How can I purify His-tagged proteins that do not bind to Ni-NTA columns?
(Thread 21037) Q2 I am purifying several eukaryotic proteins that are expressed in E. coli using Ni-NTA columns. Some of the proteins do not bind the Ni-NTA column even when they are denatured. I am using Tris buffer, pH 7.5 for binding. I also tried MOPS buffer, pH 6.5 for overnight binding, but there was no improvement. Does anyone have any suggestions for how I can purify these problematic proteins? A1 Are you sure that they still have the His-tag or could it have been cleaved? A2 Did you confirm that there is actually expression of your protein of interest? You can do this with mini-scale induction. Just spin down 50 µL of induced cells and control cells, boil them in 20 µL of 1× sample buffer, and check for a band showing the induced protein. If the band you see is small, it probably is not real and you will not see a band after the Ni-NTA spin column purification. A3 What size column are you using? Is your protein indeed overexpressed? If it is poorly expressed, you may be underl
