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Why do His tagged protein not binding to Ni beads?

There are many factors that may affect the protein binding:

0. The His tag is incorporated into the tertiary structure of the protein and inaccessible to the Ni column. Try a batch under denaturing conditions (8M Urea or 6 M Guanidine) to check this. The protein should bind to the Ni column under denaturing conditions. If you get no binding under denaturing conditions it may be that the his tag is cleaved off sometime during sample preparation.

1. Check the pH binding buffer. At low pH histidine becomes protonated and is competed off of the nickel ion. Normally the pH should greater than 7.4.

2. Too high imidazole in the binding buffer may cause the problem. Imidazole and histidine have similar ring structure that compete with each other to bind with Nickel. 

3. EDTA in the buffer. EDTA generally react with metal and interfere with his tag binding with Ni-NTA beads. While lysing the cells don't add protease inhibitors having EDTA, which chelates metal ions.

4. Proteins with 12X His binds better to which binds much stronger than Ni.

5. Check if your protein has any metal ion within it. It could be that the his tag is interacting with these within the structure and not binding to the column.

Souce: NovoPro    2018-02-21