Centrifugation – the method of separation of macromolecules And yet it moves! Orientation in the labyrinth of the methods for separation of macromolecules Features used for separation of macromolecules (generally) Molecular mass Conformation and space Charge Density Orientation in the labyrinth of the methods for separation of macromolecules Separation methods Electro-migration Chromatography Centrifugation Orientation in the labyrinth of the methods for separation of macromolecules Separation methods Electro-migration Separation of biological macromolecules by electrophoresis Chromatography Centrifugation next time Orientation in the labyrinth of the methods for separation of macromolecules Electro-migration Separation of biological macromolecules by electrophoresis Chromatography Centrifugation next time Chromatography Separation methods next time Orientation in the labyrinth of the methods for separation of macromolecules Electro-migration Separation of biological macromolecules by electrophoresis Chromatography Centrifugation Separation methods Centrifugation today´s lecture :-) Movement solid particles in gravitational field? Centrifugation? No ! Centrifugation is a process that involves the use of the centrifugal force for the sedimentation of heterogeneous mixtures with a centrifuge 7 The methods of centrifugation Principle - correction Movement of particles in liquid medium under gravitation force which arises under turning of rotor of the centrifuge The movement of the particles dependents on: 1. Features of the particles 2. Features by environment The principles of centrifugation axis of the rotor rotation turning radius The construction of centrifuges 10 The types of techniques of centrifugation Differential centrifugation Zonal centrifugation Differential centrifugation Separation of mixture of heterogeneous particles in homogenous solution A common procedure in microbiology and cytology used to separate certain organelles from whole cells for further analysis of specific parts of cells Differential centrifugation supernatant pellet Separation of nuclei, ribosomes, mitochondria, cell membranes, nucleic acids, proteins, … Differential centrifugation - praxis • Particles differ by size, weigth or density = sedimentation by different speeds • By repeating and accelerating of rpm the individual components can be separated as pellets Can I separate everything by differential centrifugation? NO! You are not able to differentiate  Different types of NAs  Ribosomal subunits  Other particles with the similar features So, what can I do? Use the zonal centrifugation Zonal centrifugation Separation of mixture of homogenous particles in gradient of solution It is used as a purifying process for differential centrifugation 17 Forces in zonal centrifugation 18 Centrifugation force Buoyancy force Zonal centrifugation Isokinetic centrifugation  Separation according to speed of sedimentation of the particles  Used to determination of sedimentation coefficient S Equilibrium (isopycnic) sedimentation  Separation according to the particle density Zonal centrifugation Homogenous solution is replaced by a solution which concentration is growing from up to bottom of centrifugation tube (gradient solution) The gradient solution is prepared from very good soluble and inert compounds – sucrose, glycerol Growing density and viscosity of gradient solution eliminate the effect of growing centrifugal acceleration (it is growing from the axis of the rotor) by which protect of growing speedy of particles sedimentation during centrifugation 20 www.coleparmer.com How to perform the zonal centrifugation growing density and viscosity Gradient eliminates the effect of growing centrifugal acceleration  size  shape  density sample Particles are stratified according to Density gradient After centrifugation the separated particles of the same features are concentrated to narrow bands in the both compositions Continuous Discontinuous 22 Preparing of continuous gradient 23http://quizlet.com/ Density gradient Commonly used compounds to density gradient preparations are Caesium chloride Sucrose Density gradient The differences in densities are about 1,0- 1,3 g/ml for the sucrose, and 1,0-1,9 g/ml for CsCl, which enables to separate and isolate for example Cell nuclei Mitochondria Nucleic acids The purity of isolated compounds is extremely high Isokinetic centrifugation This method of centrifugation is used to more detailed characterisation of particles for example to exact determination of their size Isokinetic centrifugation The 5-20% sucrose gradient is usually used in NA analysis. The concentration of sucrose changes linearly from up to bottom of tube the speed of particle sedimentation is constant during