Centrifugation
Isopycnic or isodensity Centrifugation uses what gradient?
Caesium chloride
Difference between isopycnic and zonal centrifugation:
Isopycnic we separate based on equilibrium position not sedimentation coefficient
Swinging bucket:
Longer distance to travel therefore better separation Easier to remove supernatant without disturbing pellet
Describe isopycnic centrifugation:
Mix CsCl and sample - evenly distributed Spin Molecules float or sink to a position where the density of the molecule = density of the caesium chloride gradient So separation is based on density isopycnic = same density
Order of particles:
Small or low density at top ----> Large or high density at the bottom
Zonal or Rate Zonal Centrifugation uses what gradient?
Sucrose
Density materials include:
Sucrose or caesium chloride
What rotor is best for isopycnic ?
Swinging bucket or vertical
What rotor is best for zonal?
Swinging bucket or vertical
Two types of gradient separation:
Zonal or Rate Zonal Centrifugation Isopycnic (isodensity) Centrifugation
Preparative ultracentrigution is used for:
isolation, separation and purification of a desired analyte eg: organelles, proteins and DNA
Describe zonal centrifugation:
Applied to the top of sucrose gradient (of increasing conc down the tube) Gradient provides gravitational stability Species move through and form bands based on their sedimentation coefficients (s)
Two principle uses:
1. Separate solid (pellet) from dissolved solute (supernatant) 2. Separate macromolecules of different densities or mass using a gradient
Uses of isopycnic:
DNA and proteoglycans
Major components of centrifuge:
Electric motor Drive shaft Rotor to hold tubes
What rotor is good for pelleting?
Fixed angle
Two rotor types:
Fixed angle Swinging bucket
The sedimenting force (mω²r) is opposed by:
Flotation force Frictional resistance Diffusion
For a larger molecules or more compact molecules the sedimentation coefficient is:
High
Fixed angle:
Most used Short distance before solid molecules pellet Shorter running time
Caesium chloride gradients are good for:
Nucleic acids
Sucrose gradients are good for:
Organelles
Relative centrifugal force (RCF) formula:
RCF = 1.119 x 10^-5 x (rpm)² x r Reported as number x g example: 1000 x g (indicates 1000 times earths gravitational force)
Sedimentation coefficient (s) =
Rate of movement down the tube/centrifugal force
The centrifugal force generated is proportional to:
Rate of rotation (rpm) and the distance between rotor centre and the centrifuge tube
A nomogram relates:
Rotation rate (rpm) to centrifugal force (xg)
Analytical ultracentifugation involves:
Sample spun and monitored in real time through optical detection system, helps measure physical properties including sedimentation coefficient and mass.
How is a density gradient prepared?
Using non interacting low MW solute placed in tube in a continuously decreasing conc (gradient) Highest conc in bottom, lowest at the top
Sedimenting force on particle formula:
mass x centrifugal field = mω²r where m = mass omega = angular velocity of rotor (radians/sec) radius = distance of particle from angle of rotation
Applications of zonal centrifugation:
mild, non denaturing for organelles, proteins, intact cells, dna
Ultracentrifuge speed:
up to 100,000 rpm (requires a vacuum to eliminate friction)
High speed:
up to 25,000 rpm