Differential centrifugation
Encyclopedia
Differential centrifugation is a common procedure in microbiology
and cytology
used to separate certain organelle
s from whole cell
s for further analysis of specific parts of cells. In the process, a tissue
sample is first homogenised
to break the cell membrane
s and mix up the cell contents. The homogenate is then subjected to repeated centrifugation
s, each time removing the pellet and increasing the centrifugal force. Finally, purification may be done through equilibrium sedimentation, and the desired layer is extracted for further analysis.
Separation is based on size and density, with larger and denser particles pelleting at lower centrifugal forces. As an example, unbroken whole cells will pellet at low speeds and short intervals such as 1,000g for 5 minutes. Smaller cell fragments and organelles remain in the supernatant and require more force and greater times to pellet. In general, one can enrich for the following cell components, in the separating order in actual application:
, usually a piece of porous porcelain
of the same shape and dimension as the container, is used. The container is, in most cases, a glass
boiling tube
.
The tissue sample is first crushed and a buffer solution
is added to it, forming a liquid suspension of crushed tissue sample. The buffer solution is a dense
, inert
, aqueous solution
which is designed to suspend the sample in a liquid medium without damaging it through chemical reaction
s or osmosis
. In most cases, the solution used is sucrose
solution; in certain cases brine
will be used. Then, the blender, connected to a high-speed rotor, is inserted into the container holding the sample, pressing the crushed sample against the wall of the container.
With the rotator turned on, the tissue sample is ground by the porcelain pores and the container wall into tiny fragments. This grinding process will break the cell membranes of the sample's cells, leaving individual organelles suspended in the solution. This process is called homogenization. A portion of cells will remain intact after grinding and some organelles will be damaged, and these will be catered for in the later stages of centrifugation.
. An ultracentrifuge consists of a refrigerated, evacuated chamber containing a rotor which is driven by an electrical motor capable of high speed rotation. Samples are placed in tubes within or attached to the rotor. Rotational speed may reach up to 100,000 rpm for floor model, 150,000 rpm for bench-top model (Beckman Optima Max-XP), creating centrifugal speed forces of 800,000g to 1,000,000g. This force causes sedimentation
of macromolecules, and can even cause non-uniform distributions of small molecules.
Since different fragments of a cell have different sizes and densities, each fragment will settle into a pellet with different minimum centrifugal forces. Thus, separation of the sample into different layers can be done by first centrifuging the original homogenate under weak forces, removing the pellet, then exposing the subsequent supernatants to sequentially greater centrifugal fields. Each time a portion of different density is sedimented to the bottom of the container and extracted, and repeated application produces a rank of layers which includes different parts of the original sample. Additional steps can be taken to further refine each of the obtained pellets.
Sedimentation depends on mass, shape, and partial specific volume
of a macromolecule, as well as solvent density, rotor size and rate of rotation. The sedimentation velocity can be monitored during the experiment to calculate molecular weight.
Values of sedimentation coefficient
(S) can be calculated. Large values of S (faster sedimentation rate) correspond to larger molecular weight. Dense particle sediments more rapidly. Elongated proteins have larger frictional coefficients, and sediment more slowly to ensure accuracy.
to separate particles based on their individual densities (mass/volume). It is used as a purifying process for differential centrifugation. A solution is prepared with the densest portion of the gradient at the bottom. Particles to be separated are then added to the gradient and centrifuged. Each particle proceeds (either up or down) until it reaches an environment of comparable density. Such a density gradient may be continuous or prepared in a stepped manner. For instance, when using sucrose to prepare density gradients, one can carefully float a solution of 40% sucrose onto a layer of 45% sucrose and add further less dense layers above. The homogenate, prepared in a dilute buffer and centrifuged briefly to remove tissue and unbroken cells, is then layered on top. After centrifugation typically for an hour at about 100,000 x g, one can observe disks of cellular components residing at the change in density from one layer to the next. By carefully adjusting the layer densities to match the cell type, one can enrich for specific cellular components. Caesium chloride allows for greater precision in separating particles of similar density. In fact, with a caesium chloride gradient, DNA particles that have incorporated heavy isotopes (13C or 15N for example) can be separated from DNA particles without heavy isotopes.
