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Afterward, a mixture of methanol-saturated petroleum ether and petroleum ether-saturated methanol (1:1, by volume) was sequentially added to the total fat, followed by sequential addition of petroleum ether-saturated methanol and methanol-saturated petroleum ether mixture (6:1, by volume) after shaking and homogenization

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29 (2 Suppl): S238S244

Even if youre purifying the same protein over and over, it is probably still a good idea to clean and regenerate your column about every five purifications, or earlier if you notice that the binding capacity of your beads is getting worse