom of a tube [1]. The value of k depends on the maximum angular velocity of a centrifuge, as well as the minimum and maximum radius of the rotor:
(r=radius in cm, rpm = revolutions per minute, rmax = maximum radial distance a particle can be from the rotor’s axis, rmin = minimum radial distance a particle can be from the rotor’s axis of rotation, t = time (hours), S = sedimentation coefficient in Svedberg units)
Once the centrifugation time has been calculated using this equation, the formula below can be used to calculate the run time for a new rotor.
Figure 5. Near vertical rotors – at speed, at rest inside the rotor, at rest outside the rotor
Figure 3: FA rotors – at speed, at rest in the rotor, at rest outside the rotor
Vertical tube rotors
Vertical tube rotors fix the tubes parallel to the axis of rotation, allowing bands to separate across the diameter of the tube, as shown in Figure 4. Although a reorientation of the gradient is required, the path length is the shortest possible. These rotors can therefore be used for density gradient separations where a short run time is required, but cannot be used for pelleting purposes, since the sediment will adhere to the tube wall. As specialised rotors, their most common use is for isopycnic separation, specifically for the banding of DNA in Cesium Chloride.
Matching the correct rotor to each application type can be confusing, but in order to maximise experimental efficiency, it is important that this is performed as accurately as possible. In general:
• Sample pelleting: FA rotors enable efficient reorientation of the sample solution in the tube, for a fast run time
• Isopycnic separation: FA rotors combine a shallow density gradient with reorientation. Thus, the width of sample bands decreases, while the distance between the bands increases for an easy extraction
• Rate-zonal separation: SW rotors provide a long path length for extended run times and excellent sample band resolution
• Isopycnic or rate-zonal separation requiring short run times: vertical rotors provide a shorter path length for fast run times
(Ta = run time for the new rotor; Tb = run time for the original rotor, ka = k factor for the new rotor, kb = k factor for the original rotor)
These formulae can also be manipulated to increase rotor efficiency and speed-up the complete process. Since the k factor varies directly and exponentially with the ratio of the radii of a given sample, decreasing the sample volume will in turn increase efficiency.
[1]. Schieber G L & O’Brien T W. Extraction of proteins from the large subunit of bovine mitochondrial ribosomes under non-denaturing conditions. The Journal of Biological Chemistry 1982:257(15); 8781 – 8787.
CONCLUSION
There are a great number of technical considerations which need to be understood and taken into account when selecting appropriate centrifugation techniques and accessories.
By accurately matching these options to the different application types, functionality and experimental precision can be maximised. Since each option has a subsequent impact on the selection of the next component, the experiment as a whole needs to be taken into consideration when each choice is made.
In essence, it is vital that researchers are fully aware of all available options and are able to make an informed choice so that optimal resulting data are gained from each use.
Interested in publishing a Technical Article?
Contact Gwyneth Astles on +44 (0)1727 855574 or email:
tamsyn@intlabmate.com
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