My his-tag protein has low expression level and it doesn't bind NiNTA column and elute in the flow through. Why?

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Omar Gonzalez-Ortega

How did you determine that the his-tag isn't hidden? If you denatured the protein with urea and binding occurred, then you are not using proper conditions for the native protein to bind.

Sadaf Sulman

I haven't denatured it. Its a soluble protein.

Alejandro Martin

Perhaps you misunderstood Omar's suggestion. You want to know the reason why your protein does not bind. Omar is saying that perhaps it does not bind because the affinity tag is hidden when the protein is properly folded, and is suggesting that you do a trial run under denaturing conditions to see if this is indeed the case.

Ohkay!! Thank you! I used I-tasser to find if my his-tag is masked or exposed. According to the structure its exposed but i'm not sure about the authenticity of the structure sent to me. I'll try under denaturing condition. Thanks alot!