I'm trying to express recombinant protein from mammalian cell 293F by its secretion in to the supernatant and then using the Ni-beads to enrich the His-tag protein. However, when I use 300mM Imidazole to elute the protein from Ni-NTA, Most of the protein are still on the beads.
and I also try to increase the concentration of Imidazole to 500mM, but it didn't work.
so I wander if someone can give me some suggestion or Do I need to change my construct like using other tags？
Hi there,
500mM imidazole is a lot so my guess is that your protein sticks to the resin (it might be because precipitation/agregation). To be sure of that you should try elution with EDTA. As EDTA chelates Nickel it promotes the elution of whatever protein interacting specifically with it. If your protein is still on the column then it is precipitation/agregation.
I agree with Dominique: your protein probably precipitates or sticks non-specifically to the resin. You may try various conditions to elute it: high salt, high/low pH, increasing concentrations of chaotrope (urea), increasing concentrations of ethylene glycol
What kind of matrix are you using for your column? Perhaps, something more hydrophilic would work better? You could also try to bring the pH of your buffer a little further away from the protein's pI, to increase the charge. Half a pH unit should do.
Dominique Liger Thank you, I will try to use EDTA to elute. Engelbert Buxbaum thank you.My binding buffer is 150mM NaCl /20mM Tris /20mM imidazole/PH=8.3, and my protein predicted isoelectric point is 9.53. Pierre Béguin thank you. I will try some combinations like to use 500mM NaCl and 5%ethylene glycol and PH=7.0/7.5 and 20 mM HEPES/TRIS/sodium phosphate TO see if that could do.
And I wander if the supernatant can directly load onto the HiTrap Q HP column instead of Ni-affinity ? because the supernatant(without serum) is pretty pure.
or if I could try to use Ammonium sulfate to get the protein diffused in the supernatant ?
That is always an option: purify a genetically modified protein with conventional methods, without using the tag. Just because a protein contains a His-tag, you don't have to purify it by IMAG. I had that situation once with an MBP-tagged protein, which wouldn't bind to a maltose column.
Especially if your feed is pretty pure already, you can use any capture method that works, including IEC or AS-precipitation. Note, however, that after AS-precipitation IEC is a poor choice for further purification, because of the high salt content. AS after purification by IEC makes sense, because many proteins are very stable when crystallised with AS. Just add the salt slowly until the solution becomes just slightly hazy, then allow crystallisation in the fridge o/n.
The first buffer should have a pH 8.0 or above, but the pH for elution buffer should be lower than 7 (It is better if you use pH 6.0-6.5).
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