Some proteins are mote dificult to purify, and the common adivices are: increase the imidazole concentration in the binding buffer and in the washing step or adding 5% glycerol in the buffers.

To optimice the imidazole concentration you can do a step-gradient with differents concentrations each one of at least 3cv, colect and do a western to see in wich concentration start eluting your protein, then select one step lower for the washing.

Hi Jahaziel Gasperin Bulbarela , thanks for your answer! It's true, each protein might be a different world.. What really strikes me is that all the contaminants elute in a single peak, together with the protein of interest. And also, that the same thing happens with both proteins.. I forgot to mention, the buffers also contain 10% glycerol (I had solubility issues too, and I read that would help). I'll try adding imidazole to the binding buffer! Hope you're doing well :)

If you have aggregation problems i would recommend to reduce your sample volume, i also have used 1% tween-20 in the input, but your real problem is the aggregation, because your protein becomes sticky and traps other proteins, you need to check the isoelectric point of your proteins to verify that you are working in the correct pH (its higher than pI).

Dear Julieta Beiro

Which lysis/binding buffer are you using?

Are you add some imidazole in the binding buffer?

How much bucterial pellet did you lisate for the 5ml purification?

Because to improve the protein purity in IMAC is important that you are close to the coloumn saturation. IF you are using a too big coloumn, you increase the probability of aspecific binding.

best regards

Manuele

Hi Manuele Martinelli ! I use 30 ml of binding buffer per 1000 ml of culture. Yesterday I pelleted 1.5 L of culture (final DO= 1,3), resuspended it in 45 ml of binding buffer (20 mM sodium phosphate, 500 mM NaCl, 10% glycerol) and sonicated it. After the lysis, I did a total protein quantification using Bradford, having 102 mg total in the 45 ml of binding buffer. According to the manual, the capacity of the column is 40 mg (histidine)6-tagged protein/ml. Should I decrease the amount of resin? I'll also try adding imidazole to the binding buffer!

Thank you!

Hmm, interesting Jahaziel Gasperin Bulbarela ! Do you add the Tween-20 to both binding and elution buffers? If my protein is trapping the other ones, it would explain why they're eluting all together in a single peak.. So, should I work above the PI?

No, i only use tween-20 in binding/lysis buffer. I also use at least 20 mM imidazole, but i recommended to do the gradient step elution, and if your proteins have cys, use 1 mM of bME to reduce unspecific disulfide bonds.

For imac you need to work above the pI, because below pH 7 the histag looses affinity to the Nickel.

Dear Julieta Beiro

i suggest to you to add at least 10mM of imidazole in the binding buffer.

You can try to reduce the volume of the resin since the binding capacity refer to the target protein to be purified and not the total protein.

looking to your chromatoghram, i think that you can also ttry to improve your washing or:

- insert a step where you washing the colomn (at least 10 coloum volume) with the binding buffer before to start the linear gradient

- replace the linear gradient with a 3 step gradient.

1) wash with 10mM imidazole buffer --> 20CV

2) wash with 30mM imidazole buffer --> 10CV

3) Elute with 300mM imidazole buffer

generally i'm using Tris 20mM, Nacl 300mM, imidazole xmM pH=8 buffers, but if the pH is correct i do not thin kthat phospate and highr Nacl concentration will make a great difference

good luck

Manuele

Hello :)

I know I am kinda-late but maybe I can still be of help here. Note that I did NOT read through the previous answers.

His-tag purifications as a concept are pretty impure. We usually follow up with a Size-Exclusion-Chromatography.

But the purification itself could also be further optimized in three ways IMO:

1. Increase the number of washing steps.

2. Increase imidazole concentration in the washing buffer. THe Elution buffer is NOT of importance here.

3. Switch from Ni-NTA to Co-NTA. Usually a bit less yield but purer on its own.

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

Hello, Julieta Beiro

I suggest you to add at least 20 mM of imidazole in the binding buffer.

You can try to reduce the volume of the resin since the binding capacity refer to the target protein to be purified and not the total protein.

looking to your chromatoghram, i think that you can also ttry to improve your washing step

Equlibrate the coloumn with 20CVof binding buffer or coloumn equlibration buffer.

Try Gradient washing with incresing Immidazole concentration and increase the volume of washing buffer.

3) Elute with 300mM imidazole buffer

Try different concentrations of Nacl in buffer.

good luck