In this article we will discuss about the Centrifugation Technique of Molecular Biology:- 1. Basic Principle of Centrifugation Technique 2. Types of Centrifugation Technique.
Basic Principle of Centrifugation Technique:
The principle of the centrifugation technique is to separate the particles suspended in liquid media under the influence of a centrifugal field. These are placed either in tubes or bottles in a rotor in the centrifuge. Particles differing in sizes, shape and density are separated as their sedimentation rate is different.
The centrifugal force is generated by rotating the rotor of the centrifuge at a high speed. Besides normal and high speed centrifuge there is a very high speed centrifuge known as Ultracentrifuge, which is developed by Theodor Svedberg in 1940.
This instrument is designed to produce centrifugal forces up to several hundred thousand times which can separate and purify subcellular organelles, proteins, nucleic acids and several macromolecules. Thus the ultracentrifuge has opened up a new line in many types of fundamental studies in Cell Biology, Biochemistry and Molecular Biology.
The rate of sedimentation of a particle in a centrifugal force can be shown in the following:
Now, the particles will not move if the densities of the particles and the medium are equal. If the densities of the particles are greater than the medium, they will move toward the bottom of the centrifuge tube while they will remain at the top of the tube, if the particles are lighter than the medium.
The centrifugal force produced by the centrifuge is measured by the gravity units as:
Which is generally shown as a multiple of g, i.e., gravitational field of the earth. Sometimes it is also expressed as R.C.F (Relative Centrifugal Field) which is the ratio of the weight of the particle in the centrifugal field to the weight of the same particle acted on by gravity.
On the basis of this principle of separation, particles are separated depending on their densities, size, centrifugal force, time of separation etc. Different cell components are separated in the following order—whole cells and cell debris first followed by nuclei, plastids, mitochondria, lysosomes, microsomes, fragments of endoplasmic reticulum and ribosome.
The method of separation becomes complicated when the particles are not spherical, which requires some complicated formula for calculation.
In case of Ordinary rotors as used in the preparative centrifuge, the centrifugal field does not remain uniform, because the radial dimension of a particle will vary according to the position in the centrifuge tube (rmin and rmax).
The particle will have a greater centrifugal field as it is further away from the axis of rotation. This occurs both in the fixed angle and swing- away rotor (Fig. 7.1). Hence, the centrifugal field is calculated from the average radius of rotation (rav.) of the column of liquid in the centrifuge tube.
The details of maximum and the method of calculation of relative centrifugal field (R.C.F.) are generally given in the manual of the centrifuge.
The sedimentation rate of a particle can also be expressed as sedimentation coefficient (s) which is the sedimentation rate per unit of centrifugal field. The sedimentation values depend on the solvent-solute systems.
As the sedimentation coefficient of many of macromolecules is very small, the basic unit is taken as 10~13 seconds and is designated as Svedberg unit (S). For example, the Ribosomal RNA showing sedimentation values as 5 x 106-13 seconds is said to be 5S (5 Svedberg units).
Sedimentation coefficients of some of the macromolecules are shown in Table 7.1.:
Types of Centrifugation Technique:
There are generally 4 types of centrifuges:
(1) Clinical Bench Centrifuges,
(2) High Speed Refrigerated Centrifuges,
(3) Continuous flow Centrifuges, and
The last type can again be classified into two types:
i. Preparative and
ii. Analytical Ultracentrifuges.
(i) Clinical Bench Type Centrifuge:
These are the most simple type of centrifuge used in many laboratories for routine type of work, particularly for sedimenting yeast cells, blood cells or any coarse and medium particles. The maximum speed of this type of centrifuge is between 4,000 to 6,000 r.p.m. with a ‘g’ of 3,000 to 7,000.
Sometimes the cooling arrangement can also be made in this type of centrifuge. Now some ‘Microfuges’ are available where maximum speed of 8,000 to 13,000 r.p.m. with ‘g’ value of about 10,000 can be made using small Eppendorf tubes.
(ii) High Speed Refrigerated Centrifuge:
These instruments are used to isolate organelles, to purify and isolate soluble proteins, microorganisms etc. with a speed of about 25,000 r.p.m. having ‘g’ value of 60,000. Both fixed angle and swing-out rotors can be used here. But this speed of the instrument is not sufficient for centrifuging ribosomes and viruses.
(iii) Continuous Flow Centrifuge:
It is also one type of high speed centrifuge where the rotor is slightly modified or specially designed one. In this type there is a continuous flow of the medium in the centrifuge tube. Here the cells or particles axe sedimented against the wall and the excess medium or liquid comes out through the exit tube. Cells can be harvested continuous from a large volume of the culture medium.
