#07 Biochemistry Protein Characterization Lecture for Kevin Ahern's BB 450/550

• Report rights infringement
• published: 10 Oct 2011
• views: 13169

1. Contact me at kgahern@davincipress.com / Friend me on Facebook (kevin.g.ahern) 2. Download my free biochemistry book at http://biochem.science.oregonstate.edu/biochemistry-free-and-easy 3. Take my free iTunes U course at https://itunes.apple.com/us/course/biochemistry/id556410409 4. Check out my free book for pre-meds at http://biochem.science.oregonstate.edu/biochemistry-free-and-easy 5. Course video channel at http://www.youtube.com/user/oharow/videos?view=1 6. Check out all of my free workshops at http://www.youtube.com/playlist?list=PLlnFrNM93wqyTiCLZKehU1Tp8rNmnOWYB&feature;=view_all 7. Check out my Metabolic Melodies at http://www.davincipress.com/metabmelodies.html 8. Take my courses for credit (wherever you live) via OSU's ecampus. For details, see http://ecampus.oregonstate.edu/soc/ecatalog/ecourselist.htm?termcode=all&subject;=BB 9. Course materials at http://oregonstate.edu/instruct/bb450 Protein Purification/Characterization II 1. Nucleic acids (which are negatively charged and 'rod-like' in shape) can be separated by agarose gel electrophoresis. In this technique, a 'gel' is made that consists of agarose that forms a sort of 'mesh' of holes through which the DNA molecules pass. Electric fields are used to separate macromolecules by size. The sample is loaded on the top of the gel and electrical current is passed through it such that the bottom electrode is positive and the top one is negative. Negatively charged DNA molecules at the top of the gel are driven away from the top towards the bottom. Small molecules make their way through the gel fastest and big molecules travel more slowly. 2. Proteins are usually globular in their native state and may be negatively, positively or neutrally charged. Polyacrylamide gels have smaller holes than agarose and allow separation of proteins. Sodium dodecyl sulfate (SDS) is added to the protein mixture, causing the proteins to denature, assume rod-like shapes, and be coated with the negative charge. All proteins in the mixture obtain a negative charge and can be separated just like DNA on SDS-polyacrylamide gel electrophoresis. 3. At each step of protein purification, a small sample of the protein extract is taken and the total amount of protein, and the amount of activity of the desired protein are measured. The specific activity of the protein in the tube is the amount of activity divided by the total mass of protein. The yield of the desired protein at any point in the purification process is the number of units of the desired protein at that point divided by the number of units that one started with. The purification level at any point is the specific activity at that point divided by the specific activity one started with. 4. Breaking large proteins down into smaller pieces is also important for studying them. Reagents include cyanogen bromide and enzymes known as proteases. One such protease is trypsin, which cleaves on the carboxyl side of lysine and arginine residues in a polypeptide. Thrombin is another enzyme that breaks peptide bonds. 5. Immunological techniques aid in identifying specific proteins. Antibodies recognize and bind to specific structures. The structures antibodies bind to are called antigens. Since proteins differ from each other in their structure, molecules that bind to specific structures will bind to specific proteins. 6. Antibodies can be linked to fluorescent dyes, gold particles, or enzymes to help one visualize where an antibody has bound. They are useful in binding to specific cellular structures, to identify where within a cell or within a tissue a particular protein is located. 7. A technique that employs antibodies is western blotting. In this method, a mixture of proteins is separated by SDS-PAGE. The proteins in the gel are transferred directly to a membrane, which is then treated with an antibody specific for one of the proteins. The membrane is washed to release unbound antibody and then the antibody-protein complex is visualized. 8. MALDI-TOF is a mass spectometric analysis instrument that provides molecular masses with great accuracy. It employs a laser, which, when activated, causes a polypeptide sample to volatilize in the evacuated chamber of the instrument. Volatilization causes the molecules to both ionize and to break at peptide bonds. Masses are determined by the length of time it takes for the ions to travel through the chamber to the detector. The time it takes them to make the transit is proportional to their mass. Smaller fragments move faster than larger fragments. 9. X-ray crystallography is a technique whereby a crystal of a pure protein (or DNA or RNA) is subjected to beams of x-rays. By studying the angles of deflection (on an x-ray diffraction pattern), one can, using sophisticated computer analysis determine the electron density of the material the x-rays passed through. The electron density map give 3D coordinates of atoms in the crystal to within a few Angstroms.