I recommend you to make sure that what you have cloned into the vector is right. I would sequence both ends of your portein of interest (including HIS-Tag and promoter) in order to find that out.

The thing is the His-tag results in the highest amount of purified protein, but usually not the cleanest purification results.

In protein purification this is a pay-off situation: Protein Yield vs Protein specificity/purity.

If you want to stay with the His-tag and not change your construct. Maybe do with Co-NTA instead of Ni-NTA. The yield may be lower, but the cobalt Ion is more selective when it comes to the his-tag. An overview can be found here, together with some Co-NTA alternatives to Ni-NTA.

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

If you want to stay with Ni-NTA however, you may not come around doing a SEC after the primary purification.

I was going to say that's degraded part of your protein, but if it's in the untransformed control as well... than it's nonsense.

I never got any good results with IMAC. How about using some other tag where you can use more stringent conditions? Moreover, His-tag may decrease the production of your protein, see

https://pubmed.ncbi.nlm.nih.gov/23250226/

If you want to stick with the His-tag, you may need to change the metal as suggested by Sebastian Lühn or you will need another purification step. SEC may not be sufficient though if those peptides bind and you may need to use e.g. ion exchanger.

All of this need optimization based on your will and options.

Dear KAjol

Just a doubt

How did you perform cell lysis?

Because this band resemble to me a lot the lysozyme that in some cases is added or present in some buffers used for cell lysis.

MAnuele

Hi Kajol B M Singh

I would recommend to use a different protocol for purifying his-tagged proteins, based on SDS-PAGE gel (elution fractions), your Beads lane still has a lot of non-specific proteins. I have had a great experience in purifying His-tagged proteins using Ni-NTA Agarose and different concentration of Imidazole followed by buffer exchange using Amicon centrifugal filters with a suitable cutoff.

You don't have worry about the 12-KDa non-specific as far as it doesn't get eluted with your tagged one, I agree with Tomáš Hluska that since it is detected in the negative control then its non-specific. Otherwise it could be a smaller version of your protein due to codon usage in bacteria or translation problem with your gene, for that I would check the gene sequence if its compatible with the strain that you are using or if your gene has any localization tag (mitochondrial motif or etc).

side note: I could not understand your labeling system, if you could provide us with more information, we could try to be more helpful.

Best of luck

Rabab

Dear Kajol,

As you already have seen in the given answers above you will never get a 100% pure His-tag protein using just one Ni-charged resin and also as seen in earlier answers Co charged resin will in most cases give you a his-tag protein with higher purity, but with a bit lower yield. Other possibilites for optimizing the purification can be to use Zn and/or Cu charged resins. There are very useful kits available with prepacked 1 ml and 5 ml columns packed with precharged resins with Ni, Co, Zn and Cu that will help you to find the metal that gives the highest purity of you specific His-tagged protein. After finding the best metal to use you can further optimized the imidazole concentration and perhaps add a second purification step like SEC or IEX. Check out www.bio-works.com for the BabyBio His-tag Selection kits.

Best regards,

Anna

Manuele Martinelli Thank you for mentioning about lysozyme. I avoided lysozyme this time and the non specific band issue is resolved.

But I did not understand why lysozyme band was coming with my construct and not in other people's protein expression? Also the band was binding to His antibody in western.

Anna Heijbel Rabab G. El Mergawy Tomáš Hluska Sebastian Lühn Sebastián Villar Rodriguez Thank you for the suggestions and help. Now I am able to get my protein of interest without that non specific band.

Great. What strategy did you use in the end?

Sebastian Lühn This time I did not use lysozyme in the lysis buffer and directly proceeded with sonication. So that non specific band did not appear this time. Lysozyme has a MW of 14.3kD.

I also use pet28 plasmid and have a better result with Co-NTA. Also, try to induce with lower temp 16-18... and bind the proteins in the presence of the elution factor (i.e. in my case I elute with 150mM of imidazole, I bind in the presence of 5 mM of imidazole, and make more wash steps with 10 up to 15mM of imidazole)

Hi,

Just a quick purification tips for His-tagged proteins. Ni ions (Ni-NTA resins) are so easily used by default when purifying His-tagged proteins, because it is what you see your colleagues are using and it is what you see in the references you read. Please, remember that you have several other options that can make your His-tagged more pure and also options that is better for the environment. Make sure that you have a BabyBio NTA His-tag Screening Kit (1 ml or 5 ml) in your lab and you can fast and easily find out the best metal ion to use. Using this kit you screen four different resins precharged with Ni, Co, Zn and Cu ions that are prepacked in 1 ml or 5 ml BabyBio columns. Read more on www.bio-works.com or contact me for more hints and tips, [email protected]

another option is to use pET22 which has a pelB N-terminal signal or can incorporate into whatever plasmid you like. The periplasm has a number of benefits over cytosol, yet your protein of interest will dictate which path is optimal. We use periplasmic osmotic shock for routine purification and have significantly less non-specific contamination than lysis-based purification from the cytosol.

As others have mentioned, it is quite rare to have purity using IMAC alone and you will need to use a chromatographic method in conjunction (e.g. SEC or IEX) to polish your material.