I can spot two instances of "two bands" in your gel. Not sure which one you're referring to:

First, you might be referring to the relatively low-abundant high molecular weight contaminant. This looks fairly normal, and would usually be cleaned up with a second chemistry, like SEC.

Or you might be referring to the splitting up of your protein of interest into two bands that run very close to each other, and blend together at high loading volume. This is most likely due to loss of a few terminal residues during production. This can happen to certain proteins in E. coli and there's no easy solution to it. It might not be a problem for your application, as the shortened protein would likely still be functional. It does not look possible to separate them, as the full length and shortened protein would be extremely similar. You might try to prevent this from happening, by sticking the his-tag, or another purification tag, to the affected end. Then the tag would be lost along with the few terminal residues, and you might just end up with a homogeneous full length fraction.

I can spot two instances of "two bands" in your gel. Not sure which one you're referring to:

First, you might be referring to the relatively low-abundant high molecular weight contaminant. This looks fairly normal, and would usually be cleaned up with a second chemistry, like SEC.

Or you might be referring to the splitting up of your protein of interest into two bands that run very close to each other, and blend together at high loading volume. This is most likely due to loss of a few terminal residues during production. This can happen to certain proteins in E. coli and there's no easy solution to it. It might not be a problem for your application, as the shortened protein would likely still be functional. It does not look possible to separate them, as the full length and shortened protein would be extremely similar. You might try to prevent this from happening, by sticking the his-tag, or another purification tag, to the affected end. Then the tag would be lost along with the few terminal residues, and you might just end up with a homogeneous full length fraction.

Hi,

I think you just have to increase the incubation time approx 30min of your protein of interest in wash buffer after passing of supernatant through Ni-NTA slurry. Such types of problems occur due to improper washing lead to the presence of non-specific proteins having similar molecular weight as the protein of interest.

Juan Carlos Sanchez-Bermudez Hi, You may check the concentration of protein before and after purifying the sample before loading the gel. Because of the concentration of the crude sample and the purified ones are not the same. How much concentration you are using for this gel and how much percentage this gel was?

If you are referring to the doublet band in the lower right of the gel, this could be the result of post-translational modification or proteolytic cleavage.

Hi Juan,

It looks like a contamination, this things happen since is not 100% efficient.

I would suggest you two ways of doing it:

1- use columns (like Amicin, Centricom, etc) that you can separate the proteins by size. Then, you can concentrate your sample and get rid of contaminations that present distant molecular weights

2- Alternatively, you can try to use gel filtration or ion exchange chromatography. It can help to clean the sample.

Ni-NTA purification for His-tagged proteins tends to higher protein yields but at the cost of protein purity. Maybe use Co-NTA next time. It lower in protein yield, but much purer.

Where is your tag located? N-terminus or C-terminus?

How are you so sure that is your protein? Do you have any inmunoblots using anti-His or anti-YourProtein?

I would do a Western blot and check if the bands you are seeing in the elution are isoforms of your protein or your protein of interest and a contaminant.

Have you added protease inhibitors?

How many start codons do you have after the promoter? I prefer to remove the ATG of my gene of interest in order to avoid isoforms that has two different N-terminus.

Check the protocol of E.coli growth and induction. Have you tried lower the temperature after inducing?

This could be a result of proteolytic cleavage, you may revise your protocol to avoid protein degradation.

This could be the result of post-translational modification or proteolytic cleavage. Revise your procedure or track your protocol to avoid this. Also check the concentrations of both samples before loading.

Hi Juan,

1. The band with high molecular weight should be a contamination band. There are many non-specific proteins in Ni-NTA chromatography, which is relatively normal. I usually remove the contamination band by gel filtration or ion exchange.

2. The two bands with similar molecular weight appear to be degraded or modified. According to my experience, the probability of protein degradation in the N-His construct is relatively high, and the two-terminal-His tags can effectively prevent protein degradation. Maybe you could try some other tags.

Good luck!

As often in this forum there is very little information which makes it difficult to help you. As pointed out before it is not even clear which extra band you are referring to. There are several E. coli proteins that have affinity for Ni-columns due to histidines in close proximity on the surface of the protein (I am assuming that you are expressing in E. coli - you didn't say). Some tips to increase the purity in the IMAC step are:

1 - Increasing imidazole concentration in sample and wash. This needs to be tested for each protein

2 - Increasing volume of wash. Make sure you are washing down to the baseline. The top band (or a band of similar size) seems to be present in the wash fractions also

3 - Increasing protein load. His-tag generally has higher affinity and will out-compete impurities

4 - Eluting with a gradient

In general IMAC alone will not give pure protein. Add an extra step of IEX or SEC.

Juan Carlos Sanchez-Bermudez

This is simply a band density problem. Because the band density you get is different in the run, even if you have taken the same concentration of proteins.

I can see two band in pre-purification lot at that mol. Wt.