NiNTA-purification can be a bit tricky. Generally, you can have the following contaminants in your protein:

1. Proteins having a few histidines here and there. To reduce unspecific binding of these proteins to the resin, your buffer should contain imidazole (I typically use 40 mM) and you should not use too much resin. If your resin is saturated with your protein of interest, the contaminants will not be able to compete for a place to bind and vice versa if the binding capacity of your column is largely oversized, your protein of interest will likely be dirty. I have completely switched to using Strep-Tags (WSHPQFEK) instead of His-Tags to circumvent this problem.

2. Proteins directly or indirectly interacting with your protein of interest. You can break or at least weaken these interactions by using high salt concentrations in the buffer (I typically use 500 mM, refer to your manual how far you can go - I think 1M NaCl should still work). If necessary, you might also add a little detergent (e.g. 0.1% Triton X-100). If this is insufficient, repurify using a different method.

3.  If your His-Tag is at the N-Terminus of the Protein, you will inevitably copurify all aborted variants of your protein of interest. These typically produce a ladder of bands of lower molecular weight than your protein of interest. I would generally recommend to use C-terminal affinity tags wherever possible, the purity will be much better because the plethora of aborted proteins will not have an affinity tag (this is my experience from >100 proteins). If you require an N-terminal His-Tag, then you will need to repurify your protein with an orthogonal method, whereby ion exchange chromatography is the method of choice in most cases. If you do that, mind that your elution buffer from the NiNTA-purification has a lot of salt and may cause premature elution from the IEX-column, thus either dilute your sample in the appropriate low-salt buffer, use a desalting column Prior to IEX or Dialyse. Dilution is the easist thing(I always do that because I don't have to care about the final volume), the salt concentration should be reduced to 50 mM or below.

Good luck!

Best regards,

John

First of all, mention your purification condition and lysis and wash buffer composition along with pI of your protein. then only, we can suggest you the best way to reduce non-specific binding.

still, How much salt conc. in your protein buffer is being used during purification. you try with 250mM salt conc along with 30mM Imidazole in wash buffer and give 4-5 times long washes.

I hope you will get better results in that condition.

NiNTA-purification can be a bit tricky. Generally, you can have the following contaminants in your protein:

1. Proteins having a few histidines here and there. To reduce unspecific binding of these proteins to the resin, your buffer should contain imidazole (I typically use 40 mM) and you should not use too much resin. If your resin is saturated with your protein of interest, the contaminants will not be able to compete for a place to bind and vice versa if the binding capacity of your column is largely oversized, your protein of interest will likely be dirty. I have completely switched to using Strep-Tags (WSHPQFEK) instead of His-Tags to circumvent this problem.

2. Proteins directly or indirectly interacting with your protein of interest. You can break or at least weaken these interactions by using high salt concentrations in the buffer (I typically use 500 mM, refer to your manual how far you can go - I think 1M NaCl should still work). If necessary, you might also add a little detergent (e.g. 0.1% Triton X-100). If this is insufficient, repurify using a different method.

3.  If your His-Tag is at the N-Terminus of the Protein, you will inevitably copurify all aborted variants of your protein of interest. These typically produce a ladder of bands of lower molecular weight than your protein of interest. I would generally recommend to use C-terminal affinity tags wherever possible, the purity will be much better because the plethora of aborted proteins will not have an affinity tag (this is my experience from >100 proteins). If you require an N-terminal His-Tag, then you will need to repurify your protein with an orthogonal method, whereby ion exchange chromatography is the method of choice in most cases. If you do that, mind that your elution buffer from the NiNTA-purification has a lot of salt and may cause premature elution from the IEX-column, thus either dilute your sample in the appropriate low-salt buffer, use a desalting column Prior to IEX or Dialyse. Dilution is the easist thing(I always do that because I don't have to care about the final volume), the salt concentration should be reduced to 50 mM or below.

Good luck!

Best regards,

John

If the difference between your bands is high enough, a gel permeation/size exclusion chromatography (not just desalting) could be an option as well. I also agree with the other answers that salt might also be an issue, because imac groups such as nta tend to act as ion exchange materials.

If you know the pI of your favorite protein, I would suggest an ion exchange chromatography step and then SEC to polish and desalt your purified sample.

Run a gradient of imidazole instead of stepwise protocol.  We use 20 mM to 150 mM imidazole.  Also conventional dialysis or an Amicon filter can be used to remove proteins that are smaller than your protein being purified.

http://www.emdmillipore.com/US/en/product/Amicon-Ultra-15-Centrifugal-Filter-Units,MM_NF-C7715

In either case, be sure to pick a molecular weight cut off that is well below the size of your protein of interest.

Use increasing concentrations of Imidazole to elute your protein. Run the elutes on a gel to see where maximum of your protein is coming out . 

I usually work at high salt concentration (1 M NaCl) for NiNTA and further clean by spin dialysis/concentration (vivaspin- watch out for MWCO) and Size Exclusion Chromatography.

Try to optimize the washing time

Perform an Ion Exchange..

It could be due to the washes. I work  with proteins that are either tagged with 6 or 10 His. For the 6-His proteins, I generally wash the ni-nta beads (5 bead volume, twice) with 20 mM imidazole. It could go up to 30 or 40 mM if necessary. As for the 10-His, I use either 65 or 80 mm imidazole. You can adjust the washes accordingly but if all those fail, you can perform a size exclusion chromatography to get rid of the contaminating bands.

it seems that your have encountered the problem of non-specific binding along with your protein of interest, i would suggest few things

1. if your are keeping Ni-nta beeds for binding with cell lysate. then try reducing the time you give for binging, say if you are keeping o/n for binding then try few hours.

this will reduce the non-specific binding not completely but effectively

2. here you need to optimize your elution, you can reduce unwanted protein by giving wash with higher imidazole concentration at which your protein does not elutes.

say your protein elutes at 250mM imidazole then give wash of 150 to 200 mM wash and while doing so check that your protein is not washed off, if it happens reduce the concentration of imidazole in wash

To remove non-specific binding, I would first run a small scale purification. Add some imidazole in your calibration buffer-20 mM- and use the same concentration of imidazole in your protein sample. Then collect samples at increasing concentrations of imidazole and determine where your protein of interest elutes. When you have determined the right concentration of imidazole, go on large scale purification and use a lower imidazole concentration (which you determine from your small scale experiment) to remove the unwanted junk and then elute your protein, that would get rid of most of unwanted proteins.

You can even try to use a pre-purification step, e.g. ammonium sulfate precipitation. This is helpful in most cases.

It might as well be that you are purifying not only your protein, but degraded versions. In such cases, it is helpful to include protease inhibitors in the buffer when you are extracting the protein from the cells. Don't use inhibitor cocktails containing EDTA!

Goodluck.