somewhat counter intuitively the weaker binding cobalt versions of the affinity resin can provide better purification. The Ni columns are very high affinity and as such absorb many protein species which need to be slowly washed away with extensive washing. There are also some E. coli strains which have the endogenous proteins most likely to compete with the tag knocked out. These could help. A precut with something as simple as ammonium sulfate precipitation can also sometimes knock out most of the competing impurities. Good luck!

Assuming this is Ni-NTA, if your tagged protein is abundant and binds well you could try decreasing the volume of resin you're using; large resin bed volumes may not be completely saturated by your tagged protein, giving other contaminants the opportunity to bind. You can also introduce a low concentration of imidazole into the cell lysate (~5 mM or so) to prevent contaminants from initially binding, although this can also decrease your protein yield. At elution, you can also apply a gradient of imidazole; contaminants will typically elute at lower imidazole concentrations while your tagged protein should elute at a higher concentration. This can also be done step-wise if a gradient elution can't be performed (e.g., wash with 20 mM imidazole, then 50 mM, then 100 mM, etc). You can collect these washes and monitor your tagged protein's elution via SDS-PAGE.

"30mM binding buffer" does not make sense at all

Hello :)

I assume you mean 30 mM imidazole in your Binding buffer right? Because otherwise, I am just as confused as Omar.

I can suggestion you three things here. His-tag purifications on their own just ALWAYS leave impurities behind. It is on the high-yield-low-purity side of protein purification techniques.

First suggestion: If able, perform an SEC after the His-tag purification. This is what we do here often.

Second: As Aaron Rightfully said already, use Co-NTA instead of Ni-NTA. It may have lower protein yield, but the results are way purer than Ni-NTA could hope to get on its own:

https://cube-biotech.com/products/protein-purification-products/his-tag-affinity-resins-magbeads/co-nta/

Helpful graphic:

https://cube-biotech.com/media/42/2e/24/1656592620/His-tag%20Abbildung%204.jpg

Third suggestion:

Do more washing steps with higher imidazole concentration in the washing buffer.

HI,

Some suggestions from me:

- Optimize binding& wash buffers by increasing imidazole concentration.

- Use screening kit to find the optimal metal ion(Ni2+, Co2+, Cu2+, Zn2+).

- Optimize the length of the His-tag. Longer tag will bind with higher imidazole concentration and you remove your contaminants that are not bonding.

- Optimize the elution step. Change from step to linear gradient.

- Increase the volume of the linear gradient.

- Change to a pH gradient (can be tricky).

- Use high quality imidazole (should be a white powder).

- Use NTA ligand (and not ISA ligand) for immobilization of the metal ion.

- Add a second purification step, e.g. SEC or IEX. This will help a lot for increased purity!

I have added some information of different prepacked 1 mL and 5 ml columns with different metal ions attached for easy screening for optimizing.