Purifying_LDH_from_crude_protein[1][1]

Purifying_LDH_from_crude_protein[1][1] - Purification and...

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Purification and Kinetics of Lactate Dehydrogenase Sample Lab Report October 12, 2011
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Introduction: Enzymes are used to catalyze reactions, so they can metabolize more quickly, without expending any additional energy. The majority of enzymes are proteins, and are specific to certain reactions. But because these “biological systems are very complex and difficult to study in vivo ,” (Farrell, 2006), enzymes are isolated from their systems usually through a combination of different methods and examined individually. In this experiment, Lactate dehydrogenase (from now on referred to as LDH), “an enzyme found in the cell cytosol, which makes it easy to isolate” (Farrell, 2006) will be purified through a series of centrifuging, salting out and affinity chromatography. Its subunits will be analyzed by electrophoresis and the remaining LDH will be assayed to examine its kinetics. “Enzyme kinetics is the study of enzyme rates and how these rates are affected by enzyme concentration substrates, and any inhibitors or activators,” (Farrell, 2006). Centrifuging seperates substances in a solution based on their densities. The denser a substance is the further down the test tube it falls. The more force that is applied in the centrifuge, the more substances will break apart and separate. For example, at “10,000 g mitochondria, peroxisomes, and lysosomes will spin down” (Farrell, 2006) but at 100,000 g, one can begin to break apart the endoplasmic reticulum. Centrifuging will also spin down any precipitates that may form. During the “salting out” process, a salt is added to the solution containing the enzyme to be isolated. The salt, in this experiment (NH 4 ) 2 SO 4 , is added slowly to prevent the protein from forming hydrogen bonds with the water molecules. This causes the proteins to react with each other instead of the water and precipitate out of solution. This sample can then be centrifuged again and decanted to separate the protein. Affinity chromatography uses specific resins that are chosen based on its ability to bind to the protein of interest, in this instance, LDH. The LDH binds to the resin, while the remaining proteins are eluted out. Once the remaining proteins are washed out of the column, the LDH can be eluted out by using a concentrated salt solution to “disrupt the [electrostatic] interactions and cause the bound molecule to be released,” (Farrell, 2006). Electrophoresis involves the use of an electric current running through a gel medium and moving charged particles across the medium. The velocity at which a molecule moves across the gel depends on its charge, the more negative the charge, the faster it will be. Likewise, the larger the molecule the faster it will be, this is because larger proteins are more negative Materials and Methods: Purifying LDH from crude protein: Begin the experiment by cutting up the beef heart in to small pieces, and trim off any fatty or connective tissue. Take 25g of the finely chopped beef heart and at it to a blender with 75-mL of cold sodium phosphate and blend for two minutes. From this, save 0.5-mL of the crude protein
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