Lactate Dehydrogenase Lab Report

Proteins are the most abundant macromolecules found in living organisms because they can perform multiple task in the body. The task proteins are known to carry out are compound transporters, structural support in hair and nails, protecting against infections and catalyzing reactions. However, to study a certain protein it has to go through purification because cells can contain thousands of proteins. Purification allows scientist to know what molecule substrates are present, if the enzyme activity is regulated, the size, and what subunits are in the protein. This particular experiment focuses on lactate dehydrogenase from a beef heart. Lactate dehydrogenase (LDH) catalyzes the reaction where lactate is oxidized to pyruvate and NAD+ is reduced
The first step in the process is to purify the protein by centrifuging the sample multiple times and combining it with 100 mM potassium phosphate pH 7.2 buffer at varying volumes. By doing this an initial extract, 40% extract and 60% extract is created and will be used for the remaining of steps in the following weeks. Therefore, it is important to be mindful of how the protein is stored to prevent denaturing causing skewed data. The second week is about further purification of the protein and running an activity assay on a spectrophotometer set to 340 nm. In order to achieve the objective for week two the 60% is re-suspended and stored in dialysis tube submerged in potassium phosphate buffer. After removing the sample from dialysis and transferring it to clean beaker it is ran through a Cibacron Blue affinity column to produce nine total fractions. These fractions are used to test the activity of the protein and the one with the highest activity is saved for the remaining procedures and the rest is discarded. Week three is concerned with determining the molecular weight and purity of the protein by running the sample through SDS-Page