Isolation and Identification of Subcellular Components PDF

ISOLATION AND IDENTIFICATION OF SUBCELLULAR COMPONENTS  1 INTRO The cell may be considered a "sac" full of various organelles which differ not only morphologically but also in terms of biochemical, biophysical, and physiolog...

ISOLATION AND IDENTIFICATION OF SUBCELLULAR COMPONENTS  1 INTRO The cell may be considered a "sac" full of various organelles which differ not only morphologically but also in terms of biochemical, biophysical, and physiological properties.  2 INTRO Thus, in order to study the composition and function of a certain subcellular component, it must be isolated from the rest of the cell. This would entail essentially the homogenization or destruction of cell boundaries by different mechanical or chemical procedures, followed by the separation of the subcellular fractions. The various cell fractions are then analyzed by biochemical or microscopic techniques.  3 TISSUE HOMOGENIZATION Initially, the cells come from a considerable mass of tissue, thus, they must first be broken into pieces. The cells are liberated and disrupted by mechanical means through the shearing forces created in a blender, or a tube and pestle homogenizer.  4 TISSUE AnotherHOMOGENIZATION alternative, less frequently used, is sonication wherein sound energy is applied to cells and viruses. The resulting homogenate is composed of the broken cells with the subcellular components released into a buffered medium. Debris (unseparated and unbroken cells) is separated by standing the homogenate for some time. The sediment is discarded and the supernate is subjected to centrifugation.  5 CENTRIFUGATION Though there are several techniques for separating cell components, the most commonly used is centrifugation. It can be used to separate particles that differ in density, size, and shape. The velocity of any particle in a centrifugal field depends on the angular speed of the centrifuge, mass, density, and frictional properties of the particle as well as the density of the suspending fluid.  6 CENTRIFUGATION There are two types of centrifugation that are generally used- density gradient and differential. Both provide macroscopic amounts of cell constituents for analysis.  7 CENTRIFUGATION Density-gradient centrifugation, the particles are placed in a medium with a density gradient such as a sucrose density- gradient medium. The velocity of the particles will vary according to the density of the medium in which they are suspended. The particles will tend to collect at the point where the density of the particles equals that of the medium. Particles are thus separated based on their density.  8 CENTRIFUGATION Differential centrifugation, the particles to be separated are placed in a medium of uniform density. If these particles are spun for a period of time (t), at a certain angular speed (v), the particles would travel at a distance vt. If the distance t is greater than the distance to the bottom of the centrifuge tube, the particles will be packed at the bottom of the tube forming a pellet. This fraction can then be separated from the particles that did not sediment (supernate), Particles of higher density, larger sine and of more compact shape sediment faster. Thus, particles of lower sedimentation rate can be brought down by successive centrifugation at higher speeds.  9 CYTOCHEMICAL TECHNIQUES The isolated cell fractions may be used for various purposes such as biochemical experiment and morphological studies. However, the fractions have to be identified and characterized. This could be done by using specific stains and chemical tests.  10 CYTOCHEMICAL The following areTECHNIQUES some stains that can be used to differentiate subcellular components: 1. lodine potassium iodide (I2KI) - iodine lodges inside the amylose coils of starch producing a blue-black color.  11 CYTOCHEMICAL TECHNIQUES 2. Sudan IV - it is a nonpolar stain that diffuses and accumulates in the interior of lipid droplets, imparting a scarlet red color.  12 CYTOCHEMICAL TECHNIQUES 3. Janus Green — it is a redox (reduction-oxidation) dye, which is colorless in its reduced form and turns green when oxidized; it colors cell organelles capable of performing redox reactions such as mitochondria.  13 CYTOCHEMICAL 4. Acetocarmine —TECHNIQUES it is a basic stain that preferentially binds to the DNA in the nucleus. The active component of carmine is carminic acid, which is a natural red stain.  14 CYTOCHEMICAL A chemical test TECHNIQUES that can be used to study a molecular component of the cell is the Biuret test. This test detects the presence of soluble proteins. The purple color of a positive Biuret reaction is due to the complexing of Cu2+ (Cupric) with the peptide bonds in proteins under alkaline conditions.