I would put 1 ml of Ni-NTA resin in a small glass column (e.g., 1 x 10-cm Bio-Rad Econo-column), or use a 1-mL prepacked Ni-NTA column (Cytiva), and flow the 15 ml of medium through it. I would wash the column with a buffer and elute with ~2- or 3-ml steps of the same buffer containing increasing concentrations of imidazole in 50 mM increments (pH adjusted to neutral). Then I would check each fraction for the protein and combine the best fractions. I would then concentrate the sample using a 10-kDa cutoff centrifugal ultrafiltration device.

Do not assume that this single-step chromatography will result in pure protein. It may take additional types of chromatography to get the protein pure enough. It would help to do the expression in serum-free medium so that you don't have to contend with a large amount of albumin in the medium.

Dear Apo Tax Tax

give a llook to the following post

https://proteocool.blogspot.com/2021/05/tips-of-week7small-scale-imac.html

where you can find some hints about purification with gravity flow coloumns using Nickel resin that i think can adapt well to your purpose.

best

Manuele

I only used Ni-NTA. Tried to use Co-NTA, but it tends to behave differently.

A few ideas on your Ni-NTA:

At its core, every IMAC resin does the same. Some resin ligands are more stable and thus more resistant to EDTA and DTT, which you shouldn't put into your protein buffer anyways if you paln to perform an IMAC. Seen DTT-resistant resins turn to Cola although DTT concentration in the buffer was below manufacturer limits, probably due to a prior NaOH washing step that got the chemistry a bit riled up.

As it comes to purity, as these resins essentially do the same, they will yield the same. Different metal ions may behave differently.

No matter from which organism you purify your His-tagged protein, use 10mM Imidazole in the binding buffer. This will not impede 6xHis protein binding and vastly reduce nonspecific protein binding. This eventually enhances the intended capacity of your resin, which also will result in higher elution purity.

Your average 6xHis protein will start to elute at some 30mM Imidazole (meaning low level leakage of your protein). Highly quantitative elution will happen at 120-200mM Imidazole, don't be stingy at this point, you can always dialyze your way out of it. HEK-cells tend to secrete a couple of proteins of which you can mostly get rid of at 30mM Imidazole, though not all of them. In case they may interfere with your further studies, you might consider this washing step.

Another factor to consider is the amount of IMAC resin you're using. It will preferentially bind your high-affinity His-tagged protein, but also a bunch of other histidine-rich proteins in your extract. If there is only a tiny amount of your 6xHis protein, the resin will tend to bind all of the affine host cell proteins. Thus, an excess of resin will result in impure eluted protein. In case of doubt, you're better off with consecutive purifications with a smaller resin volume, applying the flowthrough to the resin another time after elution and regeneration. This way you can reject the least pure fractions.

Hello and many many thanks to all your answers!

So from what I read, I should try to elute my x6his tag antithrombin in 50mM increments of imidazole.

Manuele Martinelli , I read that you used 20mM for 6xHis-tagged protein, is this sufficient for you? I may start with that and then I will try higher concentrations. Also you dilute your surnatant diluted 1:1 with the IMAC binding buffer, do you think I could flow my supernatant undilutet in the column as Adam B Shapiro mentioned?

Adam B Shapiro and Manuele Martinelli many thanks for your reagents recommendations. I found that HisPur™ Ni-NTA Resin from Thermo is half price compared to those from Cytiva, do you have any experience with those?

Andre Marschall (sorry for the bolded) you mention DTT and EDTA. However, I haven't seen those being used in purification protocol (I seen only the IMAC Binding Buffer that doesn't contain DTT or EDTA https://cshprotocols.cshlp.org/content/2020/6/pdb.rec105429.full?rss=1 )

Since my aim is to target antithrombin, I guess that I shouldn't use any Protease Inhibitor Cocktail, right?

Many thanks again!

/Apostolos

Dear Apo Tax Tax

it depends from the origin of the sample:

Im my experience, 20 mM of washing (with at least 20CV) is enough to purify proteins from surnantant of mammalian cells (e.g Expi CHo or Expi293) using Ni-NTA resin.

In case you purifiy proteins from E.coli citoplasm (which contain much more impurities) a wash at higher imidazole concentration could be usefull (e.g 60mM) but in some cases it can lead also to the elution of some fraction of your target protein.

i prefer dilute the supernatant 1:1 with binding buffer then load the undiluted supernantat for 3 mains reasons:

- Regulation of the pH at the optimal suitable for IMAC (about 8)

- Dilution of possibile compounds present in the surnantats that can induce the stripping of the metal from the coloumns

- Addition of some imidazole that can reduce the aspefic binding of the contaminants on the coloumn,.

i'm agree that this will strech a bit you experimental timelines but in my experience improve the experiment reproducibility.

I never used the HisPur™ Ni-NTA Resin but thermo is a good provider and i suppose it can be good. In case you would like to use it by gravity in empty PD-10 or smaller coloumns just check if it classified ad fast flow resin of not, since with gravity, since you cannot apply pressure, to have good timelines you need resins that do not tend to clog,

good luck

Manuele

Hi,

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