Purification of lactate Dehydrogenase

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Purification of Lactate Dehydrogenase
Results and Discussion
Monitoring Assays Enzyme activity assay and Bradford dye binding protein assay was used.
Ammonium Sulfate Fractionation In order to isolate and purify Lactate Dehydrogenase (LDH), we first extracted LDH from bovine muscle tissue. We minced 8 grams of meat and homogenized using blender. The homogenate was centrifuged and the pellet consisting of membranes, organelles, cytoskeletal components and structural fragments was discarded. The crude extract was subjected to 40% (NH4)2SO4 fractionation. The purpose of this is to remove molecules of lesser solubility than LDH like lipids, fats and low soluble proteins. For this, 9.24 g of was slowly added to crude extract while stirring reaching final solution saturation of 40%. The mixture was then centrifuged and the pellet consisting the contaminants was discarded. From table I, the data indicate that the yield for this was 88% with purification factor of 1.5. The total protein in 40% supernatant was 121 mg which decreased from 210 mg present in crude extract while retaining 88% of LDH suggesting some contaminant proteins were removed. Due to some discrepancies in protein data for crude extract and 40% supernatant, we re-assayed these. Even though week 3 data may not be very reliable, the crude extract assay from week 2 and 40% supernatant assay from week 3 gave the most appropriate data with least error. The 40% supernatant was then subjected to 60%
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