Cell fractionation

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In cell biology, cell fractionation is the process used to separate cellular components while preserving individual functions of each component. [1] This is a method that was originally used to demonstrate the cellular location of various biochemical processes. Other uses of subcellular fractionation is to provide an enriched source of a protein for further purification, and facilitate the diagnosis of various disease states. [2]

Contents

Homogenization

Tissue is typically homogenized in a buffer solution that is isotonic to stop osmotic damage. Mechanisms for homogenization include grinding, mincing, chopping, pressure changes, osmotic shock, freeze-thawing, and ultra-sound. The samples are then kept cold to prevent enzymatic damage. It is the formation of homogenous mass of cells (cell homogenate or cell suspension). It involves grinding of cells in a suitable medium in the presence of certain enzymes with correct pH, ionic composition, and temperature. For example, pectinase which digests middle lamella among plant cells.

Filtration

This step may not be necessary depending on the source of the cells. Animal tissue however is likely to yield connective tissue which must be removed. Commonly, filtration is achieved either by pouring through gauze or with a suction filter and the relevant grade ceramic filter.

Purification

Purification is achieved by differential centrifugation – the sequential increase in gravitational force results in the sequential separation of organelles according to their density.

See also

Media for cell separation by density:

Related Research Articles

Cell biology is a branch of biology that studies the structure, function, and behavior of cells. All living organisms are made of cells. A cell is the basic unit of life that is responsible for the living and functioning of organisms. Cell biology is the study of structural and functional units of cells. Cell biology encompasses both prokaryotic and eukaryotic cells and has many subtopics which may include the study of cell metabolism, cell communication, cell cycle, biochemistry, and cell composition. The study of cells is performed using several microscopy techniques, cell culture, and cell fractionation. These have allowed for and are currently being used for discoveries and research pertaining to how cells function, ultimately giving insight into understanding larger organisms. Knowing the components of cells and how cells work is fundamental to all biological sciences while also being essential for research in biomedical fields such as cancer, and other diseases. Research in cell biology is interconnected to other fields such as genetics, molecular genetics, molecular biology, medical microbiology, immunology, and cytochemistry.

Centrifugation Mechanical process

Centrifugation is a mechanical process which involves the use of the centrifugal force to separate particles from a solution according to their size, shape, density, medium viscosity and rotor speed. The denser components of the mixture migrate away from the axis of the centrifuge, while the less dense components of the mixture migrate towards the axis. Chemists and biologists may increase the effective gravitational force of the test tube so that the precipitate (pellet) will travel quickly and fully to the bottom of the tube. The remaining liquid that lies above the precipitate is called a supernatant or supernate.

<span class="mw-page-title-main">Differential centrifugation</span> Method of separating particles in a mixture

In biochemistry and cell biology, differential centrifugation is a common procedure used to separate organelles and other sub-cellular particles based on their sedimentation rate. Although often applied in biological analysis, differential centrifugation is a general technique also suitable for crude purification of non-living suspended particles. In a typical case where differential centrifugation is used to analyze cell-biological phenomena, a tissue sample is first lysed to break the cell membranes and release the organelles and cytosol. The lysate is then subjected to repeated centrifugations, where particles that sediment sufficiently quickly at a given centrifugal force for a given time form a compact "pellet" at the bottom of the centrifugation tube.

Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the specification of the function, structure and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity and biological activity. The pure result may be termed protein isolate.

<span class="mw-page-title-main">Fractionation</span> Method of separating components of a mixture via phase transition

Fractionation is a separation process in which a certain quantity of a mixture is divided during a phase transition, into a number of smaller quantities (fractions) in which the composition varies according to a gradient. Fractions are collected based on differences in a specific property of the individual components. A common trait in fractionations is the need to find an optimum between the amount of fractions collected and the desired purity in each fraction. Fractionation makes it possible to isolate more than two components in a mixture in a single run. This property sets it apart from other separation techniques.

<span class="mw-page-title-main">Fluorescence microscope</span> Optical microscope that uses fluorescence and phosphorescence

A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image.

<span class="mw-page-title-main">Cell disruption</span>

Cell disruption is a method or process for releasing biological molecules from inside a cell.

Synaptosome

A synaptosome is an isolated synaptic terminal from a neuron. Synaptosomes are obtained by mild homogenization of nervous tissue under isotonic conditions and subsequent fractionation using differential and density gradient centrifugation. Liquid shear detaches the nerve terminals from the axon and the plasma membrane surrounding the nerve terminal particle reseals. Synaptosomes are osmotically sensitive, contain numerous small clear synaptic vesicles, sometimes larger dense-core vesicles and frequently one or more small mitochondria. They carry the morphological features and most of the chemical properties of the original nerve terminal. Synaptosomes isolated from mammalian brain often retain a piece of the attached postsynaptic membrane, facing the active zone.

