Cell fractionation:
Cell fractionation is a procedure for rupturing cells, separation and suspension of cell constituents in isotonic medium in order to study their structure, chemical composition and function.
Cell fractionation involves 3 steps: Extraction, Homogenization and Centrifugation.
1. Extraction:
It is the first step toward isolating any sub-cellular structures. In order to maintain the biological activity of organelles and bio-molecules, they must be extracted in mild conditions called cell-free systems. For these, the cells or tissues are suspended in a solution of appropriate pH and salt content, usually isotonic sucrose (0.25 mol/L) at0-40°C.
2. Homogenization:
The suspended cells are then disrupted by the process of homogenization.
It is usually done by:
(i) Grinding
(ii) High Pressure (French Press or Nitrogen Bomb),
(iii) Osmotic shock,
(iv) Sonication (ultrasonic vibrations). Grinding is done by pestle and mortar or potter homogenizer (a high-speed blender). The later consists of two cylinders separated by a narrow gap.
The shearing force produced by the movement of cylinders causes the rupture of ceils. Ultrasonic waves are produced by piezoelectric crystal. They are transmitted to a steel rod placed in the suspension containing cells. Ultrasonic waves produce vibrations which rupture the cells. The liquid containing suspension of cell organelles and ether constituents is called homogenate. Sugar or sucrose solution preserves the cell organelles and prevents their clumping.
3. Centrifugation:
The separation (fractionation) of various components of the homogenate is carried out by a series of cemrifugations in an instrument called preparative ultracentrifuge. The ultracentrifuge has a metal rotor containing cylindrical holes to accommodate centrifuge tubes and a motor that spin the rotor at high speed to generate centrifugal forces. Theodor Svedberg (1926) first developed die ultracentrifuge which he used to estimate the molecular weight of hemoglobin.
Present day ultracentrifuge rotate at speeds up to 80,000 rpm (rpm= rotations per minute) and generates a gravitational pull of about 500,000 g, so that even small molecules like t-RNA, enzymes can sediment and separate from other components. The chamber of ultracentrifuge is kept in a high vacuum to reduce friction, prevent heating and maintain the sample at 0-4°C.
During centrifugation, the rate at which each component settle down depends on its size and shape and described in terms of sedimentation coefficient or Svedberg unit or S-value, where IS = 1 x 10-13 second.
The standard cell fractionation technique involves following methods:
(a) Differential velocity centrifugation [Velocity sedimentation or Rate zonal centrifugation):
It is the first step of cell fractionation by which various sub-cellular organelles are separated based on differences in their size. The homogenate in first filtered to remove unbroken cell clumps and collected in a centrifuge tube. The filtered homogenate when centrifuged in a series of steps at successively greater speeds, each step yields a pellet and a supernatant
The supernant of each step is removed to a fresh tube for centrifugation. For instance, at low speed (600g. for: 10 min) nuclear fraction or pellet will sediment at medium speed (15,000g x 5 min) mitochondria fraction sediment and at high speed (80,000 g. x 5 min.) micro-somal fraction sediment. The final supernant is soluble fraction or cytosol.
(b) Equilibrium Density-gradient centrifugation (Equilibrium sedimentation):
The organelle fractions (pallets) obtained in velocity centrifugation is purified by equilibrium density-gradient centrifugation. In this method organelles are separated by their density not by their size.
The impure organelle fraction is layered on the top of a gradient solution, e.g., sucrose solution or glycerol solution. The solution is more concentrated (dense) at the bottom of the centrifuge tube, and decreases in concentration gradually towards the top. The tube when centrifuged at high speed the various organelles migrate to an equilibrium position where their density is equal to the density of the medium. Meselson, Stahl and Vinograd (1957) used denser cesium chloride gradient for separation of a heavy DNA with 15N from DNA with 14N to provide evidence for semi-conservative DNA replication.
In conclusion, we may say that what one can learn about cells, depends on the tools at one’s disposed and, in fact, major advances in cell biology have frequently taken place with the introduction of new too is and techniques to the study of cell. Thus, to gain different types of information regarding cell, cell biologists have developed and employed various instruments and techniques. A basic knowledge of some of these methods is earnestly required.