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Dina Amin
Content Centrifuge.
Ultracentrifuge.
History.
Types of ultracentrifugation.
Function of ultracentrifuge’s types.
Advantages and Disadvantages
Analytical ultracentrifugation of
protein.
 A centrifuge is a device for separating
particles from a solution according to their
size, shape, density, viscosity of the medium
and rotor speed.
 Ultracentrifuge is a high-
speed centrifuge for
separating microscopic
and sub-microscopic
materials to determine
the sizes and molecular
weights of colloidal and
other small particles.
 Swedish Biochemist
Theoder Svedberg
invented the
Ultracentrifuge in 1923.
 And he won the Novel
Prize in chemistry in
1926 for his research on
colloids and protein using
the ultracentrifuge.
Ultracentrifugation
Preparative
centrifugation
Differential
centrifugation
Density gradient
centrifugation
Rate zonal
centrifugation
Isopycnic
centrifugation
Analytical
ultracentrifugation
 1. Analytical ultracentrifugation:- The aim of
Analytical ultracentrifugation is use to study
molecular interactions between macromolecules
or to analyse the properties of sedimenting
particles such as their apparent molecular
weight.
 2. Preparative ultracentrifugation:- The aim of
Preparative ultracentrifugation to isolate and
purify specific particles such as subcellular
organelles.
 Two kinds of experiments are commonly
performed on these instruments:
1. Sedimentation velocity experiments:- Aim of
SVEs to interpret the entire time-course of
sedimentation, and report on the shape and
molar mass of the dissolved macromolecules, as
well as their size distribution.
2. Sedimentation equilibrium experiments:- SEEs
are concerned only with the final steady-state
of the experiment, where sedimentation is
balanced by diffusion opposing the
concentration gradients, resulting in a time-
independent concentration profile.
 It is to isolate specific particles which can
be reused
1. Differential ultracentrifugation:- Differential
centrifugation is a common procedure in
microbiology and cytology used to separate certain
organelles from whole cells for further analysis of
specific parts of cells.
2. Density gradient ultracentrifugation:- Based on
density difference. There are two types of density
gradient ultra centrifugations under preparative
ultracentrifugation such as.
a) ZONAL or RATE
b) ISOPYCNIC
 Mixture to be separated is layered on top of
a gradient (increasing concentration down
the tube).
 Provides gravitational stability as different
species.
 Move down tube at different rates.
 Isopycnic means “of the same density”.
 Molecules separated on equilibrium position.
 Each molecule floats or sinks to position
where density.
Analytical Ultracentrifugation of protein.
Functions of analytical
and preparative
ultracentrifugation
 Uses small sample size (less than 1 ml).
 Built in optical system to analyze progress of
molecules during centrifugation.
 Uses relatively pure sample.
 Used to precisely determine sedimentation
coefficient and MW of molecules.
 Beckman Model E is an example of centrifuge
used for these purposes.
 Larger sample size can be used.
 No optical read-out collect fractions and
analyze them after the run.
 Less pure sample can be used.
 Can be used to estimate sedimentation
coefficient and MW.
 Generally used to separate organelles and
molecules. Most centrifugation work done
using preparative ultracentrifuge.
 Centrifuges have a clean appearance and
have little to no odor problems.
 Not only is the device easy to install and fast
at starting up and shutting down, but also
only requires a small area for operation.
 They can be selected for different
applications.
 The device is simple to operate .
 Centrifuge has more process flexibility and
higher levels of performance.
 The machine can be very noisy and can cause
vibration.
 The device has a high-energy consumption
due to high G-forces.
 High initial capital costs.
Analytical Ultracentrifugation of protein.
 Analytical ultracentrifugation (AUC) is a direct
biophysical analysis method.
 Analytical ultracentrifugation gives information about
purified proteins and other biomolecules in dilute
solutions.
 It shows their mass, structure, and interactions.
 This technique studies their hydrodynamic behavior
when subjected to a centrifugal field.
