1. Ultracentrifugation & Ultrafiltration By: A. Abdoli Sh. Gholami H. Sohani H. Rahmatollahi M. Abdoli Influenza Unit, Pasteur Institute of Iran Summer School 1

2. Biomolecules are purified using chromatography or Centrifugation.

3. Centrifugation Introduction: 1- Centrifugation is a technique in which solutes are separated by their different rate of travel (or sedimentation) in a centrifugal field. 2. Centrifugation is widely used in biological separation. The solutes are usually cells, Sub-cellular organelles, viruses, large molecules such as proteins and nucleic acids.

4. History and predecessors English military engineer Benjamin Robins (1707–1751) invented a whirling arm apparatus to determine drag.Benjamin Robinsdrag In 1864, Antonin Prandtl invented the first dairy centrifuge in order to separate cream from milk.Antonin Prandtl In 1879, Gustaf de Laval demonstrated the first continuous centrifugal separator, making its commercial application feasible.Gustaf de Laval 4

5. ultracentrifuge History Theodor Svedberg invented the analytical ultracentrifuge in 1925, and won the Nobel Prize in Chemistry in 1926 for his research on colloids and proteins using the ultracentrifuge. Theodor SvedbergNobel Prize in Chemistry The vacuum ultracentrifuge was invented by Edward Greydon Pickels. It was his contribution of the vacuum which allowed a reduction in friction generated at high speeds. Vacuum systems also enabled the maintenance of constant temperature.Edward Greydon Pickelsvacuumfrictiontemperature In 1946, Pickels cofounded Spinco (Specialized Instruments Corp.) and marketed an ultracentrifuge based on his design. Pickels, however, considered his design to be complicated and developed a more “foolproof” version. But even with the enhanced design, sales of the technology remained low, and Spinco almost went bankrupt. The company survived and was the first to commercially manufacture ultracentrifuges, in 1947. In 1949, Spinco introduced the Model L, the first preparative ultracentrifuge to reach a maximum speed of 40,000 rpm. In 1954, Beckman Instruments (now Beckman Coulter) purchased the company, forming the basis of its Spinco centrifuge division.SpincorpmBeckman InstrumentsBeckman Coulter 5

6. Theodor Svedberg(1884-1971),1926 Nobel prize.

7. Types of Centrifuge HIGH SPEED centrifuges SUPERSPEED centrifuges ULTRA centrifuges ultra = higher. Modern ultras have max speeds 80,000 – 150,000 rpm up to around 22,000 rpm

8. Preparation of sample for Ultracentrifuge Contaminants lipids, proteins, and nucleic acids from host cells components of the matrix such as tissue culture medium, or plasma in which the virus is suspended

9. Clarification

10. Ultra filtration Filters are defined by their pore size

11. The sample before and after centrifuge

13. Centrifugation Theory and Practice Routine centrifuge rotors Calculation of g-force Differential centrifugation Density gradient theory

14. Centrifuge rotors Fixed-angle axis of rotation At rest Swinging-bucket g Spinning g

15. Geometry of rotors bc r max r av r min r max r av r min Sedimentation path length axis of rotation a r max r av r min

16. k’-factor of rotors The k’-factor is a measure of the time taken for a particle to sediment through a sucrose gradient The most efficient rotors which operate at a high RCF and have a low sedimentation path length therefore have the lowest k’-factors The centrifugation times (t) and k’-factors for two different rotors (1 and 2) are related by:

17. Calculation of RCF and Q RCF= (1.119×10 -5 )(rpm) 2 r

19. RCF in swinging-bucket and fixed- angle rotors at 40,000 rpm Beckman SW41 swinging-bucket (13 ml) g min = 119,850g; g av = 196,770g; g max = 273,690g Beckman 70.1Ti fixed-angle rotor (13 ml) g min = 72,450g; g av = 109,120g; g max = 146,680g

20. Velocity of sedimentation of a particle v = velocity of sedimentation d = diameter of particle  p = density of particle  l = density of liquid  = viscosity of liquid g = centrifugal force

21. Differential centrifugation Density of liquid is uniform Density of liquid << Density of particles Viscosity of the liquid is low Consequence: Rate of particle sedimentation depends mainly on its size and the applied g-force.

