Dear Nagarjuna,

In addition to the above recommendations, you may try to add a high salt wash, in case that the impurities happen to bind to your protein. For example, adding a 4.5 M NaCl wash can be useful to get rid of nucleic acid contamination. Some people also add ATP to get rid of bound GroE chaperone. My experience is that with NiNTA, at pH 8 and 30 mM imidazole, contamination by endogenous E. coli proteins is minimal.

Most proteins need to go through more than one orthogonal chromatography step to be purified. While tags help, they are no all-cure. I also have found that quite a few proteins, especially from bacterial sources, are able to bind to IMAC columns without having a His-tag.

If you are reluctant to start another kind of chromatography, you could first try to optimize the Ni:NTA column. Go higher in the imidazole concentration or even add a low concentration to your loading buffer & sample.

If this does not help, try ion exchange chromatography as second step; anion exchange usually binds close to all proteins. Yuo can then try to separate your protein from impurities using a salt gradient.

Dear Nagarjuma

probably you can try to use an intermediate washing buffer as 30 or 50mM of imidazole ( do not through away the wash fraction but load it into sta page to see if your protein come out at this concentration)

as Achim told you is difficult obtain a very pure protein with just one IMAC step and other yiu need to perform at least a second purification step as SEC for example that wilk allow in parallel to improve protein purity and exchange the buffer by removing imidazole from the elution.

Is your clone contain a protease cleavage site (eg TEV or fate.xa) beetween the His tag and the protein? Because in this case you can try to digest the protein and perform a 2nd IMAC step which will remove the tag but also improve protein purity.

you can find more information about this approach on my blog: https://proteocool.blogspot.com/?m=1

ProteoCool n°13- Subctractive IMAC availble at PAGE 3.

good luck

Manuele

Dear Nagarjuna,

In addition to the above recommendations, you may try to add a high salt wash, in case that the impurities happen to bind to your protein. For example, adding a 4.5 M NaCl wash can be useful to get rid of nucleic acid contamination. Some people also add ATP to get rid of bound GroE chaperone. My experience is that with NiNTA, at pH 8 and 30 mM imidazole, contamination by endogenous E. coli proteins is minimal.

Nagarjuna Yegavinti , NEB sells a strain called NiCo(DE3) where the host proteins most frequently binding to IMAC resins have been modified through the addition of a chitin-binding domain. Hence, you can express your target protein there, do the Ni-NTA run and then clean the prep by running it through a chitin column (conveniently sold by NEB as well :-). Perhaps you could try that.

Hope it helps!

Hi,

Did you link your gene with only His tag? or if you have used pet51b (+) plasmid to express your protein, you can have strep tag at 5' end and His tag at 3' end. In my case His tag showed same problem because many proteins from bacteria can also bind to the Ni-NTA beads. and during elution they keep coming with your desired protein. if the expression level is low for your desired protein (like in my case the protein expression was low at every tested IPTG level), situation gets more worse., getting desired protein become more tough. so I changed the column (Strep tag specific), used streptiviridin beads. so its better to keep active Strep and His tag both in your plasmid.

Hi, Nagarjuna.

Two other possibilities:

1 - If you're not using protease inhibitor cocktails, you may be getting degraded parts of your own protein of interest;

2 - What is your objective? Because, sometimes, if your protein of interest is more than 80% pure, inoculation for antibody production may be performed without further purification and gives good results.

3 - Even without an AKTA System or any other chromatographic systems, you may perform a step-wise analysis with imidazole in different amounts (50, 100, 250 and 500mM) and check how they perform and even using NaCl in a specific good concentration as 100mM or other you may find in the specific bibliography. Ok?

If you're new to the area, take a look at some companies book (guides) such as QIAExpressionist. You may find it here:

https://www.qiagen.com/dk/resources/resourcedetail?id=79ca2f7d-42fe-4d62-8676-4cfa948c9435&lang=en

Hope it helps.

Hi Nagarjuna,

I don’t know specific information about your target protein, so I can only give you some basic advice.

1. As mentioned above, because the Ni-NTA resin may bind some proteins non-specifically, it is normal to have many protein bands only after affinity chromatography. You need to purify the target protein through further operations, such as gel filtration or ion exchange;

2. The probability of protein degradation of the His tag construct is relatively high, especially for some proteins with unstable properties, and it is recommended to optimize the conditions, e.g. try more constructs and mammalian cell expression systems…;

3. If these bands are endogenous and of high concentration, it is recommended that you optimize the induction conditions or try other expression systems.

Hope it helps!

thank you very much every one for your time and suggestions, they helped me.

Nice to hear that. But, If possible, Nagarjuna Yegavinti tell us what has happened. It may help others in the same situation.

All the best.