Hello,
I expressed a protein with a his tag (65 kDa) and purified it by Ni-NTA affinity chromatography. The amount of protein that I got was huge, so I didn't wonder about the aggregation in the fraction tubes overnight. The next day I spun them down and made an SDS PAGE. I repeated the SDS PAGE several times with different amounts of protein to make sure I was doing it the correct way. Everytime I get an additional band (and smear) right above my protein (the thickest band is my protein). I then diluted my protein 1:10 and purified it again by Ni-NTA affinity, but I got exactly the same result.
This puzzles me a lot, because my colleague expressed this protein many times and purified it the same way, and he never got this additional band. So now I am wondering, what could be the reason for the sudden appearance of the band?
Could it be somehow that the amount of protein is too much for the column? Was the expression too long (around 16h at 16°C)? I am not sure how to proceed now. Repeat the expression (but what should I change) or rather purify it further, e.g. with IEX?
Hi there,
What is the MW estimate of the upper band? Any MWM on gel?
Try Gel Filtration in order to define the actual behavior of the native protein prep.
Well, no matter what people say about purifying with IMAC, it is not completely selective towards the recombinant protein. There will always be native proteins also attaching to the nickel ions. That is what you are seeing, perhaps more evident given the huge concentration of your protein. IEX is regularly used to polish recombinant proteins after IMAC.
I agree with Omar and the ion exchange chromatography step; Ni-NTA has good selectivity for the recombinant protein of interest, but other proteins can always be dragged along with it. Given that some of the impurities are significantly larger than your protein of interest (and assuming your ~65 kDa protein is a monomer), size exclusion after Ni-NTA may be a good option to get rid of those higher bands; if resolution is good, it could even remove some of the immediate upper band.
Hi Mia
As others have mentioned you will not get pure protein with just IMAC. Your gel is not heavily loaded and I can see several contaminating bands. It also looks like it is not completely destained - some more weak bands may be hidden in the background stain. IEX should be the first option to test for your second step. But you should also try to optimise the IMAC step. Increase the wash stringency and/or imidazole concentration in the sample. And make sure you wash until UV is down to baseline. Overloading of the column is not likely to be the problem - generally the his-tag has higher affinity than other Ni-binding proteins so it would out-compete those when overloaded leading to purer protein after IMAC. You may also try Ni-IDA which sometimes can work better.
Unlike you I would worry about the protein aggregation overnight. With proteins that express in high amounts and elute in high concentrations we sometimes see aggregates that do not disappear completely even in reducing SDS-PAGE. i had one protein that would present as a ladder of multimers on the gel if I eluted it in too high concentration. Some proteins simply do not like staying for extended time in high imidazole concentrations so proceeding quickly to the next step can be required.
Mixed mode chromatography is another option as a second step. I have had good results with Capto adhere which can help remove aggregates. Don't be disencouraged by Cytiva marketing it as a resin for mAb purification - it is very useful for other proteins as well. But IEX usually gives better resolution so that should be tried first.
Hi Mia, as several other above say you will never get a 100% pure target protein using only a Ni-NTA resin as you have naturally cell proteins also binding to this resin.
Have you tried a cobolt charged resin instead? For example WorkBeads 40 Co-NTA also available in many different prepacked GoBio columns (from 1 mL). Co-charged resins generally gives you a more pure target protein, but the yield may go down a bit due to that the binding to the His-tag is usually weaker (therefore it binds less impurities).
Also as mentioned above you can also add another resin. We have used a high-salt tolerant IEX resin, WorkBeads 40 TREN, upstreams the Ni-NTA resin in a flow through mode and had a lot of impurities bound to this resin preventing them from running onto the Ni-NTA resin. In this way we also prolonged the lifetime of the Ni-NTA resin. The IEX resin we used is positive charged below pH 9. Let me know if you want me to send you more data.
Best regards, Anna
As Omar already said IMAC is not the way to the purest proteins there is. We often perform a SEC afterwards.
If you only want to do one purification method though I suggest you switch from Ni-NTA to Co-NTA. The protein yields may be lower to a degree but Co-NTA results in higher purity. Not to the same degree as a subsequent SEC would do, but it is definitely purer than Ni-NTA beads.
https://cube-biotech.com/products/protein-purification-products/his-tag-affinity-resins-magbeads/co-nta/
Maybe see this graph for comparison:
https://cube-biotech.com/media/42/2e/24/1656592620/His-tag%20Abbildung%204.jpg
- Badges
- Science method