French pressure cell press Apparatus used in biological experimentation

The French pressure cell press, or French press, is an apparatus used in biological experimentation to disrupt the plasma membrane of cells by passing them through a narrow valve under high pressure. The French Press can also be used for disintegration of chloroplasts, homogenates of animal tissue, and other biological particles. It is capable of disrupting cell walls while leaving the cell nucleus undisturbed. The French press was invented by Charles Stacy French of the Carnegie Institution of Washington. The press uses an external hydraulic pump to drive a piston within a larger cylinder that contains the liquid sample. The highly pressurized sample is then squeezed past a needle valve. As the sample passes through the valve, the fluid experiences shear stress and decompression, causing cellular disruption. The major components of a French press are made of stainless steel to prevent sample contamination.

In biology, autolysis, more commonly known as self-digestion, refers to the destruction of a cell through the action of its own enzymes. It may also refer to the digestion of an enzyme by another molecule of the same enzyme.

Downstream processing refers to the recovery and the purification of biosynthetic products, particularly pharmaceuticals, from natural sources such as animal tissue, plant tissue or fermentation broth, including the recycling of salvageable components as well as the proper treatment and disposal of waste. It is an essential step in the manufacture of pharmaceuticals such as antibiotics, hormones, antibodies and vaccines; antibodies and enzymes used in diagnostics; industrial enzymes; and natural fragrance and flavor compounds. Downstream processing is usually considered a specialized field in biochemical engineering, which is itself a specialization within chemical engineering. Many of the key technologies were developed by chemists and biologists for laboratory-scale separation of biological and synthetic products, whilst the role of biochemical and chemical engineers is to develop the technologies towards larger production capacities.

Fast protein liquid chromatography (FPLC), is a form of liquid chromatography that is often used to analyze or purify mixtures of proteins. As in other forms of chromatography, separation is possible because the different components of a mixture have different affinities for two materials, a moving fluid and a porous solid. In FPLC the mobile phase is an aqueous solution, or "buffer". The buffer flow rate is controlled by a positive-displacement pump and is normally kept constant, while the composition of the buffer can be varied by drawing fluids in different proportions from two or more external reservoirs. The stationary phase is a resin composed of beads, usually of cross-linked agarose, packed into a cylindrical glass or plastic column. FPLC resins are available in a wide range of bead sizes and surface ligands depending on the application.

Chromatography is a physical method of separation that distributes the components you want to separate between two phases, one stationary, the other moving in a definite direction. Cold ethanol precipitation, developed by Cohn in 1946, manipulates pH, ionic strength, ethanol concentration and temperature to precipitate different protein fractions from plasma. Chromatographic techniques utilise ion exchange, gel filtration and affinity resins to separate proteins. Since the 1980s it has emerged as an effective method of purifying blood components for therapeutic use.

Blood plasma fractionation are the general processes of separating the various components of blood plasma, which in turn is a component of blood obtained through blood fractionation. Plasma-derived immunoglobulins are giving a new narrative to healthcare across a wide range of autoimmune inflammatory diseases. This widespread applicability is anticipated to leverage market prospects for plasma fractionation, pegged to witness a noteworthy 7% CAGR. COVID-19 pandemic is expected to generate growth opportunities for the plasma fractionation market.

Homogenization (biology)

Homogenization, in cell biology or molecular biology, is a process whereby different fractions of a biological sample become equal in composition. It can be a disease sign in histopathology, or an intentional process in research: A homogenized sample is equal in composition throughout, so that removing a fraction does not alter the overall molecular make-up of the sample remaining, and is identical to the fraction removed. Induced homogenization in biology is often followed by molecular extraction and various analytical techniques, including ELISA and western blot.

Electrofiltration is a method that combines membrane filtration and electrophoresis in a dead-end process.

Membrane technology covers all engineering approaches for the transport of substances between two fractions with the help of semi-permeable membranes. In general, mechanical separation processes for separating gaseous or liquid streams use membrane technology.

Single cell oil, also known as Microbial oil consists of the intracellular storage lipids, triacyglycerols. It is similar to vegetable oil, another biologically produced oil. They are produced by oleaginous microorganisms, which is the term for those bacteria, molds, algae and yeast, which can accumulate 20% to 80% lipids of their biomass. The accumulation of lipids take place by the end of logarithmic phase and continues during station phase until carbon source begins to reduce with nutrition limitation.

A separation process is a method that converts a mixture or a solution of chemical substances into two or more distinct product mixtures. In other words, it's a scientific process of distinguishing to two or more substance in order to obtain purity. At least one product mixture of the separation is enriched in one or more of the source mixture's constituents. In some cases, a separation may fully divide the mixture into pure constituents. Separations exploit differences in chemical properties or physical properties between the constituents of a mixture.

A ligand binding assay (LBA) is an assay, or an analytic procedure, which relies on the binding of ligand molecules to receptors, antibodies or other macromolecules. A detection method is used to determine the presence and extent of the ligand-receptor complexes formed, and this is usually determined electrochemically or through a fluorescence detection method. This type of analytic test can be used to test for the presence of target molecules in a sample that are known to bind to the receptor.

References

  1. Alberts, B; Johnson, A (2002). "Fractionation of Cells". Molecular Biology of the Cell. 4th edition.
  2. Ninfa, Alexander J. (2010). Fundamental Laboratory Approaches for Biochemistry and Biotechnology. United States of America: John Wiley & Sons, INC. p. 209. ISBN   978-0-470-08766-4.