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Douglass, J., Weiss, K

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Ni-Penta 6FF resins were added to 20 volumes of E.coli lysate (~0.5 mg/mL containing a His-tagged protein of 26 kDa in size), in the presence of various dilutions of reducing agents, including TCEP, DTT, and -ME respectively. Protein binding was then performed at room temperature for 2 hours. After washing 5 times with 20mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole, pH7.4, the target His-tagged protein was eluted with 20mM sodium phosphate, 0.5 M NaCl, 250 mM imidazole, pH7.4. Protein concentration was measured by UV absorbance at OD280