I'm using Ni-NTA resin (Qiagen) column to purify my his-tagged protein. Over a period of time the color of my column has changed to color less. I'm using the concentration you can see in the attached file. Please help me with the problem.
Have you run a sample of your Induced culture? I did not see that in the gel map. You should compare UnInduced vs Induced samples. Perhaps your protocol to express your protein failed.
Have you cleaned and recharged your column? There is a simple protocol to do that. I can share it with you if you want.
If the column loses its blue color, it is because you have stripped off the bound metal ions. This would happen if your sample or washing/elution buffer contains a chelating agent such as EDTA or citrate. The solution is to remove the chelator from the protocol.
In addition to above-mentioned comments, if your column turned colorless you could regenerate the column to re-charfe it. It is highly recommend to avoid reducing agents such as 2ME, etc. from the purification reagents. Moreover, for the efficient protein purification you should carefully determine the suitable pH for all buffers based on your protein pI.
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