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The important techniques involved in the study of molecular biology of proteins are:- 1. Purification from Cell Extracts 2. Using Column Chromatography 3. Affinity Chromatography 4. Separation of Proteins on Polyacrylamide Gels 5. Antibodies can be Visualized Electrophoretically: Separated Proteins 6. Sequencing Protein Molecules.
Technique # 1. Purification from Cell Extracts:
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Purification of proteins is a major part of understanding their function. Purification of protein is designed to exploit its unique characteristic, including size, charge, shape and in many instances, function. The purification of protein requires two major aspects; first a specific assay e.g. interpretation assay second, preparations of cell extracts containing active proteins.
Technique # 2. Using Column Chromatography:
This includes:
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(1) Techniques of ion exchange chromatography on the basis of surface ionic charge using beads.
(2) Gel filtration chromatography which separates proteins on the basis of size and shape.
Technique # 3. Affinity Chromatography:
This exploits the specific nature of proteins to bind e.g. ATP binding proteins or DNA binding proteins. One of the most common forms of protein affinity chromatography is immunoaffinity chromatography in which an antibody which is specific for target protein is attached to beads.
The bound protein hence can be eluted from the column using salt or in some cases mild detergent. In this some other purposes include recognition and characterization of specific epitopes i.e., a sequence of 7 to 10 amino acids which can be attached to any protein. This method permits the modified protein to be purified using the immunoaffinity purification and a heterologous antibody which is specific for the added epitope. Some specific proteins can be precipitated rapidly by attaching the antibody to the same type of bead used in column chromatography; this process is known as immuno precipitation.
Technique # 4. Separation of Proteins on Polyacrylamide Gels:
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It is used by using strong ionic detergent sodium dodecyl sulphate (SDS) and a reducing agent like mercaptoethanol. After electrophoresis the proteins can be visualized with a stain like coomassie brilliant blue which binds to protein.
Technique # 5. Antibodies can be Visualized Electrophoretically: Separated Proteins:
The proteins are altogether different from DNA and RNA but the process has been called immunoblotting, by which an individual protein can be seen squinting amongst thousands of other proteins which is analogous incomplete to southern and northern blot hybridization. Southern, northern and immunoblotting have in common the use of selective reagents to visualize particular molecule in complex mixtures.
Technique # 6. Sequencing Protein Molecules:
Sequencing of protein molecules is more complex than sequencing of nucleic acids which can be done using:
(a) Edman degradation and
(b) Tandem mass spectrometry (MS/MS).
Edman degradation is a chemical reaction in which the amino acid residues are sequentially released for the N-terminus of a polypeptide chain. The N-terminal of most amino acids in chain can be modified specifically using a chemical reagent called phenylisothiocyanate (PITC) which possess the potential to modify free a – amino group. The amino acid derivoltized in this way can be easily determined by its elution profile using a column chromatography method called High Performance Liquid Chromatography or just HPLC.
Mass spectrometry is a method in which the mass of a very small sample of material can be known on the principle that material travels through an instrument (in a vacuum) in a manner which is sensitive to its mass/charge ratio. The Tandem mass spectrometry or MS/MS has played a wonderful role in protein sequencing and identification.