(a) Preparative Ultracentrifuge:
This is a type of instrument where actual isolation, purification of macromolecules or cell organelles can be done. It operates in refrigerated condition under vacuum to avoid frictional resistance of the rotor caused by the spinning air.
The whole system is sophisticated with continuously monitoring system of the rotor temperature (temperature sensor). There is also one over speed disk system which checks the rotor so that it does not exceed its maximum allowable speed.
It operates through some photoelectric devices. For ultra smooth and quiet performance of the rotor, it is attached directly to the motor. Most rotors are fabricated from Titanium or Aluminum alloys. Titanium rotor has one advantage that it is quite resistant to corrosion. This instrument can attain a maximum speed of 80,000 r.p.m. and can produce a centrifugal field of 600,000 g.
(b) Analytical Ultracentrifuge:
This instrument has many applications in the fundamental studies of macromolecules showing the molecular weight, purity and shape of the material. It runs at a speed of about 70-80,000 r.p.m. with about 500,000 g and consists of a specially designed rotor in a special rotor chamber which remains under vacuum at low temperature.
There is an arrangement of a special optical system to determine the concentration distributions within the sample during centrifugation.
There are two special optical cells on the rotor, known as the Analytical cell and the Counterpoise cell (Fig. 7.2). There are two holes (Reference holes) in the counterpoise cell for the calibration of distances in the analytical cell. The rotor chamber has an upper and lower lens and the upper lens is joined with a camera lens which emits lights on the photographic plate. Light from the light source comes through the bottom.
The principle of monitoring in this system is done either through the ultraviolet absorption system or by noting the differences in the refractive index. If the concentration is uniform, light passes through it without any deviation. But if the light passes through a solution of different density zones, it is refracted at the boundary between these zones.
By measuring the refractive index (Fig. 7.3) between the reference solvent and the solution, the concentration of solute at any point can be measured. In recent models, the photographic plate system has been replaced by electronic scanning system which can directly measure and plot the concentration of the sample at all points in the analytical cell.
Centrifuge tubes are either manufactured in hard glass or with polypropylene, polycarbonate, stainless steel and nylon materials.
Generally, centrifugation is done to separate the particles or cells on the basis of their size, length or mass. But in some cases, separation is done on the basis of the density of the particles. When the shape (size) and density of some macromolecules are same then these macromolecules can be separated from each other according to mass.
This type of separation through centrifugation is known as Rate Zonal Centrifugation [Fig. 7.4(a)]. As the name signifies, different-sized molecules will occupy different zones in a centrifuge tube after centrifugation.
This separation of macromolecules at different zones is stabilized by using Sucrose gradient. So, this technique is also known as Sucrose Density Gradient Centrifugation. This is used for separating all types of particles and organelles.
In analytical ultracentrifuge the rate of sedimentation can be measured by taking photographs of the moving boundaries of sedimenting particles. To separate particles of different densities, they are labelled with H3 or C14 and then centrifuged in Sucrose density gradient. Particles labelled with H3 have the greater mass and sediment faster than those labelled with C14.
The second type of density gradient centrifugation is known as Equilibrium Density Gradient Centrifugation [Fig. 7.4(b)] where the density gradient is formed during centrifugation. The material used in this process is the aqueous solution of Caesium chloride (CsCl).
After a run in the ultracentrifuge, it will form 0.92g/ml heavier at the bottom than at the top permitting the separation of particles which differ in density by even a fraction of 0.02g/ml.
The densities of protein, DNA and RNA are 1.3, 1.6 to 1.7 and 1.75 to 1.8 g/ml, respectively. The same chemical CsCl can separate the different macromolecules like DNA and RNA due to the fact that Cs+ binds to DNA at phosphate groups, while it binds to RNA both at phosphates and at the hydroxyl groups of sugar thus increasing the density of RNA more than that of DNA.
Different isotopes are used with different labelling precursors to alter their densities which will be helpful for the separation of these macromolecules on the basis of density.
Gradient forming chemicals commonly used are Caesium and Rubidium chloride, Sucrose, some proteins and polysaccharides, colloidal silica (Percoll, Ludox), Metrizamide, Nycodenz, Renograffin etc. (Table 7.2).
Of them, Sucrose is most commonly used in Density Gradient centrifugation because of being very viscous even at 10% concentrations. Ficoll (copolymer of Sucrose and epichlorhydrin) is used for the separation of whole cells and cellular organelles.
CsCl solutions are used for Equilibrium Density Gradient or isopycnic separation of nucleic acids. After density gradient centrifugation, generally the visual bands separating the particles are collected with the help of the hypodermic needle or syringe.
Sometimes the centrifuge tube is punctured at the base by a fine needle. As the drops of the liquid come out through the needle they may be collected and analysed using ultraviolet spectrophotometer.