ultracentrifugation - nilamel · 2019-02-27 · analytical ultracentrifugation analytical...
TRANSCRIPT
![Page 1: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/1.jpg)
ULTRACENTRIFUGATIONULTRACENTRIFUGATION
![Page 2: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/2.jpg)
It is the apparent outward force that draws a rotating body away from the centre of rotation. It is caused by the inertia of the body as the body's path is continually redirected.
(Inertia - it is the resistance of any physical object to a change in its state of motion or rest, or the tendency of an object to resist any change in its motion.)any change in its motion.)
![Page 3: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/3.jpg)
Centrifugation Theory and
Practice
�Routine centrifuge rotors
�Density gradient theory
![Page 4: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/4.jpg)
Centrifuge rotorsaxis of rotation
At rest
Swinging-bucket
Spinning g
Fixed-angle
At rest
g
Spinning g
![Page 5: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/5.jpg)
Density Barrier Discontinuous Continuous
Density gradient centrifugation
![Page 6: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/6.jpg)
How does a gradient separate
different particles?
Least dense
Most denseMost dense
![Page 7: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/7.jpg)
1
3 Formats for separation of different density portions in a
testtube.
2
3
![Page 8: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/8.jpg)
1
2
Equilibrium
density banding5
3
4
![Page 9: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/9.jpg)
An important tool in biochemical research is the centrifuge, which through rapid spinning imposes high centrifugal forces on suspended particles, or even molecules in solution, and causes separations of such matter on the basis
ULTRACENTRIFUGATION
causes separations of such matter on the basis of differences in weight.Example:Red cells may be separated from plasma of blood, nuclei from mitochondria in cell homogenates, and one protein from another in complex mixtures
![Page 10: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/10.jpg)
� . Proteins are separated by ultracentrifugation—very high speed spinning; with possibility of appropriate photography of the protein layers as they form in the centrifugal field, it is possible to determine the molecular weights of proteins.
� The ultracentrifuge is a centrifuge optimized for spinning a rotor at very high speeds, capable of generating acceleration as high
as 19600 km/s²as 19600 km/s²
(around 50000 rpm).
![Page 11: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/11.jpg)
Analytical Ultracentrifugation� Analytical Ultracentrifugation (AUC) experiments give
us a method for the direct measurement of basic thermodynamic properties of macromolecules in solution. solution.
� Since sedimentation relies on the principal property of mass and centrifugal force, it is a valuable technique for a wide variety of solution conditions.
![Page 12: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/12.jpg)
� 1) What can be done with AUC to characterize a sample?
� a) Determine number of components and number of species; detection of impurities
� b) Molar mass of each species
� c) Kinds and stoichiometry of chemical reactions present in solution, including association with ligands, present in solution, including association with ligands, self-association.
� d)Shape and charge of the molecules, as inferred from their sedimentation frictional behavior.
![Page 13: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/13.jpg)
� 2) Examples of molecules that can be analyzed:� a) Proteins � b) Polysaccharides� c) Nucleic acids � d) Small molecules: drugs, ligands, gasses � e) Large aggregates: viruses, organelles
� 3) Kinds of buffers and additional solutes:� a) BME, DTT, � a) BME, DTT, � b) Triton X100. Tween-80 � b) Nucleotides � c) Salts and neutral molecules that significantly affect
density � d) PEG, glycerol affect viscosity and desnity strongly � e) 6 M Gdn and 8 M Urea
![Page 14: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/14.jpg)
Analytical ultracentrifugeIn an analytical ultracentrifuge, a sample being spun can be monitored in real time through an optical detection system, using ultraviolet light absorption and optical refractive index sensitive system.
�Working:-
#As the rotor turns, the images of the cell (proteins)#As the rotor turns, the images of the cell (proteins)
are projected by an optical system on to film or a
computer. The concentration of the solution at
various points in the cell is determined by absorption
of a light of the appropriate wavelength. This can be
accomplished either by measuring the degree of
blackening of a photographic film or by the deflection
of the recorder of the scanning system and fed into a
computer.
![Page 15: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/15.jpg)
This allows the operator to observe the separation of the sample concentration versus the axis of rotation ( lateral axis of the tube)as a result of the applied centrifugal field. Two kinds of experiments are commonly performed on these instruments: sedimentation velocity experiments instruments: sedimentation velocity experiments and sedimentation equilibrium experiments.
![Page 16: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/16.jpg)
![Page 17: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/17.jpg)
![Page 18: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/18.jpg)
# Sedimentation velocity experiments aim 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.Sedimentation velocity experiments can also be used to study reversible chemical equilibria between macromolecular species, by monitoring the number macromolecular species, by monitoring the number and molar mass of macromolecular complexes.#Sedimentation equilibrium experiments 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.
