Hello I am doing his-tag protein. I got soluble protein but some the protein does not bind to the Ni-NTA column. I used Na-Phosphate pH8, 0.5M Nacl, 20mM Imidazole for my binding protein but the pH of my flow through was about 7.2. I used pET28 which can provide 5 amino acids linker bewteen my protein and histag and I saw histag is ok by Western blot. Would you know what could be a possible problem?

I would recommend going to Qiagen's website, look at the Qiaexpressionist manual under troubleshooting

they offer about 1/2 dozen things you can try and some possible reasons; even if the vector is different, the technology is the same

good luck