LDH Purification lab Report

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PURIFICATION OF LACTATE DEHYDROGENASE FROM CHICKEN MUSCLE TISSUE Abstract The enzyme lactate dehydrogenase (LDH) catalyzes the last step of anaerobic glycolysis that is important for the normal function of the body. Purification of LDH is essential to understand its structure and function. The purpose of this experiment was to extract and purify LDH enzyme from chicken muscle tissue using a variety of various. Analytical methods such as activity and protein assay were employed to determine the presence and purity of LDH. The cells were initially disrupted and proteins were solubilized. LDH was purified from the ammonium sulfate precipitated protein mixture by affinity chromatography and its activity was studied by…show more content…
10 microliters of the sample is then added and the assay absorption is measured at 340nm. If absorbance was above 1.5, samples were diluted. Protein Assay: The Pierce BCA Protein Assay (Thermo Scientific) is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein concentration. A series of standard solution of Bovine Serum Albumin (BSA) ranging from 0-2000 µg/ml was prepared from a stock solution of 2 mg/ml BSA. 25ul of diluted crude (1:500, 1:250), desalted (1:100, 1:50), and 6 peak fractions from cibarcon blue column (1:10, 1:5) were loaded in microplate along with 175ul of BCA working reagent. Microplate was incubated for 30min at 370C and then the absorbance was measured at 562nm. Results/Discussion The purpose of this experiment was to extract and purify LDH enzyme from chicken muscle tissue using a variety of techniques including homogenization, ammonium sulfate precipitation, dialysis, and affinity chromatography. Activity and Protein assay were used to track the overall amount of LDH present in the samples. Crude Extraction: Chicken muscle tissue was homogenized in a blender with cold extraction buffer in order to lyse cells, releasing LDH into

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