Hello everyone, I am trying to purify a 13 KDa membrane protein using Ni NTA. The protein is solubilised in Triton X 100, 20 mM phosphate buffer, 150 mM NaCl and binds very well to the column. However, it also turns the column brownish. The protein contains 4 cysteine residues so I suspect that this causes cross-linking with other proteins and thus brownish precipitation on the column. So I included 5 mM beta-ME in my buffer to prevent disulfide bond formation but this doesn't help. I tried 1 mM DTT and this ruined the column. Help!! Is there anyway to prevent this brownish problem?
Thanks a lot in advance Kien
