name the steps involved in the isolation of cellular components?
Answers
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.
A preparative ultracentrifuge
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.
Cells fractionation
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.