![Page 19: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/19.jpg)
�Analytical centrifugation involves measuring thephysical properties of the sedimenting particlessuch as sedimentation coefficient or molecularweight.
![Page 20: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/20.jpg)
•Preparative Centrifuge:
�Preparative ultracentrifuges are available with a wide variety of rotors suitable for a great range of experiments.� Most rotors are designed to hold tubes that contain the samples.� Swinging bucket rotors allow the tubes to hang on hinges so the tubes reorient to the horizontal as the rotor initially accelerates.accelerates.
![Page 21: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/21.jpg)
�Fixed angle rotors are made of a single block of metal and hold the tubes in cavities bored at a predetermined angle.
�Zonal rotors are designed to contain a large volume of sample in a single central cavity rather than in tubes. Some zonal rotors are capable of dynamic loading and unloading of samples while the rotor is spinning at high speed.
![Page 22: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/22.jpg)
�They can also be used for gradient separations, in which the tubes are filled from top to bottom with an increasing concentration of a dense substance in solution.� Sucrose gradients are typically used for separation of cellular organelles.� Gradients of caesium salts are used for separation of � Gradients of caesium salts are used for separation of nucleic acids. �After the sample has spun at high speed for sufficient time to produce the separation, the rotor is allowed to come to a smooth stop and the gradient is gently pumped out of each tube to isolate the separated components.
![Page 23: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/23.jpg)
�The other form of centrifugation is calledpreparative ultracentrifugation and the objectiveis to isolate specific particles which can be reused.Two types.
1)Differential ultracentrifugation: Differentialcentrifugation is a common procedurein microbiology and cytology used to separatecertain organelles from whole cells for further analysis ofcertain organelles from whole cells for further analysis ofspecific parts of cells. In the process, a tissue sample isfirst homogenised to break the cell membranes and mix up thecell contents. The homogenate is then subjected torepeated centrifugations, each time removing the pellet andincreasing the centrifugal force. Finally, purification may bedone through equilibrium sedimentation, and the desiredlayer is extracted for further analysis.
![Page 24: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/24.jpg)
2) Density gradient. Based on denstiydifference�There are two types of density gradientcentrifugations under preparativecentrifugation such as:�ZONAL(or)RATE
� ISOPYCNIC� ISOPYCNIC
![Page 25: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/25.jpg)
•ZONAL(or)RATEcentrifugation:� Mixture to be separated is layered on top of a
SUCROSE, or FICOLL, GRADIENT(increasing concentration down the tube)
� Provides gravitational stability as different species move down tube at different rates.
� Forming separate bands.� Forming separate bands.
![Page 26: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/26.jpg)
�Species are separated by differences in �SEDIMENTATION COEFFICIENT (S)=Rate of �SEDIMENTATION COEFFICIENT (S)=Rate of movement down tube/ Centrifugal force�S is increased for particle of LARGER MASS�S is also increased for MORE COMPACT STRUCTURES of equal particle mass.�Mild, non-denaturing procedure, useful for protein purification, and for intact cells and organelles.
![Page 27: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/27.jpg)
ISOPYCNIC centrifugation:� Isopycnic means "of the same density."
� Molecules separated on EQUILIBRIUM POSITION, NOT by RATES of sedimentation.
� Each molecule floats or sinks to position where density equals density of CsCl solution.
� Then no net sedimenting force on molecules.
� Isopycnic = Equal density and separation is on basis of � Isopycnic = Equal density and separation is on basis of DIFFERENT DENSITIES of the particles.
![Page 28: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/28.jpg)
•The term "isopycnic" is also encountered in biophysical chemistry and usually in reference to a process of separating particles, sub cellular organelles, or other substances on the basis of their density.• Isopycnic centrifugation refers to a method wherein a density gradient is either pre-formed or wherein a density gradient is either pre-formed or forms during high speed centrifugation, after this gradient is formed particles move within the gradient to the position having a density matching their own . This technique is extremely powerful.•Following example shows the isopycnic centrifugation:
![Page 29: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/29.jpg)
•Viruses purified by isopycnic centrifugation.• A density gradient was formed during high speed centrifugation of a solution of caesium chloride and the two virus types come to rest at the two virus types come to rest at points corresponding to their density.• The tube is about 10cm long.
![Page 30: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/30.jpg)
BENCHTOP CENTRIFUGE
![Page 31: ultracentrifugation - Nilamel · 2019-02-27 · Analytical Ultracentrifugation Analytical Ultracentrifugation (AUC) experiments give us a method for the direct measurement of basic](https://reader033.vdocuments.site/reader033/viewer/2022042412/5f2ac3b868f52475bb7502ea/html5/thumbnails/31.jpg)
Thank You