Laboratory methods in the summer semester

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1 Taking a capillary blood sample Laboratory methods in the summer semester Vladimíra Kvasnicová Centrifugation Centrifugation = separation of particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed centrifugal force: P = m x r x ω 2 m - mass of the particle r - radius of the centrifuge ω - angular speed horizontal or fixed-angle rotor the centrifuge cups in the rotor must be balanced The figures were found at and (Feb 2008)

2 Centrifugation Centrifugation speed: revolutions per minute (rpm)! the same rpm doesn t mean the same centrifugal force in different centrifuges! centrifugal force: relative centrifugal force (RCF) RCF = 1.12 x 10-5 x r x (rpm) 2 r = radius of the centrifuge (cm) units: G (= how many times is the acceleration higher than the gravitational acceleration, G = 9.81 m x s -2 ) The figure was found at (Feb 2008) Centrifugation a) simple centrifuges (usual in a chemistry) (up to 10,000 G) b) high-speed refrigerated centrifuges (up to 50,000 G) c) ultracentrifuges (refrigerated + vacuum) (up to 500,000 G) example: RCF at least 1000 G for 10 min will give good separation of clotted blood from serum The figures were found at and (Feb 2008)

3 A) Preparative centrifugation separation of particles from a solution two fractions are formed: 1. sediment (pellet, solid phase) 2. supernatant (liquid phase) The figures were found at and (Feb 2008) The figure was found at (Feb 2008) differential centrifugation (= moving boundary, rate-zonal centrifugation) Differential centrifugation special kind of the preparative centrifugation it is used for separation of cell organelles which differ in size and density large, dense structures form a sediment (pellet) in a centrifuge tube faster (low RCF is enough for the separation) than small, less dense ones do supernatant obtained from a low speed centrifugation is centrifugated again (a number of time) using a higher RCF The figure was found at (Feb 2008)

4 B) Analytical centrifugation it involves a measuring of physical properties of the sedimenting particles (sedimentation coefficient, MW) ultracentrifugation is optimal molecules are observed by optical system during centrifugation and projected on to a film or a computer Electrophoresis = an analytical method based on movement of charged particles because of an external electric field velocity of a particle depends on the: a) size, shape and charge of the particle b) given applied voltage Electrophoresis anion - negatively charged ion, it moves to the anode (+) cation - positively charged ion, it moves to the catode (-) amphoteric - a substance that can have a positive, zero, or negative charge, depending on conditions (e.g. proteins) Classification of electrophoretic techniques 1. free-boundary electrophoresis separation is carried out entirely in a liquid phase, i.e. no support is used (capillary electrophoresis) 2. electrophoresis in a supporting medium paper, gel (agarose, polyacrylamide) it can be done horizontally or vertically

5 Capillary electrophoresis Capillary electrophoresis The figure was found at (Feb 2008) The figure was found at (Feb 2008) Gel electrophoresis - horizontal Gel electrophoresis - vertical SDS-PAGE animation The figure was found at (Feb 2008) The figure was found at (Feb 2008)

6 Effects of electrophoretic parameters on separation ph changes charge of analyte and hence its mobility, it can affect structure of analyte (denaturing, dissociating) ionic strength changes voltage or current: increased ion. str. usually reduces migration velocity and increases heating temperature: overheating can denaturate (precipitate) proteins; lower t. reduces diffusion but also reduces migration velocity, no effect on resolution current: too high current causes overheating voltage: migration velocity is proportional to voltage time: resolution (separation of bands) increases linearly with time, but dilution of bands (diffusion) increases with the square root of time medium: major factors are endosmosis and pore-size effects, which affect migration velocities 1. sample application Process of electrophoresis 2. adjustment of voltage or current - DIRECT CURRENT! (gel-electrophoresis about volts, capillary electrophoresis about 20,000 volts) 3. separation time: minutes (e.g. gel-electrophoresis of serum proteins 30 min.) 4. electrophoresis in supporting medium: fixation, staining 5. evaluation: qualitative (standards) quantitative (densitometry) Equipment used for the gel electrophoresis in the practical training A1 power suply (direct current) electrophoresis chamber containers for staining and destaining gel applicator Electrophoresis examples from clinical medicine separation of serum proteins, isoenzymes, nucleic acids immunoelectrophoresis (immunoglobulins) The figure was found at (Feb 2008)

7 Electrophoresis examples from clinical medicine The use of protein electrophoresis in diagnostics of diseases separation of serum proteins, isoenzymes, nucleic acids immunoelectrophoresis (immunoglobulins) electrophoretic patern is constant under physiological conditions (intensity of bands) spectrum of plasma proteins changes under various diseases (their ratio) evaluation of electrophoretic patern (bands or peaks) The figure was found at (Feb 2008) Principal proteins of each fraction Electrophoresis of serum proteins on agarose gel 6 bands hypergammaglobulinemia α 2 -macroglobulin haptoglobin immunoglobulins: IgG, IgA, IgM normal patern α 1 -antitrypsin orosomucoid transferrin C3-complement The figure is from (Feb 2007)

8 Electrophoresis of serum proteins on agarose gel 5 bands Evaluation by densitometry - peaks A. normal patern B. acute response C. paraproteinemia D. fraction of fibrinogen if plasma is analyzed instead of serum 60% 3% 9% 12% 16% The figures are from and respectivelly (Feb 2007) The figure is from textbook: Devlin, T. M. (editor): Textbook of Biochemistry with Clinical Correlations, 4th ed. Wiley-Liss, Inc., New York, ISBN The figure is from textbook: Devlin, T. M. (editor): Textbook of Biochemistry with Clinical Correlations, 4th ed. Wiley-Liss, Inc., New York, ISBN

9 The figure is from (Feb 2007) The figure is from (Feb 2007) IMMUNOFIXATION - paraprotein specification (monoclonal Ig) The figure is from (Feb 2007)

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