the centrifugation Isokinetic centrifugation The speed, in which any particle sediments depends on Size of the particle Shape of the particle Density of the particle And is influenced by Features of the environment Conditions of the centrifugation Isokinetic centrifugation 29http://quizlet.com/ Sedimentation coefficient It characterises the speed of particle moving during centrifugation => It is defined as the ratio of a particle's sedimentation velocity to the acceleration that is applied to it S = sedimentation coefficient vt = terminal velocity r = distance from the axis of rotor ω = rotational speed (angular velocity) m = weight of particle η = viscosity of the medium r0 = radius of the particle 1 Svedberg = 10-13 s 23S-rRNA, 16S-rRNA, ribosomal units 30S, 50S 30 𝐒 = 𝐯𝐭 𝐫𝛚 𝟐 = 𝐦 𝟔𝛑𝛈𝐫𝟎 Examples of sedimentation coefficients So 20,wDensity http://jpkc.scu.edu.cn Isopycnic centrifugation Equilibrium sedimentation uses a gradient of a solution such as caesium chloride to separate particles based on their individual densities (mass/volume) Centrifugation to equilibrium Isopycnic centrifugation Density gradient is formed spontaneously during centrifugation the concentration gradient Particles of lysed cells move by the both directions (up and down) so long until they receive the position in which the density of the solution is the same as the density of the particles Isopycnic centrifugation The density determined by this manner is named as floating density The parameters of the floating density are influenced by interaction of the particles with ions in solution and they are usually higher than the density of the particles directly in cell Isopycnic centrifugation Centrifugation lower density higher density CsCl solution which contains a mixture of particles MESELSON, M; STAHL, FW. The replication of DNA in E. coli. Proc. Natl Acad. Sci. USA, 1958, vol. 44, pp. 671-682. Copyright © motifolio.com Rate-zonal centrifugation versus equilibrium density gradient centrifugation Sample Stabilizing sucrose gradient Slow-sedimenting component Fast-sedimenting component Centrifugation Fractionation Sample Steep sucrose gradient Low buoyant-density component High buoyant-density component Determination of the floating density by isopycnic centrifugation 25 °C = 10,8601 × nD 25 °C – 13,4974 nD 25 °C = refractive index of CsCl solution Isopycnic centrifugation - praxis Stahl Meselson experiment 1958 MESELSON, M; STAHL, FW. The replication of DNA in E. coli. Proc. Natl Acad. Sci. USA, 1958, vol. 44, pp. 671-682. Meselson-Stahl experiment - design 39 palyapbio.edu.glogster.com Copyright © motifolio.com The Meselson-Stahl experiment – predicted results Conservative Dispersive Semiconservative Parent DNA (15N) New DNA (14N) Parent double helices Daughter molecules Expected bands Granddaughter molecules Expected bands Meselson-Stahl experiment - results 41palyapbio.edu.glogster.com Isopycnic centrifugation - praxis Stahl Meselson experiment 1958 Calculation of (G+C) content • The composition of base pairs in dsDNA influence the floating density • This is used for determination of the GC content in DNA samples according to the rule % 𝐆 + 𝐂 = 𝛒 − 𝟏. 𝟔𝟔 𝟎. 𝟎𝟗𝟖 × 𝟏𝟎𝟎 ρ = floating density of dsDNA 43 (G+C) content versus floating density Pneumococcus G+C(%) Floating density Salmon sperm DNA E. coli Separation of different DNA forms  A special example how to use the isopycnic centrifugation is separation of different structural forms of DNA in the gradient of CsCl in the presence of ethidium bromide (EtBr)  After binding of EtBr to DNA the floating density of the DNA is significantly lowered. The amount of bonded EtBr and lowering the DNA density depends on the structural form of the DNA  It enables to separate and to isolate different DNA forms, for example covalently closed circles of plasmid DNA from opened plasmid and linear chromosomal DNA molecules Isopycnic centrifugation - praxis I would like to separate linear nuclear DNA from circular mitochondrial DNA (or plasmids from bacterial chromosome) by centrifugation. How to do it? Isopycnic centrifugation - praxis 6M CsCl,  = 1,7 Extract of bacterial cells Mix Centrifugation for 48 hours Isopycnic centrifugation - praxis Molecules stop their movement in the place with the same density as is the density of the solution Density in g/ml 1,80 1,65 Proteins Chromosomal DNA Plasmid DNA Isopycnic centrifugation - practice CsCl density 1,65 1,80 RNA Chromosomal DNA Proteins 49 Isopycnic centrifugation – real picture Chromosomal DNA Mitochondrial DNA 50 https://www.mun.ca centrifugation for 10 hrs at 100,000 rpm (450,000 x g) Centrifugation – useful rule RCF = relative centrifugal force (g) rpm = repeats per minute r = radius (cm) RCF = 1,119 × 10-5 × rpm2 × r