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Hydrodynamic Techniques electrophoresis centrifugation Types of Centrifuges Ultracentrifuge -Analytical -Preparative High Speed Table Top Clinical Microfuges.

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Presentation on theme: "Hydrodynamic Techniques electrophoresis centrifugation Types of Centrifuges Ultracentrifuge -Analytical -Preparative High Speed Table Top Clinical Microfuges."— Presentation transcript:

1 Hydrodynamic Techniques electrophoresis centrifugation Types of Centrifuges Ultracentrifuge -Analytical -Preparative High Speed Table Top Clinical Microfuges F c = m 2 r F b = -m o 2 r F f = -fv

2 v = 2 rm ( p - s )/f The greater the centrifugal force ( 2 r), the faster the particle sediments. The more massive a particle, the faster it moves in a centrifugal field. The denser a particle, the faster in moves in a centrifugal field. The denser the solvent, the slower the particle will move in a centrifugal field. The particle velocity is 0 when the solvent density is greater than the particle density. The greater the frictional coefficient (factors such as solvent viscosity, particle shape, etc.), the slower the particle will move.

3 Sedimentation Coefficient (s) is the velocity per F c, or s = v/ 2 r units are the Svedberg (S), where 1 S = sec = m ( p - s )/f Analytical vs Preparative Centrifugation

4 collect material cells precipitated macromolecules subcellular fractionation

5 Differential Centrifugation Relative Centrifugal Force expressed as x gravity RCF = F c /F g = 2 r/980 [ = (rpm)/30] RCF = x 10 5 (rpm) 2 r [radius in cm] prepare cell lysate subject to centrifugation centrifugal force time (g ·min) tube size and shape rotor angle separate supernatant and pellet re-centrifuge supernatant

6 Problems contamination large particles contaminated with smaller particles resolution particles of similar sizes not separated vibrations and convection currents Density Gradients centrifugation in a dense medium increases stability provides greater resolution common media: sucrose, CsCl, Ficoll ®, Hypaque ®, Percoll ®

7 Density Gradient Centrifugation Two Types: 1) Rate Zonal p > s separates primarily by mass 2) Isopycnic p < s equilibrium separates by density

8 Example of Isopycnic Separation Percoll, self-forming gradient light vs. heavy fraction

9 General Procedures 1) prepare gradient step continuous self-forming (isopycnic) 2) apply sample layer on top bottom (isopycnic) dispersed (self forming) 3) centrifuge

10 Measuring Density marker beads refractometer 4) collect and analyze fractions manually aspirate bands fraction collector


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