Lecture 5. Tuesday, September 12. Protein Purification. Principles of Protein Structure, contd.

Lecture 5. Tuesday, September 12. Protein Purification. Principles of Protein Structure, contd.

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Source: Matthew Young Outline: • Proteins have different physical properties These properties can be used to isolate populations of specific proteins from one another Purification of Proteins Chem130/MCB100A Fall 2006 University of California, Berkeley
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Protein properties Different solubilities Different lengths of amino acid chain Different charges Different 3D shapes Different surface features/ligand binding Purifying proteins takes advantage of these differences to isolate a population of a protein of interest
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Protein Purification Flowchart cell Membranes, insoluble matter Soluble matter DNA, RNA, etc. proteins 99% of all of the proteins in the cell A population of one protein of interest Darnell, Lodish, Baltimore, Molecular Cell Biology
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Protein properties Different solubilities Different lengths of amino acid chain Different charges Different 3D shapes Different surface features/ligand binding
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Releasing proteins from the cell Physical methods – Shearing open the cells using pressure or strong sound waves Chemical methods – Detergents, or enzymatic digestion Centrifuge to separate insoluble membranes and proteins from soluble matter and proteins
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Protein properties Different solubilities Different lengths of amino acid chain Different charges Different 3D shapes Different surface features/ligand binding
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Gel electrophoresis SDS (sodium dodecyl sulfate) – Detergent – Unfolds proteins – Gives all protein chains a net negative charge samples • Porous gel: – polyacrylamide • Voltage – Top: (-) – Bottom: (+) • “run to the red”
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SDS-Page gel Proteins travel at a rate inversely proportional to their size (number of residues) only – Shorter proteins travel down the gel faster Proteins must be stained to see them Size (KDa) Direction of travel (-) (+)
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UV Absorbance λ max Trp 280 Tyr 274 Phe 257
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Chromatography
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The beads are much bigger than proteins A column is packed with millions of tiny beads: Sample flow
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