Most imidazole has an appreciable A280nm, are you sure you are seeing protein off the column at 5M imidazole or just an increase in A280nm?  The protein started out soluble when it flowed onto the column, and it was not in the flow through (FT) correct? Are you following a band on a gel? Imidazole > 0.5M are almost never needed to elute His tagged proteins from Ni2+ chelate columns, as their binding Kd is only in the micromolar range.

my protein has GFP and I see my protein in column. 

No, I do not see my protein in FT or in washing step.

If your protein is not eluting using 500 mM imadazole, there is something wrong. simply, your protein is not bound on the column. Increase in imadazole concentration do have a significant absorption at 280 nm. Do a quick bradford of all the elution, which you think has your protein, to make sure you got your protein of interest.

Hi -

I have a few questions-

1. As Edward mentioned, what are you using to detect protein?  Are you sure it isn't something else?  Are you getting no protein with 500 mM or just some?

2. Is your Imidazole pH adjusted?  If not, you might be changing the pH with such high concentrations.

3. Are you overloading your column?  Can you elute it with more 500 mM Imidazole washes or not?

4. You could try a Cobalt column or a Ni IMAC with a different support (your HisTrap column uses sepharose).

5. Edward mentioned solubility - you could spin your lysate down in an ultra before loading it into your column to try to get rid of any odd stuff.

Hi Amanda

1. I have GFP fused protein and I see it , if some proteins come out. No, I do not get any with 500 mM.

2.with 500 mM, it is adjusted. but, i do not adjust the pH after adding more imidazol to the elution buffer,

3.I do not think so. I did not try with more 500 mM.

4. it is Ni IMAC.

 http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences-de/17524701

5. I do that.

1. That is very unusual!  Do you have another His tagged protein that you can use that is known to elute normally?  Then you could make sure the 500 mM buffer is working fine.

2. You can pH your imidazole stock so that it is less likely to alter your elution buffer pH.  Check the pH of your 5 M imidazole buffer to see if it could affect your protein or the column.

3. Can you do a smooth gradient instead of step?  Maybe that could help?

4. Maybe Cobalt would work better (binds less tightly)?  I saw yours was a Ni IMAC, but I thought you could try for a different support (agarose or chitin maybe?  do they make that?) to make sure your protein wasn't binding the support instead of the nickel.

5. Well no help there I guess!

I would try Amanda's suggestion to exchange Ni to Co, I witnessed it works well (one of my colleagues did).

An easier way try is change the pH to around 5.8 or so. That is well below the pKa of Histidine (thus it will be protonated and not be able to bind). Like Amanda has suggested, adjust your imidazole stock to around 7.0 by HCl then there will be no change in pH of your buffer. Imidazole is very basic, the pH of its solution is about 10-11. If you add a lot (500 mM, even for 100 mM), the pH of your buffer will substantially change (your buffer is about what, 25-100 mM?). The farther away the pH above the Histidine pKa, the stronger the protein will be bound. So, increasing imidazole concentration concomitant with pH increase is not effective.

Last but not least, ensure your protein doesn't make additional interaction with the resin; normally salt concentration above 150 mM is recommended to minimize/diminish but try higher if it doesn't work.

Hope these helps, good luck.

If your protein is pH resistant (and considering its isoelectric point), you might consider to elute your protein with lower pH: wash at pH 6 and elute at pH 5.0.

Alternatively, you can strip of the Ni and the protein with EDTA to recover your protein, at least if the chromatography is a true IMAC and not aspecific binding to the NTA resin.

Hi,

I have this same problem with supercharged proteins that I work with. I find that my super positively charged protein binds so tightly to the column (probably ionic interactions with the matrix rather than with the nickel) that standard edition procedures do not work. I got around this problem by including up to 2M NaCl in the Lydia buffer and all subsequent wash and elution buffers. My proteins act normally under these conditions.

Good luck!

By edition I mean elution, and by Lydia I mean lysis! iPhone autocorrect not great with science terminology!!!!

Hi, I agree with James Alexander Joseph Arpino. This is not normal situation and usually His6-tagged protein must eluate by 0.2 – 0.3 M imidazole concentrations. In this case hydrophobic or electrostatic interactions are interfering the most likely. I don’t know nothing about your buffer compositions, but I would try to elute by 50 mM EDTA and 500 mM NaCl after chromatography anyway and analyze all fractions by means of SDS-PAGE. Hydrophobic interactions may delete by isopropanol solution (50%) and electrostatic interactions may delete by high salt concentration, but high salt may increase chelating interactions strong. I guess that pH decreasing will be helpful anyway.

Hi there,

1.It could be your protein is not at all expressed or its in insoluble form which hinder the His-tag accessibility to Nickel-NTA bead.

2. It could also be the case that the protein expression is hindered by codon bias, promoter mutation, stop codon in the gene by random mutation, low expression.,etc.

3.You can also look for any other components in your buffer that could chemically hinder the His-tag protein binding to Ni-NTA if your protein indeed expressed. 

4.I would suggest you to do a mini induction in 5 ml or 10 ml culture and subject to SDS-PAGE, comassie staining and also silver staining (low expression) to check indeed protein is expressed are not.

5. For low expression you can scale up the volume of culture or play with IPTG concentration for ideal induction and also try induction at low temperature.

I hope this was useful to you to address you problem.

Rajkumar