The Applications and Design of Analytical Ultracentrifugation
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 4
0
C.
•
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
Analytical ultracentrifugation, a powerful technique in biochemistry, allows for precise measurement of sample properties and characterization of macromolecular complexes. The method is widely used for determining sample purity, equilibrium constants, and assembly mechanisms of biological complexes. The design of analytical ultracentrifugation instruments includes optical systems for measuring sample concentration using absorbance or Rayleigh interference. Preparative centrifugation methods, such as cell fractionation, are also essential for isolating specific organelles in biological samples.
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Presentation Transcript
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: Optical systems Absorbance optical system: measurement of sample concentration at 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) wavelengths
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