Microbiology
Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell , cell clusters or no cell at all . This includes eukaryotes, such as fungi and protists, and prokaryotes...
and cytology
Cell biology
Cell biology is a scientific discipline that studies cells – their physiological properties, their structure, the organelles they contain, interactions with their environment, their life cycle, division and death. This is done both on a microscopic and molecular level...
used to separate certain organelle
Organelle
In cell biology, an organelle is a specialized subunit within a cell that has a specific function, and is usually separately enclosed within its own lipid bilayer....
s from whole cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
s for further analysis of specific parts of cells. In the process, a tissue
Tissue (biology)
Tissue is a cellular organizational level intermediate between cells and a complete organism. A tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function. These are called tissues because of their identical functioning...
sample is first homogenised
Homogenization (biology)
Homogenization is a process that involves breaking apart cells — releasing organelles and cytoplasm.When the purpose is to extract organelles, it is frequently done in two steps; first using a blender to break the tissue up, and then with an ultrasonic or mechanical tissue disruptor. The...
to break the cell membrane
Cell membrane
The cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...
s and mix up the cell contents. The homogenate is then subjected to repeated centrifugation
Centrifugation
Centrifugation is a process that involves the use of the centrifugal force for the sedimentation of mixtures with a centrifuge, used in industry and in laboratory settings. More-dense components of the mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture...
s, each time removing the pellet and increasing the centrifugal force. Finally, purification may be done through equilibrium sedimentation, and the desired layer is extracted for further analysis.
Separation is based on size and density, with larger and denser particles pelleting at lower centrifugal forces. As an example, unbroken whole cells will pellet at low speeds and short intervals such as 1,000g for 5 minutes. Smaller cell fragments and organelles remain in the supernatant and require more force and greater times to pellet. In general, one can enrich for the following cell components, in the separating order in actual application:
- Whole cells and nuclei;
- Mitochondria, lysosomes and peroxisomes;
- Microsomes (vesicles of disrupted endoplasmic reticulum); and
- Ribosomes and cytosol.
Sample preparation
Before differential centrifugation can be carried out to separate different portions of a cell from one another, the tissue sample must first be homogenised. In this process, a blenderBlender (device)
A blender is a kitchen and laboratory appliance used to mix, puree, or emulsify food and other substances. A stationary blender consists of a blender jar with blade at the bottom, rotated by a motor in the base...
, usually a piece of porous porcelain
Porcelain
Porcelain is a ceramic material made by heating raw materials, generally including clay in the form of kaolin, in a kiln to temperatures between and...
of the same shape and dimension as the container, is used. The container is, in most cases, a glass
Glass
Glass is an amorphous solid material. Glasses are typically brittle and optically transparent.The most familiar type of glass, used for centuries in windows and drinking vessels, is soda-lime glass, composed of about 75% silica plus Na2O, CaO, and several minor additives...
boiling tube
Boiling tube
frame|A boiling tube looks much like a test tube.A boiling tube is a small cylindrical vessel used to strongly heat substances in the flame of a Bunsen burner...
.
The tissue sample is first crushed and a buffer solution
Buffer solution
A buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. It has the property that the pH of the solution changes very little when a small amount of strong acid or base is added to it. Buffer solutions are used as a...
is added to it, forming a liquid suspension of crushed tissue sample. The buffer solution is a dense
Density
The mass density or density of a material is defined as its mass per unit volume. The symbol most often used for density is ρ . In some cases , density is also defined as its weight per unit volume; although, this quantity is more properly called specific weight...
, inert
Inert
-Chemistry:In chemistry, the term inert is used to describe a substance that is not chemically reactive.The noble gases were previously known as inert gases because of their perceived lack of participation in any chemical reactions...
, aqueous solution
Aqueous solution
An aqueous solution is a solution in which the solvent is water. It is usually shown in chemical equations by appending aq to the relevant formula, such as NaCl. The word aqueous means pertaining to, related to, similar to, or dissolved in water...
which is designed to suspend the sample in a liquid medium without damaging it through chemical reaction
Chemical reaction
A chemical reaction is a process that leads to the transformation of one set of chemical substances to another. Chemical reactions can be either spontaneous, requiring no input of energy, or non-spontaneous, typically following the input of some type of energy, such as heat, light or electricity...
s or osmosis
Osmosis
Osmosis is the movement of solvent molecules through a selectively permeable membrane into a region of higher solute concentration, aiming to equalize the solute concentrations on the two sides...
. In most cases, the solution used is sucrose
Sucrose
Sucrose is the organic compound commonly known as table sugar and sometimes called saccharose. A white, odorless, crystalline powder with a sweet taste, it is best known for its role in human nutrition. The molecule is a disaccharide composed of glucose and fructose with the molecular formula...
solution; in certain cases brine
Brine
Brine is water, saturated or nearly saturated with salt .Brine is used to preserve vegetables, fruit, fish, and meat, in a process known as brining . Brine is also commonly used to age Halloumi and Feta cheeses, or for pickling foodstuffs, as a means of preserving them...
will be used. Then, the blender, connected to a high-speed rotor, is inserted into the container holding the sample, pressing the crushed sample against the wall of the container.
