Lecture6

Lecture6 - #6 Recombinant DNA technology (III) 1. 2. 3. 4....

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#6 Recombinant DNA technology (III) 1. Protein purification 2. Protein analyses 3. Protein expression in E. coli 4. Protein expression in animal cells
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1. Protein Purification How to separate proteins from other molecules? How to separate one protein from others How to detect or “see” a specific protein ….. Many methods can be used to analyze proteins Centrifugation and chromatography SDS-PAGE, 2D gel, and 2D-DIGE Immunological techniques
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Break-up tissues and cells ultrasound French press detergent homogenize
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Centrifugation Differential centrifugation separates matters according to mass and density, it can be used to separate organelles and large protein complexes
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(3) Affinity chromatography: -Separate according to ability to bind to ligands -cylindrical columns filled with water permeable solid matrix (also called bead, resin or gel) of different properties. -Different proteins behavior different in different columns, so that to be separated from each other. Separation of proteins by chromatography (1) Gel filtration chromatography (or sizing column): -Separate according to size (2) Ion exchange chromatography: -Separate according to charge Anion exchange resins—binding to anions (-) Cation exchange resins—binding to cations (+)
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Components of a column Chromatography apparatus : (1) A cylindrical column filled with resins (also called matrix, beads, etc). The type of chromatography is determined by the chemical nature of the resin (2) A pump to push solution flow through the column (3) A fraction collector to collect solutions come out from the column (4) An UV detector to detect proteins in different fractions A simple chromatography used in a lab Large chromatography used to purify pharmaceutical proteins
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Separation, collection, detection Proteins in each fraction are detected by their absorption of UV(280nm) light Proteins in the mixture are separated into individual fractions after running through a chromatographic column
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Gel filtration chromatography Gel filtration separate molecules according to their size. The larger molecules migrate faster in the gel filtration column. The smaller molecules migrate slower because they travel through pores of the porous beads
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Ion exchange chromatography Ion exchange chromatography separate molecules according to their charge. Cation exchangers (beads negatively charged, e.g. CM) or anion exchangers (beads positively charged (e.g. DEAE) preferentially bind to cations or anions, respectively. Proteins are bound to beads according to their different charges, and eluted from the beads by gradually increasing salt concentration in the elution buffer that out-compete the bound proteins to beads.
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chromatography separate molecules according to their affinity to specific ligand, such as antibody or Ni. Bead can be covalently linked to the ligand that the target protein will bind to.
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This note was uploaded on 02/20/2009 for the course LS 3 taught by Professor Lin during the Spring '06 term at UCLA.

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Lecture6 - #6 Recombinant DNA technology (III) 1. 2. 3. 4....

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