Other proteins rich in histidine may also bind. Are you using imidazole only in the elution buffer. You may consider washing the column with 10-20 mM imidazole before elution. Alternatively, you may perform several step elutions at different imidazole concentrations to determine when does your protein of interest elute.

I suggest you to perform a washing step with wash buffer having 30 mM imidazole. In elution step you may not get a single band due to non-specific binding of proteins rich in histidine residues. Collect eluate in five or ten fractions. Usually non-specific proteins will come in first two or three fractions along with desired protein. In rest of the fractions you will get purified proteins. You can dialyse impure fractions and pass it again through the column.

Alternately, you can perform size exclusion chromatography of the partially purified protein for better results. Some of the undesired proteins can be removed by using Amicon cut off filters.

Good luck

I have used imidazole in my wash buffer also and collecting eluate in fractions also but still i am getting faint extra bands.

The column is to be regenerated first atleast 10-15CV(column volumes) and then equilibrate the column with the imidazole buffer for next 20CV. when you do elution the pH supposed to be very narrow and specific for the desired protein. The try to regulate the flow rate so as to get the fractions one by one. If you still face problems then pre purification steps like GFC or HIC are suggested.

Few thing I would like to add:

1.Use freshly prepared Imidazole and buffers.

2. Check the pH of your buffers (pH; 8.0) and don't store them for longer periods. There may be pH variations upon storage for longer periods.

Hello,

I am going to try and help you with my own experience and problem I have solved. It may be useful or useless for you because you probably know that purification is not an exact science and that your protein doesn't react like another.

After a His-tag purification, I have never observed a single band of my protein on a SDS-PAGE. Actually, other His-rich proteins and sometimes fragments of your protein bind on the column. I add another step of purification everytime (exclusion or ion exchange or both ...).

1/ Wash well

If you are using an Akta system, it is very simple to follow the different steps of the purification. If no, you'll have to follow your instinct!

In a first time, be sure that your column is well washed by passing 0.2N NaOH (some columns accept up to 1N NaOH) until the DO come back to 0. If you don't have Akta, then you can try with 5-6 CV. It will remove the protein stuck at your column and also other molecules that may interfere with the purification. Then rinse well the column with 10CV of water.

After that, equilibrate the column with 6CV (if you want to be really sure, you can pass 10CV, more than that is useless).

2/ Inject well

First of all you have to resuspend your sample in a buffer added with 20mM Imidazole (Is your protein soluble or not?).Be sure that your sample is clear, because membrane fragments can block the column. Do not hesitate to centrifugate twice or three times at 10000G during 15'. Your column will appreciate.

Once your column is ready you can pass your sample at 0.5 to 1mL/min.

The next washing step is very important and can be the critical point of your problem. You have to wash the column with your buffer added with 20mM imidazole until the DO reach 0. If you can't check it, then wash with at least 15CV and keep the flow through à 4°C.

3/ Separate well

Then come the real purification. You have to check the "behaviour" of your protein and of the contaminants during the elution. In a first time you can apply a linear gradient from 20mM to 300mM on 20CV. It is not perfect at all but it can give you an idea on how you could continue the purification. You can also try a step-by-step gradient: you begin with 20mM of imidazole and you rise the concentration by step of 30mM (for example) every 5CV. It is longer than the first one but it gives you more indications on how the different proteins bind to your column.

Once you know the concentration of imidazole that permits to purify your protein (or at least the less contaminants possible) it is all good.

4/ Don't cry and continue!

If you still have contaminants, then continue with an ion exchange column (anion or cation depending on the pI of your protein), a gel filtration or try a differential precipitaion with ammonium sulfate. A cut off membrane can also be useful with low weight contaminants.

I hope it will help you.

Good luck

I agree with the previous comments, you might need to add an additional purification step. If you try size exclusion chromatography it might be worth your time to run the column manually versus using an FPLC system (this is based on my personal experience). You might also want to consider adding a protease inhibitor to your buffers if the extra bands are of a lower mass than your protein of interest. Finally, you should consider trying a different cell line for expression. I've seen cases where individuals working with different proteins observe identical contaminating bands that were due to the cell line used for expression.

Hello,

I can offer a bit of advice as I found it to be quite frustrating trying to purify from the supernatant. I posted a similar question and after following this procedure (courtesy of Birendra Singh) it worked extremely well to purify my protein:

- Prepare a binding/wash buffer with 50mM tris, 500mM NaCl, and 50mM imidazole, pH 7.5

- Prepare elution buffer with 50mM tris, 500mM NaCl, and 250mM imidazole, pH 7.5

Dilute your supernatant 1:1 with the binding/wash buffer, then add your fresh (or recharged) Ni-NTA resin. Incubate this overnight with shaking at 4C. The next day, allow the resin to settle then decant the supernatant slowly. Keep this fraction as the "flow through". Transfer the settled resin into a fritted polypropylene column, and use a vacuum flask to settle the resin. Add your binding/wash buffer and allow the column to elute via gravity. Collect this fraction as the "wash fraction". (you may need to use a glass pipette or similar to stir the resin to remove air bubbles after packing). Finally, add your elution buffer to the column and collect the "eluted fraction". Of course, it will be wise to do all those over ice (or in a cold room if you have one). I've used this method several times with results similar to the gel shown below (from L-R: crude, flow through, wash, elution). It has worked well for me, even with as high as 10% FBS in the media.

Good luck!

I think Jason told you a perfect protocol. you can add 30-80 mM imidazole (depending on affinity of your protein to bind Ni-resin) in media during binding of His-tag protein. It can omit the low affinity Ni-resin interacting contaminants.

This question is long since closed, but there is a strain of E. coli called LOBSTR that's been optimized to reduce contaminations when prepping out his-tagged proteins.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4086167/