What is your buffer composition ?

This can be very helpfull. As David said presence of reducing or chelating agents, imidazole concentration and buffer pH (histidine protonation is a critical point).

Hi Parismita,

Many components in the buffer system (DTT, EDTA, pH of the buffer) can hinder the binding process. Go through the pdf below for more information.

https://tools.thermofisher.com/content/sfs/manuals/ninta_system_man.pdf

Regards

Sunil Singh

Hi:

After binding in the flow through step, the flow rate need to be very slow. I always use 1 drop/second to let the protein fully binding with beads. This can help at least gain 20% amount.

That's all my experience. 

Eddie

Eddie has a good pint. How long do you let the protein bind to the resin? I usualy incubate the protein/resin mixture for 30-60min on a tube rotator 4°C.

You can also lower the Immidazole concentration in your sample and wash buffer

Dear Parismita,

When I am using simple peristaltic pomp for my bench purification using NiNTA I usually do recirculation of flow through to increase chances of binding my protein. As Sunil said if you have any reducing agent, chelating agent or too high imidazole in your buffer binding of your protein will be reduced or even impossible. 

Is it possible that the His-tag was somehow cleaved from your protein during purification steps? Have you added some protease inhibitors? Have you used anti-his tag antibody or some other?

Good luck with purification,

Best,

Dawid

What is your buffer composition ?

This can be very helpfull. As David said presence of reducing or chelating agents, imidazole concentration and buffer pH (histidine protonation is a critical point).

Dear Xavier,

The composition of my buffer is 50mM phosphate (pH-8.0), 300mM NaCl, 1mMPMSF. I am not using imidazole in my buffer.

Dear Parismita,

Your buffer seems OK, even if I usually add 10-20mM Imidazole to have a more stringent buffer. It is also possible to increase NaCl concentration.

But your binding issue looks more like an issue of the poly-His (cleavage or accessibility). First, check carefully your sequencing, especially the in-frame of the C-ter poly-His (pET-23). The second possibility is to clone your poly-His in N-ter if possible.

Thank you.