 Biological areas of interest amenable to
analytical ultracentrifugation study
include:
 protein mass, shape and stoichiometry.
 protein-protein/lipid/nucleic acid
interactions.
 membrane proteins and their complexes.
 glycosylated proteins and their complexes.
 Our complete analytical
ultracentrifugation service includes:
 formulation of the initial model
 building any prior knowledge into the model
 fitting all start-to-end experimental data to the
model
 treating all sedimentation experiments globally
 reconciling sample sedimentation /
hydrodynamic analysis data with other
biophysical analysis such as x-ray/neutron small
angle scattering, dynamic light scattering,
calorimetry, etc.
 Analytical centrifugation offers two highly
complementary methodologies.
1. Sedimentation velocity.
2. Sedimentation equilibrium.
 Sedimentation velocity is an experiment which
measures how fast macromolecules move in
response to centrifugal force.
 Measuring changes in sedimentation boundary
movement gives us information about the mass and
shape of macromolecules.
 Sedimentation velocity experiments are performed
at high speeds of 30,000 - 50,000 rpm.
 They can address sample-related questions
concerning:
 homogeneity / heterogeneity in protein mass and
conformation.
 determining sedimentation coefficients of
solutes.
 detecting the presence of aggregates and their
size distribution.
 determination of overall shape/asymmetry of
macromolecules.
 define molecular mass and stoichiometry.
 Sedimentation equilibrium is based on balancing
the opposing forces on a particle of centrifugal
sedimentation and diffusion out of the resulting
concentrated solute phase. This ends in a
gradient which remains constant for an
indefinite period of time.
 Such experiments are generally performed at
lower rotation speeds. They can take several
days to complete.
 The technique permits determination of:
 molecular mass and subunit stoichiometry
 molecular dissociation constants (KD)
 second virial coefficient (B) – the measure of non-
ideality of the solute (due to protein shape or charge)
 All samples should be thoroughly dialyzed
against buffer.
 Gel-filtration buffer is also suitable.
 The buffer should contain at least 100mM
salt. The best choice of initial experimental
buffer is either phosphate or Tris-buffer
containing about 150mM NaCl.
 Analytical ultracentrifugation is a non-
destructive method. You can get your sample
back at the end if you wish.
Thank You

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Analytical Ultracentrifugation of protein.

  • 2. Content Centrifuge. Ultracentrifuge. History. Types of ultracentrifugation. Function of ultracentrifuge’s types. Advantages and Disadvantages Analytical ultracentrifugation of protein.
  • 3.  A centrifuge is a device for separating particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed.
  • 4.  Ultracentrifuge is a high- speed centrifuge for separating microscopic and sub-microscopic materials to determine the sizes and molecular weights of colloidal and other small particles.
  • 5.  Swedish Biochemist Theoder Svedberg invented the Ultracentrifuge in 1923.  And he won the Novel Prize in chemistry in 1926 for his research on colloids and protein using the ultracentrifuge.
  • 7.  1. Analytical ultracentrifugation:- The aim of Analytical ultracentrifugation is use to study molecular interactions between macromolecules or to analyse the properties of sedimenting particles such as their apparent molecular weight.  2. Preparative ultracentrifugation:- The aim of Preparative ultracentrifugation to isolate and purify specific particles such as subcellular organelles.
  • 8.  Two kinds of experiments are commonly performed on these instruments: 1. Sedimentation velocity experiments:- Aim of SVEs to interpret the entire time-course of sedimentation, and report on the shape and molar mass of the dissolved macromolecules, as well as their size distribution. 2. Sedimentation equilibrium experiments:- SEEs are concerned only with the final steady-state of the experiment, where sedimentation is balanced by diffusion opposing the concentration gradients, resulting in a time- independent concentration profile.
