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Relative to other affinity interactions, the binding between GST and glutathione is pretty slow, so youll want to incubate your protein of interest with the beads for around 30 minutes to an hour before uncapping the column and letting the flow-through drip out the bottom

Figure 2. Retained His-tagged protein binding capacity in presence of EDTA. 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 2 mM, 5 mM, 10 mM EDTA 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

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