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Ultrcentifugation: Basic Training

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Ultrcentifugation: Basic Training, some slides out of Thermo Scientific education material, published with the permission of Richard Siccard.

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Ultrcentifugation: Basic Training

  1. 1. Ultracentrifugation: Basic Training Thermo Laboratory Products
  2. 2. Applications Smallest particles need strong g- forces for separation. Superspeeds are not sufficient (only 50-100,000 xg max) Ultras separate tiny particles with forces over 800,000 xg!! Microultras separate small volumes - forces over 1,000,000 xg!! 2
  3. 3. Applications Important Terms in Ultracentrifugation Abbreviation What it means RPM Revolutions per Minute: The number of times a rotor spins completely around in a single minute. As RPM increases, RCF also increases. RCF Relative Centrifugal Force: G-force developed in a rotor while it spins. Many protocols use RCF instead of RPM. As g-force increases, pelleting or separation time decreases. K K-Factor: Pelleting efficiency of a rotor. The smaller the K-factor, the more efficient the rotor, or the less time it takes to pellet particles. S Sedimentation Coefficient: Depends on diameter, shape, density. The larger the S value, the faster a particle separates. 3
  4. 4. Applications • Smaller particles also have very small S- Density (g/ml) values. • High RCF needed to pellet or separate • Ultras required! Sedimentation Coefficient (S) 4
  5. 5. Applications – As particle size/weight decreases, more force needed to “pellet” Differential – Smaller particles remain in “supernatant” Pelleting – Fixed Angle rotors work best • Ultra: T-8100, T-880, T-1270, etc. • Microultra: S150-AT, S120-AT2, etc – Example protocol - subcellular fractionation • Spin 1: 1000xg to pellet nucleus • Spin 2: 10000xg to pellet mitochondria • Spin 3: 100000xg to pellet microsomes • Spin 4: 900,000xg to isolate proteins 5
  6. 6. Applications Rate zonal density gradient centrifugation Before After • Good for particles with Low similar densities, but Density Sample different masses • Example particle: Proteins • Example gradient material: sucrose High Density 6
  7. 7. Applications Isopycnic (Equilibrium density gradient ) centrifugation Before • Separates particles according After to density • Example particles: – DNA, RNA, Plasmid DNA – Viruses – Organelles • Example gradient: Cesium Chloride 7
  8. 8. Applications Centrifugation Method Versus Rotor Type Rotor Separation Considerations Type Swinging Longer separation time due to longer bucket pathlength. Especially good separation for Rate-Zonal separation. Beware of Cesium Chloride quot;point loadsquot; during isopycnic centrifugation. Fixed Shorter separation time due to shorter Angle pathlength. Excellent for simple pelleting. Vertical Shortest pathlength. Fastest separation, especially for Isopycnic separation. Pathlength may be too short for rate-zonal separation. Pellet smears along wall in simple pelleting. 8
  9. 9. Applications Type of Rate-zonal Rate-zonal rotor Pelleting sedimentation flotation Isopycnic Fixed- Excellent Limited Good VariableA angle Ex. T-880, S120-AT2 Vertical NS Good Good Excellent Ex. Stepsaver, S120-VT Swinging Inefficient Good Excellent GoodB Bucket Ex. TH-641, S55-S For Excellent rotor-applications, a common ultra and microultra rotor are listed NS = not suitable AGood for macromolecules, poor for cells and organelles BGood for cells and organelles, caution needed if used with CsCl 9
  10. 10. Applications Most Typical Typical Appropriate Separation Sample Applications Rotor Method DNA and RNA Sequencing, Vertical and CsCl gradient, gene therapy, Fixed angle 400,000xg; cloning, gene Ethidium expression Bromide staining Viruses Vaccines, gene Swinging bucket Sucrose therapy vector gradients, 100,000xg Proteins Protein structure Fixed angle Rate-zonal studies, separation, Proteomics, 600,000xg HDL/LDL studies Cells and Cell function, Fixed angle Differential organelles membrane pelleting. Low biology, speeds for cells. mitochondrial High speeds - DNA smaller organelles. 10
  11. 11. Applications - Tube Selection • Selection of the appropriate Properties of 4 popular tube plastics ultracentrifuge tube Chemical – Prevents sample leakage or Plastic type Clarity Resistance loss Polypropylene (PP) Opaque Good – Ensures chemical compatibility Polyallomer (PA) Opaque Good – Allows easy sample recovery Polycarbonate (PC) Clear Poor • Major factors in selection of a Polyethelyene Clear Poor tube (plastic) material: Terephthalate (PET)* – Clarity – Chemical resistance PET is also in Polyclear™, Clearcrimp®, – Sealing mechanism (if Ultraclear & other tubes needed) 11
  12. 12. Applications - Tube Selection Tube type must be carefully matched with rotor type to prevent sample loss and/or tube failure Fixed Swinging Tube type Puncture Angle Bucket Vertical or slice Rotor Rotor Rotor Thin wall Yes No Yes No open top Thick wall No Yes Yes No open top Thin wall Yes Yes Some Yes sealed tube types Oak Ridge No Yes No No 12
  13. 13. Applications - Sample Collection • Sample collection from tubes (depends on application, gradient, etc) – Pour liquid from top if simple pellet – Draw liquid from top with pipette – Use syringe to puncture tube & draw out sample – Cut tube with tube slicer – puncture bottom & collect droplets (fractions) 13
  14. 14. Applications - Rotor Types Review • Fixed Angle – differential pelleting • Vertical - isopycnic separation (density) • Swinging Bucket – isopycnic or rate-zonal separation (density or size) • Others –Zonal - rate zonal separation (size) –Continuous-Flow – large volumes of sample –Near Vertical – isopycnic separations (density) 30

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