CENTRIFUGATION

Dr. Vinod K.V.

 Types of centrifugation
• Based on purpose
• ANALYTICAL & PREPARATIVE
• Analytical- measuring the physical properties
of the sedimenting particles such as
sedimentation coefficient or molecular weight
• Preparative- Isolate specific particles which
can be reused
 Analytical Ultracentrifugation –
Applications
• determine sample purity
• characterize assembly and disassembly mechanisms of
biomolecular complexes
• detect and characterize macromolecular confirmational
changes
• measure equilibrium constants and thermodynamic
parameters for self- and hetero-associating systems

 characterize the solution-state behavior of

macromolecules under various conditions
 Analytical Ultracentrifugation –
Design
• analytical ultracentrifuge = preparative
ultracentrifuge + optical detection system
 measure sample concentration inside the centrifuge
cell during or after sedimentation
• centrifugation parameters and data
acquisition under computer control
 experiments lasting many days performed with
minimal operator intervention
Analytical Ultracentrifugation –
Design
 Analytical Ultracentrifugation –
Design: Optical systems
• Absorbance optical system:
 measurement of sample concentration at wavelengths
from 200 to 800 nm
 detection of macromolecules containing strong
chromophores
• Rayleigh interference optical system:
 measurement of sample concentration based on
refractive index changes
 analyze macromolecules lacking intense
chromophores (eg, polysaccharides) and samples that
contain strongly absorbing buffer components
(eg, ATP/GTP)
 PREPARATIVE CENTRIFUGATION
• Many types
• Differential centrifugation (cell fractionation)
• The process of separation of cell organelles is
known as cell fractionation
• To isolate a specific organelle, the organs are
homogenised in suitable medium at 40C.
• The resulting suspension is called homogenate
• Fractionation is done by differential centrifugation
• This method is based upon the differences in the
sedimentation rate of particles of different sizes and
density
• Uses a series of centrifugation steps at successively
greater speeds
• Each step yield a pellet & supernatant
• The supernatant from each step is subjected to
centrifugation in the next step
• Provides four pellets- nuclear, mitochondrial, lysosomal
& microsomal fractions
• At each step, the pellet is washed several times by
resuspending in the homo.medium followed by
centrifugation under the same conditions
 Density gradient centrifugation
• Uses a medium that has gradients
• Eg: Caesium chloride, C. sulphate, sodium
bromide, Glycerol, dextran...
• Separation depends upon the buoyant
densities of the particles
• Two types
• Rate –Zonal technique &
• Isopycnic technique
 Rate Zonal Centrifugation
• The gradient used has a maximum density at the bottom
but density is less than the most dense sedimenting
particle to be separated
• density gradient is shallow
• Sample is taken at the top as a zone
• Centrifugation is performed at low speed for short time
• Depending on the sedimentation rate the samples form
discrete zones
• Must be terminated before the zones reaching the
bottom
 Application
• Useful for separating proteins with nearly
identical densities but differing slightly in size
• Used for separation of RNA-DNA hybrids,
ribosomal subunits, and subcellular organelles
 Isopycnic centrifugation
• Depends on the buoyant density of the particle
• Does not depend on shape, or size of the
particle
• Independent of time
• Maximum density of the gradient always
exceeds the density of the densest particle
• At the point of isodensity, no further
sedimentation occur
 Application
• Used to separate particles of similar size, but
of differing density
• Subcellular organelles such as golgi apparatus,
mitochondria can be effectively separated by
this method