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After loading, matrix washed with same urea buffer and started eluting the impurities with elution buffer containing 20mM Imidazole followed by 250mM Imidazole to give elute having pure His6-AV- Ubiquitin-semapeptide

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You hit weeks 3-4 of your microdose schedule, feel absolutely nothing, and conclude the dose is too low to be doing anything useful