You have got success in the purification of one protein from the same vector, so the possibility it getting cleaved is very low. To rule out try purification in denaturing buffer once like with 6 M Guanidium HCl or 8 M urea (at 4 degrees it may phase out, so avoid cold temperature) to see if that is the case indeed. If that works, an on-column refolding protocol can be tried, where denaturant is brought to zero, then eluted normally.

I also agree that your His-Tag is rather not cleaved, since you were able to detect the protein of the correct size in the supernatant. To be absolutely sure, run the WB using anti-6His antibodies (I'd say this is a must experiment).

What I suspect is that those two proteins don't bind the NiNTA because their His-Tags are buried due to the protein fold. As Mangal Singh Panwar suggests you can try purifying under the denaturing conditions. However, renaturation is usually very tricky, takes loads of time to optimize, and the 100% native form is not guaranteed. Instead, I would advise to simply put the His-Tag on the other end of the protein, or modify the linker. But first, do the anti-His WB. His-Tag is small, so if it gets cleaved, the change of mass of protein is almost undetectable on gel.

Best of luck!

Dear Sara

The size in SDS-page is not informative, because it not change a lot if you have degradation at N- or C-term. As Anton suggested you, you can run a WB using anti His antibody to understand if the tag is removed or just not well exposed (sometimes it may happen in protein those tend to form High molecular weight aggregates; eg contain hydrofobic regions? it is rich of cysteines? )

However protease cleavage during culture may happen expecially if the N- or C-term domain of the protein where you add the His tag are not structured and exposed to protease cleavage.

Which expression conditions are you using? in terms of temperature and induction time?

Because if you are performing induction at high temperature, eg 37°C, before work on the clone you can just try to decrease induction temperature (eg 25°C or 17°C) and time to see if the degradation issue decrease.

On the contrary, if you alredy using low induction temperature and it is not able to solve, at least partially your problem you can re-desing the clone and:

- Move the His tag at the C-term

- Use a shorter linker beetween TEV and his TAG

- Re-desing the N-term domain of the protein (eg remove some aa) to remove the unstable region, if it present.

which is the best of those 3 possibilities, it depends from the specific protein sequence that you cloned into the vector.

ciao

Manuele

have you considered the structure of your protein may cause an N-term HisTag to be unavailable for binding? C-term HisTag is an option (unfortunately more cloning and no way to remove it easily).

Hi Sara,

You have got a lot of good suggestions above!

I also want to say that maybe you have to run the Ni-charged resin in denaturing conditions to open up the structure of your proteins and get the His-tag available, but this is not easy and also to do the refolding of the proteins. The yield and actitivity of your protein are often very low.

Another option is to not use the His-tag for purifications of these two proteins, but instead use ion exchange chromatography. Depending on the Ip on your protein you go for either an anion exchanger e.g. WorkBeads 40Q or a cation exchanger, WorkBeads 40S. These two resins are also convenient available in small 1 ml and 5 ml prepacked columns so it is very easy to test. You can check this web page for more info www.bio-works.com or contact me directly if you need more detailed help, [email protected]

Best regards, Anna

I agree with all previous answers,

You succeded to purify one so maybe His-tag cleavage not highly suggested

If possible you can His-detect kit to can evaluate that His-tag presents or not.

Moreover their some loss in the amount of the protein using AKTA system so you can modify the method and condition.

Hi,

As commented above, I also think that the protein folding affects the His-tag bound to the nickel column. I suggest that you place the His-tag at the C-terminus of the protein or change other tags.

Good luck!

Hello,

It seems to be folded into the protein core and couldn't be detected either by NiNTA or antibody,

I am afraid if you used denaturing condition that you may lose some protein during the refolding process (as usually happens with the insoluble proteins), especially if higher protein amount required for crystallography or something.

So, you may extend a (GGS)n linker between the His-tag and the cleavage site to let the tag to be exposed for detection.

Good luck!

Hi Sara,

I do think you have the correct answer in one of the above. Like others say you previously, I think your protein still with the 6xHis tail. You could try whatever, just remember what,s your porpouse (if you need high activity, if you dont need activity, if you want for diagnostic or totally folded), due to there are many things you could do like you could see.Purification under denaturing conditions could be a quickly way to check if there is a 6xHis tail. good luck

Sara, could you elaborate on your purification procedure: Are you using the same column for all purifications? How do you elute? Do you re-charge the column? How do the yields develop over purification run?

Plenty of good answers and troubleshooting. When you check things make sure u start from checking induced protein on gel. Since you isolated on other vectors I would not doubt your protein purification protocol. Few considerations. I would check the pi of the protein and the buffers you try isolating. Your proteins become more soluble when ur buffers have a different pi as the protein. Normally on SDS gels while using lameli buffer u always get positive bands and western works. But when u use native conditions in buffers with closer pI you will lose the protein in membrane fractions.

Sara Heidarpanah

I suggest you to use 2-3 detection methods . It is highly likely that errors are producing when you do western blot.