I am purifying a recombinant protein (host Rosetta) with his tag. I am getting reasonably good yield (4-5 mg/liter of culture). However after affinity purification using Ni-NTA column, during concentration protein is precipitating. I am using Amicon 10 KDCO centricons for concentration and I could observe that even at 0.4mg/ml concentration protein is getting precipitated. Protein buffer is 50 mM tris pH 7.5, 500mM NaCl, 5% glycerol and 10mM beta ME and pI is ~6.0. Protein is eluting at 250mM Imidazole, is imidazole causing the precipitation?

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