1st Year SNL (2023-2024) Cell Biology Tutorial 2 PDF

1st Year SNL (2023-2024) / Cell biology module / Tutorials / Tut - 2 Fractionation and separation of cellular constituents The biochemical or structural study of cellular constituents requires prior separation. Indeed, the separation of cellular constituents begins w...

1st Year SNL (2023-2024) / Cell biology module / Tutorials / Tut - 2 Fractionation and separation of cellular constituents The biochemical or structural study of cellular constituents requires prior separation. Indeed, the separation of cellular constituents begins with a homogenization step to dissociate the organelles and suspend them in an appropriate medium in order to obtain a cellular homogenate. 1. Homogenization To obtain a homogenate, the cells are placed in a test tube containing an isotonic solution; this suspension will be fractionated by one of the following treatments: - Physical: with ultrasound or a hypotonic solution. - Chemical: with detergents. - Mechanical: grinding and crushing by a piston. In order to have good homogenization (especially for tissues) it is important to combine one of the previous methods with enzymatic digestion depending on the nature of the cells. ; For example: lysozyme for bacteria (degradation of the bacterial wall), trypsin for animal tissues (degradation of the extracellular matrix) and cellulase for plants (degradation of the cellulosic wall). 2. Separation by ultracentrifugation 2.1. Centrifugation principle Centrifugation consists of the separation of particles put into solution (examples: cellular constituents and biomolecules) under the effect of a significant gravity field produced by rotation of the centrifuge. At the end of the centrifugation, the large particles sediment and form a pellet, the other particles remain in suspension and form a supernatant. The speed of movement and sedimentation of the particles during centrifugation depends on: - The acceleration generated by the centrifuge (centrifugation speed expressed in rpm/min or in g) - The mass of the particle - The difference between the density of the particle and that of the solvent - Centrifugation duration There are classic centrifuges which perform low rotation (around 3000 rpm/min) such as those used to fractionate plasma from formed blood elements. However, Ultracentrifuge is a sophisticated and advanced centrifuge that operates at an extremely high speed and separates smaller molecules that cannot be separated from the traditional centrifuges. The speed of the rotors in ultracentrifuge can range from 60,000 rpm to 150,000 rpm. 2.2. Differential Ultracentrifugation (DUC) Differential ultracentrifugation (DUC) is a series of centrifugations at increasing speeds. Its aim is to separate the cellular organelles and biological macromolecules contained in a homogenate according to their difference in molecular weight. Indeed, thanks to a series of increasingly longer centrifugations and at increasingly higher speeds, the initial extract is divided into a series of pellets and supernatants with different compositions (Figure 1). 1  1st Year SNL (2023-2024) / Cell biology module / Tutorials / Tut - 2 Figure 1: Cell fractionation by differential ultracentrifugation (DUC) 2-3-Density gradient ultracentrifugation (DGUC) : It is ideal when the goal is to isolate particles of similar sizes, but different densities. This is the case for mitochondria, lysosomes and peroxisomes. The (DGUC) consists of using a solvent whose density will vary depending on the position in the tube: the gradient (Sucrose gradient is the most commonly used) (Figure 2). After centrifugation, each constituent will reach the zone of density equivalent to its own and the contents of the tube can be recovered separately. 2  1st Year SNL (2023-2024) / Cell biology module / Tutorials / Tut - 2 Figure 2: Cell fractionation by density gradient ultracentrifugation (DGUC) - Figure 3 summarizes a protocol for separating different cellular constituents: -What is the cell type used in the experiment? Justify your answer -Complete the diagram; why did they use ultrasounds for the purification of nucleoli? 3