Describe the methods of seperating macromolecules on the basis of their size,cgarge,solubility,and molecular affinity
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Over the years, a number of sophisticated analytical and preparative techniques have been developed for separating, analyzing, and isolating the various macromolecular constituents of cells and tissues.
Various forms of electrophoresis, chromatography, and ultracentrifugation are now in routine use in most laboratories engaged in molecular biological studies and have greatly increased our understanding of the chemistry and properties of the cellular macromolecules.
Most of the methods that are used to separate and isolate different members of a class of macromolecules simultaneously provide information concerning their chemistry because parameters such as molecular size, shape, density, net and absolute charge, differential solubility, and so forth are used as the basis for the separation.
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Nearly all the techniques that are used for separating and isolating macromolecules require that these substances initially be in a soluble state.
Consequently, macromolecules present in extracellular fluids such as plasma, lymph, and hormonal and digestive secretions are most easily isolated and until recently were the subject of the most intensive studies.
Now, however, a variety of procedures are available to solubilize macromolecular constituents of membranes and other particulate components of cells. Therefore, in recent years, there has been a concerted effort to isolate and chemically characterize these macromolecular constituents as well.
A number of physical methods were described for disrupting cells. Often, more vigorous or more extensive applications of the same procedures to whole cells or to isolated organelles will also free some of the constituent macromolecules. For example, extended sonification or homogenization of mitochondrial suspensions solubilizes many of the mitochondrial enzymes and other constituents, and nucleic acids may be released from isolated nuclei under similar conditions.
Various forms of electrophoresis, chromatography, and ultracentrifugation are now in routine use in most laboratories engaged in molecular biological studies and have greatly increased our understanding of the chemistry and properties of the cellular macromolecules.
Most of the methods that are used to separate and isolate different members of a class of macromolecules simultaneously provide information concerning their chemistry because parameters such as molecular size, shape, density, net and absolute charge, differential solubility, and so forth are used as the basis for the separation.
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Nearly all the techniques that are used for separating and isolating macromolecules require that these substances initially be in a soluble state.
Consequently, macromolecules present in extracellular fluids such as plasma, lymph, and hormonal and digestive secretions are most easily isolated and until recently were the subject of the most intensive studies.
Now, however, a variety of procedures are available to solubilize macromolecular constituents of membranes and other particulate components of cells. Therefore, in recent years, there has been a concerted effort to isolate and chemically characterize these macromolecular constituents as well.
A number of physical methods were described for disrupting cells. Often, more vigorous or more extensive applications of the same procedures to whole cells or to isolated organelles will also free some of the constituent macromolecules. For example, extended sonification or homogenization of mitochondrial suspensions solubilizes many of the mitochondrial enzymes and other constituents, and nucleic acids may be released from isolated nuclei under similar conditions.
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These are the techniques to separate macro-molecules on the basis of
- Molecular size: Gel filtration Chromatography, Centrifugation, Gel Electrophoresis using SDS is the technique to separate macro-molecules on the basis of molecular size.
- Solubility: on the basis of solubility, salting out technique can be used
- Molecular affinity: Affinity chromatography is used to separate molecules on the basis of their molecular affinity.
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