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Biochemistry Lecture No. 9.docx

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Biochemistry 3380G

Biochemistry Lecture No. 9: Protein Purification th Tuesday September 25 , 2012 Introduction: -Protein purification as a process is important in many ways, these include: For medical use (like human growth hormones, insulin and chemotherapy), to study structure and function (difficult to understand proteins in mixtures) and for use as a reagent in the laboratory (like enzymes for manipulating DNA). The Purification Process: -Proteins are purified/isolated from their environment by using their unique properties to differentiate them from everything else. These include the protein’s: size and shape, charge, location, and surface hydrophobicity. Before The Purification Process Begins: -An assay method is needed prior to the purification process. One must be able to detect the protein in a mixture as well as measure some property of the protein of interest. A starting material (human tissues, plants, or bacteria must also be decided upon. Throughout the process, the protein will have to be isolated (preparative method) and its purity determined (analytical). Homogenization (the break-up of material) as well a good quantity of proteins (much is needed) will be key steps of the process. Preparative & Analytical Methods: -Both of these methods are used to purify proteins, starting with the preparative method, followed by the analytical methods. Preparative methods are used to homogenize (“bust up”) material at a large scale (mg-kg) and divide the mixture into “fractions.” Analytical methods are used to detect a specific protein of interest at smaller scale (mg ≥) with small samples of fractions for analysis. Homogenization: -The first step of protein purification uses different forms of homogenization in order to break open tissue or cell in order to release protein. These include: sonication (high frequency sound waves), grinders of various types, blenders/homogenizers, exertion of pressure, and detergents. A compatible buffer solution is also necessary to maintain the protein’s native conformation. Centrifugation: -Administering samples into chamber turning at very high speeds. Particles are separated by size and density. Differential centrifugation requires that the samples spin at various speeds; different debris will pellet out or sink to the bottom. At low speed nuclei and whole cells pellet, at medium speed slightly smaller organelles pellet and at maximum speed viruses, ribosomes and large macromolecules pellet. -It is a quick and easy way of getting rid of things you don’t need. Column Chromatography: rd -Often the 3 step of protein purification, column chromatography further separates based on characteristics like charge and size. It requires two components: a mobile phase (an appropriate aqueous buffer solution) and a solid phase (usually small beads). The solution is held in a cylinder or 'column.' Proteins in mobile phase interact (or not) with the matrix as a continuous flow of the buffer solution carries unbound proteins along. Eluting Columns: -The effluent is collected (or eluted) in 'fractions.' Generally a constant flow rate is maintained, and a fraction is collected every few minutes with many fractions from a single run. Each fraction is tested to detect the specific protein of interest and to measure the total amount of protein. Absorbance at 280 nm wavelength measures total protein since aromatic amino acids (W, F and Y) absorb at 280 nm. Analyzing Fractions (Elution Diagram): -The following figure would represent some 50 test tubes. In measuring the A280, the A280 does not tell you which protein you want since it gives all the protein in the sample. The protein of interest comes out in only a few tubes. Types Of Column Chromatography: -There are three kinds of column chromatography: ion exchange chromatography (beads that carry a charge/separating amino acids according to charge), gel-filtration chromatography (beads contain tiny holes that small proteins get stuck in; “size exclusion.”; big proteins come f
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