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Affinity chromatography affinity chromatography is

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Affinity ChromatographyAffinity chromatography is used to purify molecules, proteins in particular,by using a support that has a particular molecule covalently linked to thebeads that the molecule or protein of interest has an “affinity” for and willbind. See page 217 of the texbook for a figure of affinity chromatography.The protein of interest can be released from the column by adding extra ofthe same molecule that is covalently linked to the support and will flow outin later fractions. Molecules are separated by this method based on theirbinding affinity.
High Performance Liquid Chromatography (HPLC)High performance liquid chromatography (HPLC), also called high pressureliquid chromatography, is used to purify or separate molecules based ontheir polarity. The supports are composed of tiny beads that are packed verytightly, so that high pressure is needed for solvents and buffers to flowthrough. The supports may be polar (normal phase separation) in whichnon-polar molecules flow through followed by elution of polar molecules inlater fractions; or non-polar (reverse phase separation), where polarmolecules are eluted first, followed by non-polar molecules.2.Demonstrate an understanding of the function of histidinetagging to purify proteins.Histidine ColumnsOne of the approaches a scientist may take to study a particular protein, ifthey already know the gene sequence, is to have that protein cloned andexpressed in a plasmid. We will cover cloning in another lesson, but thepoint of doing this is to have a pure protein with multiple copies to test.Once this protein is cloned into a plasmid and grown in a bacterium suchasEscherichia coli, it is necessary to purify this protein from the bacterialcells. The best way to do this is to use histidine tagging and run the proteinon a column. This is achieved by adding nucleotide sequences to yourprimers for cloning that after transcription and translation will result in sixhistidine residues added to the recombinant protein. After obtaining a celllysate, the his-tagged protein can be purified relatively easily by applyingthe sample to a protein with a support of cobalt or nickel, to which thehistidine residues will bind. All other proteins in the lysate will not stick andwill flow through the column. The his-tagged protein can then be releasedfrom the support by adding imidazole to recover the pure protein from thecolumn. The first figure on page 218 of the textbook illustrates histidinetagging and column recoveryStudy Questions1.What is the basis for chromatography? What are the generalcomponents? How does it generally work? What is thepurpose of this technique?Chromatography allows the separation of molecules to a higher resolutionand may allow purification of one molecule such as a protein. It isperformed in columns or tubes containing a material used for separationbased on size, charge, polarity, or molecular interactions. A buffer is added,and the sample is then added and allowed to flow through the support

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Term
Fall
Professor
N/A
Tags
Cellular Respiration, Glycolysis, Adenosine triphosphate

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