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Cell supernatants (up to 200 g of glycoprotein in a volume of 35 l) were denatured with 10 l of 250 mM phosphate buffer and 2.5 l of 2% SDS with 1 M 2-mercaptoethanol, heated at 100 C for 5 minutes and cooled

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Intracellularly, copper is tightly bound by proteins like metallothioneins or glutathione (GSH), preventing the presence of free ions, which are cytotoxic