22. Size of major cell organelles Nucleus4-12  m Plasma membrane sheets3-20  m Golgi tubules1-2  m Mitochondria0.4-2.5  m Lysosomes/peroxisomes0.4-0.8  m Microsomal vesicles 0.05-0.3  m

24. Differential centrifugation of a tissue homogenate (I) 1000g/10 min Decant supernatant 3000g/10 min etc.

25. Differential centrifugation of a tissue homogenate (II) 1.Homogenate – 1000g for 10 min 2.Supernatant from 1 – 3000g for 10 min 3.Supernatant from 2 – 15,000g for 15 min 4.Supernatant from 3 – 100,000g for 45 min Pellet 1 – nuclear Pellet 2 – “heavy” mitochondrial Pellet 3 – “light” mitochondrial Pellet 4 – microsomal

26. Differential centrifugation (III) Expected content of pellets 1000g pellet: nuclei, plasma membrane sheets 3000g pellet: large mitochondria, Golgi tubules 15,000g pellet: small mitochondria, lysosomes, peroxisomes 100,000g pellet: microsomes

27. Differential centrifugation (IV) Poor resolution and recovery because of: 1- Particle size heterogeneity 2- Particles starting out at r min have furthest travel but initially experience lowest RCF 3- Smaller particles close to r max have only a short distance to travel and experience the highest RCF

28. Differential centrifugation (V) Fixed-angle rotor: Shorter sedimentation path length g max > g min Swinging-bucket rotor: Long sedimentation path length g max >>> g min

29. Differential centrifugation (VI) Rate of sedimentation can be modulated by particle density. Nuclei have an unusually rapid sedimentation rate because of their size AND high density. Golgi tubules do not sediment at 3000g, in spite of their size: they have an unusually low sedimentation rate because of their very low density: (  p -  l ) becomes rate limiting.

30. Purification by ultracentrifugation The solute in the gradient is usually sucrose, caesium chloride, caesium sulfate, potassium sodium tartrate. These reagents are chosen for their high solubility, and the high density of the resulting solutions.

31. Purification by ultracentrifugation SoluteSaturated solution w/w (g per 100 g solution) Saturated solution w/v (g per 100 ml solution) Density (g cm-3) Sucrose67.990.91.34 Potassium sodium tartrate 39.7151.91.31 Caesium chloride65.71261.92 Caesium sulphate64.5129.82.01

32. Density BarrierDiscontinuousContinuous Density gradient centrifugation

33. A discontinuous sucrose density gradient is prepared by layering successive decreasing sucrose densities solutions upon one another. Influenza Unit, Pasteur Institute of Iran summer 2011 33

34. How does a gradient separate different particles? Least dense Most dense

35. When  p >  l : v is +ve When  p =  l : v is 0 Predictions from equation (I)

36. When  p <  l : v is -ve Predictions from equation (II)

37. Summary of previous slides A particle will sediment through a solution if particle density > solution density If particle density < solution density, particle will float through solution When particle density = solution density the particle stop sedimenting or floating

38. Buoyant density banding Equilibrium density banding Isopycnic banding 1 5 2 3 4

39. 1 2 3 3 Formats for separation of particles according to their density When density of particle < density of liquid V is -ve

40. Discontinuous Resolution of density gradients ContinuousDensity Barrier III

41. Problems with top loading

42.  p >>  l : v is +ve for all particles throughout the gradient Separation of particles according to size

43. http://www.sciencegateway.org/tools/rotor.htm 43

45. Influenza Unit, Pasteur Institute of Iran summer 2011 45

50. Mechanical stress Always ensure that loads are evenly balanced before a run. Always observe the manufacturers maximum speed and sample density ratings. Always observe speed reductions when running high density solutions, plastic adapters, or stainless steel tubes. Corrosion rotors are made from either titanium or aluminum alloy, chosen for their advantageous mechanical properties. While titanium alloys are quite corrosion-resistant, aluminum alloys are not. When corrosion occurs, the metal is weakened and less able to bear the stress from the centrifugal force exerted during operation. The combination of stress and corrosion causes the rotor to fail more quickly and at lower stress levels than an uncorroded rotor