With the rotator turned on, the tissue sample is ground by the porcelain pores and the container wall into tiny fragments. This grinding process will break the cell membranes of the sample's cells, leaving individual organelles suspended in the solution. This process is called homogenization. A portion of cells will remain intact after grinding and some organelles will be damaged, and these will be catered for in the later stages of centrifugation.
Ultracentrifugation
The homogenised sample is now ready for centrifugation in an ultracentrifugeUltracentrifuge
The ultracentrifuge is a centrifuge optimized for spinning a rotor at very high speeds, capable of generating acceleration as high as 2,000,000 g . There are two kinds of ultracentrifuges, the preparative and the analytical ultracentrifuge...
. An ultracentrifuge consists of a refrigerated, evacuated chamber containing a rotor which is driven by an electrical motor capable of high speed rotation. Samples are placed in tubes within or attached to the rotor. Rotational speed may reach up to 100,000 rpm for floor model, 150,000 rpm for bench-top model (Beckman Optima Max-XP), creating centrifugal speed forces of 800,000g to 1,000,000g. This force causes sedimentation
Sedimentation
Sedimentation is the tendency for particles in suspension to settle out of the fluid in which they are entrained, and come to rest against a barrier. This is due to their motion through the fluid in response to the forces acting on them: these forces can be due to gravity, centrifugal acceleration...
of macromolecules, and can even cause non-uniform distributions of small molecules.
Since different fragments of a cell have different sizes and densities, each fragment will settle into a pellet with different minimum centrifugal forces. Thus, separation of the sample into different layers can be done by first centrifuging the original homogenate under weak forces, removing the pellet, then exposing the subsequent supernatants to sequentially greater centrifugal fields. Each time a portion of different density is sedimented to the bottom of the container and extracted, and repeated application produces a rank of layers which includes different parts of the original sample. Additional steps can be taken to further refine each of the obtained pellets.
Sedimentation depends on mass, shape, and partial specific volume
Partial specific volume
The partial specific volume or PSV is a measure of the density of the particle using its calculated volume and mass information. The PSV is usually written in terms of milliLiters per gram .Partial specific volume is an inverse of density....
of a macromolecule, as well as solvent density, rotor size and rate of rotation. The sedimentation velocity can be monitored during the experiment to calculate molecular weight.
Values of sedimentation coefficient
Sedimentation coefficient
The sedimentation coefficient s of a particle is used to characterize its behaviour in sedimentation processes, notably centrifugation. It is defined as the ratio of a particle's sedimentation velocity to the acceleration that is applied to it .The sedimentation speed v_t is also known as the...
(S) can be calculated. Large values of S (faster sedimentation rate) correspond to larger molecular weight. Dense particle sediments more rapidly. Elongated proteins have larger frictional coefficients, and sediment more slowly to ensure accuracy.
Equilibrium (isopycnic) sedimentation
Equilibrium sedimentation uses a gradient of a solution such as cesium chloride or sucroseSucrose
Sucrose is the organic compound commonly known as table sugar and sometimes called saccharose. A white, odorless, crystalline powder with a sweet taste, it is best known for its role in human nutrition. The molecule is a disaccharide composed of glucose and fructose with the molecular formula...
to separate particles based on their individual densities (mass/volume). It is used as a purifying process for differential centrifugation. A solution is prepared with the densest portion of the gradient at the bottom. Particles to be separated are then added to the gradient and centrifuged. Each particle proceeds (either up or down) until it reaches an environment of comparable density. Such a density gradient may be continuous or prepared in a stepped manner. For instance, when using sucrose to prepare density gradients, one can carefully float a solution of 40% sucrose onto a layer of 45% sucrose and add further less dense layers above. The homogenate, prepared in a dilute buffer and centrifuged briefly to remove tissue and unbroken cells, is then layered on top. After centrifugation typically for an hour at about 100,000 x g, one can observe disks of cellular components residing at the change in density from one layer to the next. By carefully adjusting the layer densities to match the cell type, one can enrich for specific cellular components. Caesium chloride allows for greater precision in separating particles of similar density. In fact, with a caesium chloride gradient, DNA particles that have incorporated heavy isotopes (13C or 15N for example) can be separated from DNA particles without heavy isotopes.