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#06 Biochemistry Protein Purification Lecture for Kevin Ahern's BB 450/550

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Published on Oct 7, 2011

1. Contact me at kgahern@davincipress.com / Friend me on Facebook (kevin.g.ahern)
2. Download my free biochemistry book at http://www.davincipress.com/freeforal...
3. Take my free iTunes U course at https://itunes.apple.com/us/course/bi...
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6. Check out all of my free workshops at http://oregonstate.edu/dept/biochem/a...
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8. My courses can be taken for credit (wherever you live) via OSU's ecampus. For details, see http://ecampus.oregonstate.edu/soc/ec...
9. Course materials at http://davincipress.com/bb450.html

Highlights Protein Purification/Characterization
1. Proteins have amino acids in them that are chemically modified. Chemical modification of amino acids in proteins occurs AFTER the protein is synthesized. Examples include hydroxyproline and hydroxylysine, gamma carboxyglutamate, and phosphoserine.
2. Purification of proteins exploits differences in charge, size, shape, and affinity for specific compounds. Centrifugation (artificial gravity) provides a means of precipitating cellular components. The amount of centrifugal force applied (speed of spin) determines the rate with which a given substance will be driven towards the bottom of the tube.
3. Dialysis provides a means of separating large molecules (like proteins, DNA, etc) from small molecules (like salts, etc.) by encasing the protein/salt mixture in a membrane. The membrane has holes (pores) big enough to let out small molecules, but too small to let out the big molecules. Thus, with this technique, one can effectively remove the salt from a protein.
4. Gel filtration (gel exclusion chromatography) provides another way to separate large molecules from smaller ones. It employs beads with tunnels in them. The beads are packed in a column. The beads have buffer running through them and the openings to the tunnels are a fixed size (called an exclusion limit). The size of the opening determines the maximum size of molecule that can enter it. Molecules smaller than the exclusion limit enter the beads and travel a longer path than molecules larger than the exclusion limit. Thus, when large and small proteins are applied to a column the large proteins come through first and the small ones come last.
5. Ion exchange chromatography uses beads in a column also, but instead of tunnels, the beads are coated with a molecule having either a positive or negative charge. If a mixture of molecules with positive and negative charges is added to the column, the negative molecules will "stick" to the column when the beads are coated with positive charges.
6. Affinity chromatography exploits the tendency of many proteins to 'bind' to molecules. For example, many proteins bind ATP. If one takes beads and coats them with ATP and passes a protein mixture through the column, only those proteins that bind to ATP will stick to the column.
7. HPLC employs densely packed columns containing material with chemical groups on them that interact with molecules as they are pumped through the column. Reverse phase chromatography, which uses column material that is very non-polar.
8. Gel electrophoresis separates molecules using an electric field across a support (gel). In this method, molecules separate by size. The smallest ones move the fastest and the largest ones move the slowest. There are several considerations for this method and I will explain this in more detail in the next lecture.
9. Isoelectric focusing separates molecules on the basis of their pI. It is performed in tubes containing special compounds (polyelectrolytes) that migrate to specific points in the tube when in the presence of an electric field. This effectively creates a pH gradient from one end of the tube to the other. If proteins are added to the tube as the gradient is getting established, they will migrate to the point in the tube where the pH corresponds to their pI.
10. 2D gel electrophoresis is a powerful tool for proteomics that combines the techniques of isoelectric focusing with SDS-PAGE. In this method, proteins are first separated according to their pI by isoelectric focusing. Then the tube from the isoelectric focusing is applied to the top of an SDS-PAGE gel and the proteins are separated by size. The result is a two dimensional separation of virtually every protein in the cell.

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