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6 centrifugation (1)

6 centrifugation (1)






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    6 centrifugation (1) 6 centrifugation (1) Document Transcript

    • Centrifugation Centrifugation is a process used to separate particles or concentrate materialssuspended in a liquid medium. In biology, the particles are usually cells, subcellularorganelles, viruses, large molecules such as proteins and nucleic acids. Thetheoretical basis of this technique is the effect of gravity on particles (includingmacromolecules) in suspension. Two particles of different masses will settle in a tubeat different rates in response to gravity. Centrifugal force (measured as xg, gravity) isused to increase this settling rate in the centrifuge. The two common types of centrifugation are analytical and preparative; thedistinction between the two is based on the purpose of centrifugation. Analytical centrifugation involves measuring the physical properties of thesedimenting particles such as sedimentation coefficient or molecular weight. Optimalmethods are used in analytical ultracentrifugation. Molecules are observed by opticalsystem during centrifugation, to allow observation of macromolecules in solution asthey move in gravitational field. The samples are centrifuged in cells havingwindows that lie paralleled to the plan of rotation of the rotor head. As the rotorturns, the images of the cell (proteins) are projected by an optical system on to filmor a computer. The concentration of the solution at various points in the cell isdetermined by absorption of a light of the appropriate wavelength (Beers law isfollowed). The other form of centrifugation is called preparative and the objective is toisolate specific particles which can be reused. There are four basic types ofcentrifuges that you are likely to encounter in the laboratory:1. Clinical centrifuges - inexpensive tabletop centrifuges that can run at a speed of up to 3000 rpm. These can pellet cells, but not organelles or biomolecules. 1
    • 2. Microfuge - these can typically run at a speed of up to 14,000 rpm, sufficient speed to pellet nucleic acids and denatured proteins. Microfuges are specially adapted for small volumes of sample.3. High speed centrifuges - can run at speeds up to 25,000 rpm, sufficient to pellet cell nuclei and most biomolecules. These are often refrigerated.4. Ultracentrifuges - The ultracentrifuge is capable of reaching even greater velocities and requires a vacuum to reduce friction and heating of the rotor. It can run at speeds up to 75,000 rpm, sufficient to allow fractionation of biomolecules, for example: plasmid DNA, chromosomal DNA, and RNA. Ultracentrifuges are usually refrigerated and are very expensive and delicate pieces of machinery.RotorsRotors for a centrifuge are either fixed angles, swinging buckets, continuous flow, orzonal. Fixed angles generally work faster; substances precipitate faster in a givenrotational environment. The most common is a rotor holding 8 centrifuge tubes at anangle of 34 °C from the vertical (such as the Sorvall SS-34 rotor or the Beckman JA-20).Fixed angle rotors: In a fixed angle rotor, the materials are forced against the side ofthe centrifuge tube, and then slide down the wall of the tube. This action is theprimary reason for their apparent faster separation, but also leads to abrasion of theparticles along the wall of the centrifuge tube.Swinging bucket (horizontal rotors): For swinging bucket rotors, the materials musttravel down the entire length of the centrifuge tube and always through the media 2
    • within the tube. Since the media is usually a viscous substance, the swinging bucketappears to have a lower relative centrifugal force, which is it takes longer toprecipitate anything contained within. If, however, the point of centrifugation is toseparate a molecules or organelles on the basis of their movements through viscousfield, then the swinging bucket is the rotor of choice.Moreover, if there is a danger or scraping off an outer shell of a particle (such as theouter membrane of a chloroplast), then the swinging bucket is the rotor of choice.Most common clinical centrifuges have swinging buckets. Since the buckets are easyto interchange, this type of rotor is extremely versatile.Cell biologists employ zonal rotors for the large scale separation of particles ondensity gradients. Zonal rotors can contain up to 2 liters of solution and can workwith tissue samples measured in ounces (or even pounds).Rotor tubes:For biological work, the tubes are divided into functional groups: made of regularglass, Corex glass, or nitrocellulose. Regular glass centrifuge tubes can be used atspeeds below 3,000 RPM that is in a standard clinical centrifuge. For work in thehigher speed ranges, centrifuge tubes are made of plastic or nitrocellulose.Preparative centrifuge tubes are made of polypropylene (sometimes polyethylene)and can withstand speeds up to 20,000 RPM. These tubes should be carefullyexamined for stress fractures before use. A tube with a fracture will hold fluidsbefore centrifugation, but the cracks will open under centrifugal force. 3
    • Relative Centrifugal Force (RCF) The centrifugal force generated by a centrifuge is determined by the speed ofrotation and the distance of the sample from the axis of rotation. The centrifugal force isusually compared to the force of gravity and is reported as the relative centrifugal force(RCF) in g units. (A force of 1 g is equal to the force of gravity at the earths surface)The following formula relates the RCF to the rotor speed and diameter: RCF = (1.119 x 10-5) * (rpm)2 * rwhere r is the radius of rotation (the radius of the rotor) in cm and rpm is the centrifugespeed in revolutions per minute. The number "1.119 x 10-5" is a conversion factor thatallows us to use "rpm" and "g" units.The RCF is dependent upon the radius of the rotation of the rotor, the speed at which itrotates, and the design of the rotor itself (fixed angle or swinging bucket). Rotor speedand design can be held constant, but the radius will vary from the top of a centrifuge tubeto the bottom. If a measurement for the radius is taken as the mid-point, or as an averageradius, and all forces are mathematically related to gravity, then one obtains relativecentrifugal force, labeled as xg.Three r-values given (by the manufacturer) for a rotor: the maximum, from the center ofrotation to the bottom, top and middle of minimum and average r. These correspond tothe distances for the sample tube.Practical pointers1. Centrifuge sample containers come in a variety of shapes, sizes, and materials. Be careful to use the correct tubes. Glass tubes always require an adapter to cushion them because direct contact between the glass and a metal rotor will result in a 4
    • cracked or shattered tube. Adapters may slide over the tube like a sleeve or may be cushions that sit in the bottom of the rotor slot. (The "cushion" adapters may be found in clinical centrifuges but are not used in high speed centrifuges.)2. Tubes that are over-filled can spill their contents inside the rotor. For fixed-angle rotors it is especially important not to fill the tubes more than 2/3 to 3/4 full. Spills need to be wiped up immediately to prevent corrosion of the rotor. It is always a good practice to wash out a rotor when you are finished with it; even when theres no apparent spill, liquid from the tubes often spatters. Rinse the rotor with distilled water, dry with paper s to let any moisture drain. Wipe up any liquid in the chamber towels, and store upside down on top of paper towel of the centrifuge, then use a paper towel (dampened with distilled water) to wipe down the chamber. Dry the chamber well; if the refrigeration has been used, dont leave the door open any longer than necessary because condensation will build up on the chamber walls.3. Most important of all, a rotor must be balanced: tubes in opposite positions should be equal in weight. The tubes can be balanced by adding or removing liquid. An unbalanced centrifuge can vibrate violently, and horror stories abound about unbalanced ultracentrifuges crashing through walls.4. Finally, note that it is important to keep your samples cold during the lab by using chilled tools and glassware and by keeping the samples on ice whenever possible. Procedures that disrupt cell structure release degradative enzymes, which are less active at colder temperatures. 5