CELL FRACTIONATION Useful technique for studying cell structure

CELL FRACTIONATION � Useful technique for studying cell structure and function of cellular structures � Make the cell apart and separates organelles and other subcellular structures � Equipment used is centrifuge � Principle is to spin the samples with different speeds that isolate the cell organelles/molecules � Samples are placed in tubes, where after centrifugation pellet is formed at the bottom of the tube

CELL FRACTIONATION � At lower speeds, the pellet contains of large components � At higher speed, the pellet contains of smaller components � Cell fractionation enables researches to prepare specific cell components in bulk and identify their functions � i. e. Biochemical test of the fraction of mitochondria shows the presence of respiratory enzymes while EM revealed large number of mitochondria. � It determines that mitochondria is the site of cellular respiration

DIFFERENTIAL CENTRIFUGATION Differential centrifugation is a procedure used in microbiology and cytology to separate certain organelles from whole cells for further analysis. � In the process, a tissue sample is first homogenized to break the cell membranes and mix up the cell contents. � The homogenate is subjected to repeated centrifugation, each time removing the pellet and increasing the centrifugal force. � Finally, purification may be done through equilibrium sedimentation, and the desired layer is extracted for further analysis. � Separation is based on size and density, with larger and denser particles pelleting at lower centrifugal forces. �

DIFFERENTIAL CENTRIFUGATION

ISOLATION OF DNA

SEPARATION OF LIQUIDS

TABLE TOP MICROCENTRIFUGE (SIGMA)

TABLE TOP CENTRIFUGES

ULTRA CENTRIFUGATION � The homogenized sample is centrifuged in an ultracentrifuge � An ultracentrifuge consists of a refrigerated, low -pressure chamber containing a rotor which is driven by an electrical motor capable of high speed rotation. � Samples are placed in tubes within or attached to the rotor. Rotational speed may reach up to 100, 000 rpm for floor model, 150, 000 rpm for bench-top model (Beckman Optima Max-XP or Sorvall MTX 150), creating centrifugal speed forces of 800, 000 g to 1, 000 g. This force causes sedimentation of macromolecules, and can even cause non-uniform distributions of small molecules.

CONSTRUCTION OF ULTRACENTRIFUGE

PRINCIPLE OF ULTRACENTRIFUGATION

TABLE TOP ULTRA CENTRIFUGE

ULTRACENTRIFUGE

ROTORS OF ULTRACENTRIFUGE

ROTERS OF ULTRACENTRIFUGE

DAMAGE OF ULTRACENTRIFUGE

ISOPYCNIC CENTRIFUGATION � Isopycnic centrifugation (density gradient centrifugation or equilibrium sedimentation) separates molecules on the basis of buoyant density. Typically, a "selfgenerating" density gradient is established via equilibrium sedimentation, and then analyte molecules concentrate as bands where the molecule density matches that of the surrounding solution. � For fractionation of nucleic acids, the mixture of Caesium chloride and DNA is centrifuged for several hours at high speed. Caesium chloride concentration of 1. 6 to 1. 8 g/m. L is similar to the density of DNA. � A gradient of Caesium ions is formed, due to two opposing forces: diffusion and centrifugal force. The caesium ions will sediment away from the rotor, and become more concentrated near the bottom of the tube. The diffusive force arises due to the concentration gradient of solvated caesium chloride and is always directed towards the center of the rotor. The balance between these two forces generates a stable density gradient in the caesium chloride solution, which is more dense near the bottom of the tube, and less dense near the top.

ISOPYCNIC CENTRIFUGATION � The DNA molecules will be separated based on the relative proportions of AT (adenine and thymine base pairs) to GC (guanine and cytosine base pairs). � An AT base pair has a lower molecular weight than a GC base pair and thus, for two DNA molecules of equal length, the one with the greater proportion of AT base pairs will have a lower density � Different types of nucleic acids will also be separated into bands, e. g. RNA is denser than super coiled plasmid DNA, which is denser than linear chromosomal DNA.

SUCROSE GRADIENT CENTRIFUGATION � Sucrose gradient centrifugation is used to purify enveloped viruses (with densities 1. 1 -1. 2 g/cm³), ribosome, membranes and so on. � This method is also used to purify exosomes. � There are two methods - equilibrium centrifugation and non-equilibrium centrifugation. � Typically in equilibrium centrifugation, a sucrose density gradient is created by gently overlaying lower concentrations of sucrose on higher concentrations in a centrifuge tube. For example, a sucrose gradient may consist of layers extending from 70% sucrose to 20% sucrose in 10% increments