Dear CCP4 users, Thank you all of your advices which help me to solve this problem. I believe that all of your advices will help me and others having the same problem. This is the summary of experienced advices about how you will do if the protein having His-tag don't bind to NTA resin.
1. The protein is aggregated or misfolded in such as way as to hide the His tag, try adding things like glycerol or detergents to try to prevent aggregation 2. Use mono-q or mono-s as a first step. After that, running the NTA resin. This way is helpful when dealing with insect cells. 3. Dialyze the lysate to remove contaminants that prevent binding. This is very common for Bv expression, but somewhat unusual for EC 4. Partial unfold of protein by including 1-3 M Urea in the buffer A 5. Incubate the protein with the resin in an end-over-end nutator to increase the interaction time or run the flow-through over a fresh batch of resin and do this several times until getting enough protein 6. Change the tag from e.g the N-terminus to the C-terminus ? Or if you have a structural homolog you could add the His tag into a loop, which is exposed. 7. Introduce a linker between the protein and the his-tag or create a 8xhis or 10xhis tag to enhance binding to the nta matrix 8. Using the Strep-II instead of His-tag My best regards, TriNgo Sungkyunkwan University
