The purpose of this experiment is to separate a mixture of proteins from muscle tissue using SDS-polyacrylamide gel electrophoresis (PAGE). Proteins are a macromolecule in cells that assist in conformation, function, and behavior. They result from a two-step process of gene expression. This happens when DNA of a gene is copied into RNA and RNA is copied into the protein. Cells control protein production so that proteins that are not needed will not be detrimental to an organism. SDS-PAGE is a technique that uses electrical current to separate proteins. The SDS detergent is used first and it is heated to denature proteins in a solution. The primary structure of the protein is maintained but the secondary, tertiary, and quaternary structures are destroyed. This is important for complex protein conformations because proteins can appear bigger or smaller than determined in gel electrophoresis. The SDS detergent coats the proteins with a negative charge so that they all move in the same direction down the gel. Once the proteins have been run through gel electrophoresis, the proteins are stained. The staining on the proteins will make the bands appear so that the different proteins can be identified.
Materials and Methods
1 cm3 pieces of B. Taurus, G. gallus, and Tilapia were obtained from the grocery store. Each sample is to be handled separately or by a different group member. Be sure to use gloves and wash hands between handling each sample to prevent cross contamination. Obtain a mortar and pestle to grind each cube of meat, separately. Add three mL of diH2O to each meat sample. Before grinding a new cube of meat, clean the mortar with soap and water. After grinding the samples add three mL of buffer to each mortar and continue to use the pestle to mix. Use a spoon to scrape the meat samples into their own fifteen mL centrifuge tubes. Label each tube with your group name or number and a way to identify the sample. Let the tubes centrifuge for five minutes. Be sure to balance the centrifuge before turning it on. Once the tubes have been taken out of the centrifuge, use a pipet to transfer the liquid contents to a new fifteen mL centrifuge tube. Be sure to label these tubes with group name and sample number. The tubes with the original sample with the remaining tissue can be discarded. Now, obtain the unknown sample from an ice bucket. There should be four samples: three knowns and one unknown. Transfer thirty µL of sample to separate centrifuge tubes. Add ten µL of blue buffer to the microcentrifuge tube. Do not heat the...