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The elution was concentrated using an Amicon Ultra Centrifugal Filter (MWCO 100 kDa) and subjected to size-exclusion chromatography (SEC) on a Superdex 200 Increase 10/300 column that was pre-equilibrated with 20 mM HEPES pH 7.4, 100 mM NaCl, 2 mM MgCl 2 , 1 M peptide, 0.01% (w/v) LMNG and 0.0006% (w/v) CHS

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This pattern allows any potential receptor adaptation to normalize while preventing dependence on the peptide for sleep