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Lab Documentation 1 I. STUDENT HANDOUT CHEM 3501L Experiment: Examining Protein Quaternary Structure Using Gel Electrophoresis Kennesaw State University, Kennesaw, GA 30144 BACKGROUND READING: ±Quaternary Structure,² pp. 102-104. Horton, Moran, Ochs, Rawn, and Scrimgeour. Principles of Biochemistry, 3 rd edition. 2002. Prentice Hall, Upper Saddle River, NJ 07458. INTRODUCTION : Gel electrophoresis is a separation technique that separates molecules based on size and charge differences. In polyacrylamide gel electrophoresis (PAGE), protein samples are placed on a highly cross-linked gel matrix and an electric current is applied. The cross-linked polyacrylamide molecules act as molecular sieves, retarding the passage of large molecules more than small molecules. Gels can be made in the lab or bought from a supplier. Precast polyacrylamide gels come in a variety of concentrations, such as 7.5%, 12% and a gradient gel (4-15%). The higher the percentage acrylamide, the smaller the pores will be. Thus, the % gel used is selected based on the protein size(s) in the sample of interest. You will be using the 4- 15% gradient gel because it allows for separation of a larger range of protein sizes (10 kDa ³250 kDa). In native PAGE techniques, protein migration is affected by size and charge. To accurately determine a protein´s molecular weight, a method is needed in which charge does not vary from protein to protein. The technique that is typically used is SDS-PAGE. When sodium dodecyl sulfate (SDS), a negatively charged detergent, is added to the protein samples prior to placing on the gel, the protein is denatured as the hydrophobic ends of SDS molecules associate with the hydrophobic side chains of the amino acid residues in the protein. The negatively charged end of each SDS molecule points out toward the surface, as in a micelle arrangement, so each protein molecule is surrounded by an overwhelming number of negative charges. The migration of the protein is now dependent on the molecular weight (size) of the protein. Native PAGE techniques allow proteins to be separated in their native conformation. However, the addition of SDS denatures the protein causing secondary, tertiary, and quaternary structure to be altered. If a protein commonly containing two protein subunits held together by noncovalent interactions is subjected to SDS-PAGE, the subunits should dissociate completely. Thus, a protein with a molecular weight of 100 kDa comprised of two identical chains of 50 kDa each would appear as one single band in the loaded lane, with a molecular weight of approximately 50 kDa. Likewise, a protein with a molecular weight of 100 kDa made of two nonidentical chains of molecular weights 30 kDa and 70 kDa would show up as two separate bands of 30 kDa and 70
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Lab Documentation 2 kDa, respectively, in an SDS-PAGE experiment. Finally, a protein of 100 kDa comprised of one
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This note was uploaded on 03/28/2008 for the course CHEM 3501L taught by Professor Staff during the Spring '08 term at Texas A&M.

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