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The protein was then incubated with a buffer consisting of 20 mM HEPES, pH 7.4, 100 mM NaCl, 10% (v/v) glycerol, 5 mM MgCl 2 , 1 mM MnCl 2 , 25 M TCEP, 50 M tirzepatide, 20 g/mL Nb35, 0.03% (w/v) LMNG, 0.01% (w/v) GDN, 0.008% (w/v) CHS and 30 g/mL His-tagged TEV protease on the column overnight at 4C