Biochemistry in the summer semester - MODULE A -

1. Biochemistry in the summer semester- MODULE A - Credit • 4 practicals (A1 – A4) • 3 oral testings: • Respiration, Blood and Body Fluids • Urogenital system, Acid-Base Balance • Digestion and Resorption, Intermediary Metabolism

3. Laboratory methodsin the summer semester Vladimíra Kvasnicová

4. Taking a capillary blood sample

5. 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2 m - mass of the particle r - radius of the centrifuge  - angular speed

6. Centrifugation • horizontal or fixed-angle rotor • the centrifuge cups in the rotor must be balanced The figures were found at http://www.all-science-fair-projects.com/science_fair_projects_encyclopedia/Centrifuge and http://www.flickr.com/photos/gonzales2010/9624402/ (Feb 2008)

7. Centrifugation The figure was found at http://www.flickr.com/photos/businesscheese/1803417133/ (Feb 2008)

8. 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 10-5x 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)

9. 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

10. The figures were found at http://www.chem.arizona.edu/tpp/chemt/CAn/Graphics/centrifuge/blood%20centrifugation.png and http://www.academic.marist.edu/~jzmz/HematologyI/Intro3.html (Feb 2008)

11. The figures were found at http://science.tjc.edu/Course/BIOLOGY/bott/anatomy/2402/summer%202402%20%20blood%20notes/c18_02.jpg and http://faculty.washington.edu/kepeter/119/images/hematocrit_tube_trio.jpg(Feb 2008)

12. A) Preparative centrifugation • separation of particles from a solution • two fractions are formed: • sediment(pellet, solid phase) • supernatant(liquid phase) The figure was found at http://www.steve.gb.com/science/molecular_biology_methods.html (Feb 2008)

13. differential centrifugation(= moving boundary, rate-zonal 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

14. Differential centrifugation The figure was found at http://fig.cox.miami.edu/~cmallery/150/protein/c7x3fractionation.jpg (Feb 2008)

15. 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

16. 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

17. 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)

18. Classification of electrophoretic techniques • free-boundary electrophoresis • separation is carried out entirely in a liquid phase, i.e. no support is used (capillary electrophoresis) • electrophoresis in a supporting medium • paper, gel (agarose, polyacrylamide) • it can be done horizontally or vertically

19. Capillary electrophoresis The figure was found at http://www.hood.edu/images/content/academics/instruments/Agilent_Capillary_Electrophoresis_System.JPG(Feb 2008)

20. Capillary electrophoresis The figure was found at http://en.wikipedia.org/wiki/Capillary_electrophoresis (Feb 2008)

21. Gel electrophoresis - horizontal The figure was found at http://www.mun.ca/biology/desmid/brian/BIOL2250/Week_Three/electro4.jpg (Feb 2008)

22. Gel electrophoresis - vertical SDS-PAGE – animation The figure was found at http://fig.cox.miami.edu/~cmallery/150/protein/page.jpg (Feb 2008)

23. 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 squareroot of time • medium: major factors are endosmosis and pore-size effects, which affect migration velocities

24. Process of electrophoresis • sample application • adjustment ofvoltage or current - DIRECT CURRENT ! (gel-electrophoresis about 70 - 100 volts,capillary electrophoresis about 20,000 volts) • separation time: minutes(e.g. gel-electrophoresis of serum proteins 30 min.) • electrophoresis in supporting medium: fixation, staining • evaluation: • qualitative (standards) • quantitative (densitometry)

25. Equipment used for the gel electrophoresisin the practical training A1 power suply (direct current) containers for staining and destaining gel electrophoresis chamber applicator

26. Electrophoresis– examples from clinical medicine • separation ofserum proteins, isoenzymes,nucleic acids • immunoelectrophoresis (immunoglobulins) The figure was found at http://www.sebia-usa.com/images/controlGel1.jpg (Feb 2008)

27. Electrophoresis– examples from clinical medicine • separation ofserum proteins, isoenzymes,nucleic acids • immunoelectrophoresis (immunoglobulins) The figure was found at http://www.sebia.com/V2/php/index.php?tpc=1&nv=0,2&page=contenu&id_prod=39#(Feb 2008)

28. The use of protein electrophoresis in diagnostics of diseases • 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)

29. Principal proteins of each fraction immunoglobulins: IgG, IgA, IgM 2-macroglobulin haptoglobin 1-antitrypsin orosomucoid transferrin C3-complement

30. Electrophoresis of serum proteinson agarose gel – 6 bands hypergamma- globulinemia normal patern The figure is from http://www.sebia-usa.com/products/proteinControl.html (Feb 2007)

31. Electrophoresis of serum proteinson agarose gel – 5 bands • normal patern • acute response • paraproteinemia • fraction of fibrinogen if plasma is analyzed instead of serum

32. Evaluation by densitometry - peaks 60% 3% 9% 12% 16% The figures are from http://www.sebia-usa.com/products/hyrys2.htmland http://erl.pathology.iupui.edu/LABMED/GENER27.HTMrespectivelly (Feb 2007)

33. The figure is from textbook: Devlin, T. M. (editor): Textbook of Biochemistry with Clinical Correlations, 4th ed. Wiley‑Liss, Inc., New York, 1997. ISBN 0‑471‑15451‑2

34. The figure is from textbook: Devlin, T. M. (editor): Textbook of Biochemistry with Clinical Correlations, 4th ed. Wiley‑Liss, Inc., New York, 1997. ISBN 0‑471‑15451‑2

35. The figure is from http://www.sebia-usa.com/products/reagents.html (Feb 2007)

36. The figure is from http://www.sebia-usa.com/products/reagents.html (Feb 2007)

37. IMMUNOFIXATION- paraprotein specification (monoclonal Ig) The figure is from http://www.sebia-usa.com/products/reagents.html (Feb 2007)