Ultracentrifugation Flashcards
How fast can an ultracentrifuge go?
Up to 1,000,000xg
How is friction minimised and cooling of the rotor and sample allowed?
By holding the rotor in a vacuum
What are the 2 types of ultracentrifugation?
Preparative and analytical
How do the high speeds permit separation?
Buoyant density and sedimentation velocity
Who is Theodore Svedburg?
Won Nobel prize for work on colloids and the invention of the ultracentrifuge
What is a Svedberg unit equal to?
S= 10^-13 seconds= 100fs
Equations for sedimentation coefficient
s=vt/a
vt=mrw^2/6pinr0
s=vt/rw^2=m/6pinr0
How to work out the sedimentation coefficient for protein complexes
Quoted for a protein in water at 20 degrees Celsius
Is non-additive which is why a 30s ribosome and a 50s ribosome make a 70s ribosome
Describe preparative ultracentrifugation
Needs a large sample size
Gives no optical readout
Gives estimates of the sedimentation coefficient and the molecular weight
What is preparative ultracentrifugation used for?
Usually used for sub-cellular fractionation of organelles, virus particles and large protein complexes
How is preparative ultracentrifugation differential?
The separation of the samples is based on the differential sedimentation speeds of particles of different sizes
How is a density gradient created in an ultracentrifuge?
A gradient of dense solute is used to isolate particles of similar sizes but different gradients
What solute is used for DNA separation?
Caesium chloride
What solute is used for protein complex separation?
Sucrose
Describe analytical ultracentrifugation
Needs a small sample size
Needs high concentrations of highly pure samples
Gives an optical readout of the absorbance, interference and fluorescence of the sample
Measures the sedimentation coefficient
Determines sizes of particles in the solution
