I have DNA binding protein and I am getting a conc, of 50um from 50ml BL21 DE3 culture (I will be scaling this up to 1mM). The protein is Hexa-His tagged at C-terminus and to elute the protein from Ni-NTA column I am using 1M imidazole as even at lower concentrations [even at 500mM] protein does not elute out at lower concentrations of imidazole.

I am doing dialysis in two steps. 5ml of protein elute (1M imidiazole, 400mM NaCl, 20mM Tris, pH 8) against 1L of 500mM Imdiazole, 20mM phosphate, 400mM NaCl, pH 7.5, followed by second step of dialysis against 1L buffer containing 20mM phosphate, 400mM NaCl, 50mM EDTA, and 2mM DTT, pH 7.5. [I am adding EDTA and DTT because I read the imdidazole might leach out Ni2+ and removal of imidazole results in precipitation]

While concentrating the protein in Millipore centricon tubes protein precipitates out. I cannot increase salt beyond 500mM NaCl as this will cause problems with NMR experiments. Any suggestions on how I can modify my final step (or the rest of the protocol) to prevent protein precipitation will be greatly appreciated.