  • 9.  It is to isolate specific particles which can be reused 1. Differential ultracentrifugation:- Differential centrifugation is a common procedure in microbiology and cytology used to separate certain organelles from whole cells for further analysis of specific parts of cells. 2. Density gradient ultracentrifugation:- Based on density difference. There are two types of density gradient ultra centrifugations under preparative ultracentrifugation such as. a) ZONAL or RATE b) ISOPYCNIC
  • 10.  Mixture to be separated is layered on top of a gradient (increasing concentration down the tube).  Provides gravitational stability as different species.  Move down tube at different rates.
  • 11.  Isopycnic means “of the same density”.  Molecules separated on equilibrium position.  Each molecule floats or sinks to position where density.
  • 13. Functions of analytical and preparative ultracentrifugation
  • 14.  Uses small sample size (less than 1 ml).  Built in optical system to analyze progress of molecules during centrifugation.  Uses relatively pure sample.  Used to precisely determine sedimentation coefficient and MW of molecules.  Beckman Model E is an example of centrifuge used for these purposes.
  • 15.  Larger sample size can be used.  No optical read-out collect fractions and analyze them after the run.  Less pure sample can be used.  Can be used to estimate sedimentation coefficient and MW.  Generally used to separate organelles and molecules. Most centrifugation work done using preparative ultracentrifuge.
  • 16.  Centrifuges have a clean appearance and have little to no odor problems.  Not only is the device easy to install and fast at starting up and shutting down, but also only requires a small area for operation.  They can be selected for different applications.  The device is simple to operate .  Centrifuge has more process flexibility and higher levels of performance.
  • 17.  The machine can be very noisy and can cause vibration.  The device has a high-energy consumption due to high G-forces.  High initial capital costs.
  • 19.  Analytical ultracentrifugation (AUC) is a direct biophysical analysis method.  Analytical ultracentrifugation gives information about purified proteins and other biomolecules in dilute solutions.  It shows their mass, structure, and interactions.  This technique studies their hydrodynamic behavior when subjected to a centrifugal field.
  • 20.  Biological areas of interest amenable to analytical ultracentrifugation study include:  protein mass, shape and stoichiometry.  protein-protein/lipid/nucleic acid interactions.  membrane proteins and their complexes.  glycosylated proteins and their complexes.
  • 21.  Our complete analytical ultracentrifugation service includes:  formulation of the initial model  building any prior knowledge into the model  fitting all start-to-end experimental data to the model  treating all sedimentation experiments globally  reconciling sample sedimentation / hydrodynamic analysis data with other biophysical analysis such as x-ray/neutron small angle scattering, dynamic light scattering, calorimetry, etc.
  • 22.  Analytical centrifugation offers two highly complementary methodologies. 1. Sedimentation velocity. 2. Sedimentation equilibrium.
  • 23.  Sedimentation velocity is an experiment which measures how fast macromolecules move in response to centrifugal force.  Measuring changes in sedimentation boundary movement gives us information about the mass and shape of macromolecules.  Sedimentation velocity experiments are performed at high speeds of 30,000 - 50,000 rpm.
  • 24.  They can address sample-related questions concerning:  homogeneity / heterogeneity in protein mass and conformation.  determining sedimentation coefficients of solutes.  detecting the presence of aggregates and their size distribution.  determination of overall shape/asymmetry of macromolecules.  define molecular mass and stoichiometry.
  • 25.  Sedimentation equilibrium is based on balancing the opposing forces on a particle of centrifugal sedimentation and diffusion out of the resulting concentrated solute phase. This ends in a gradient which remains constant for an indefinite period of time.  Such experiments are generally performed at lower rotation speeds. They can take several days to complete.  The technique permits determination of:  molecular mass and subunit stoichiometry  molecular dissociation constants (KD)  second virial coefficient (B) – the measure of non- ideality of the solute (due to protein shape or charge)
  • 26.  All samples should be thoroughly dialyzed against buffer.  Gel-filtration buffer is also suitable.  The buffer should contain at least 100mM salt. The best choice of initial experimental buffer is either phosphate or Tris-buffer containing about 150mM NaCl.  Analytical ultracentrifugation is a non- destructive method. You can get your sample back at the end if you wish.