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Production of Crude Extract

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Crude Extract
In order to produce crude extract, bovine tissue was obtained, precisely minced to exclude extra fat, and then blended with pH 7.2 phosphate buffer. The purpose of blending the tissue with the buffer was to pulverize the cells, causing them to release their contents evenly—most importantly lactate dehydrogenase (LDH)—into the solution. Since other cell components such as proteases which reduce LDH were also released from the lysed cells, the slurry was kept on ice to minimize their kinetic activity. The solution was then centrifuged, creating a pellet made of cellular debris and leaving behind the crude product in the supernatant. Using data from the enzyme assay, the crude extract revealed 155±7 mg total protein, 4980±80 units of total activity, 32±2 units/mg of specific activity, a 100% yield, and a purification factor of 1 fold (Table 1).

40% Supernatant
The purpose of the 40% ammonium sulfate fractionation step is to extract impurities with low solubility—such as lipids—from the solution. Because ammonium sulfate salt is extremely soluble, it hydrates competitively against weakly soluble impurities causing them to precipitate out of the solution as a pellet. Hence, LDH is left behind in the 40% supernatant. This step revealed a slight drop in total protein amount from 155±7 to 124±5 mg and comparatively no change in total activity from 4980±80 to 4900±100 units (Table 1). This data agrees with the salting out theory which states that since only weakly
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