Dear All,
A large amount of soluble protein of my interest could be harvested in E. coli. Yet, the protein seems to have rather low binding capacity with Ni-NTA column. The addition of 20 mM imidazole in washing buffer catalyze the elution of my protein together with other proteins. Both 50 mM Tri-Hcl and 50 mM NaH2PO4 have been tried for using as the buffering conditions. pH 7.4 and pH 8.0 have also been attempted, so did the concentration of NaCl. To avoid the possibility that the His-tag might have been masked, I tried to induce my protein into inclusion bodies and purified it in 8 M urea. The same problem appeared. Even under denatured conditions, 20 mM imidazole lead to the elution of my protein from the column. Then, I tried to modify my protein by adding two more His tags on its carboxyl end. This newly expressed protein was expressed as inclusion bodies, and it shows a higher binding capacity with Ni-NTA. 30 mM imidazole result in its elution from the column with other proteins. So my problem here is that Ni-NTA affinity chromatography seems not to be able to be used as a method for purifying my target protein. Can anyone give me some help on affinity purification of His-tagged protein?
Hi,
I had a similar problem and the following youtube video was a huge help and solved my problem. I think the amount of imidazole you are using is too low, esp the 20mM concentration. I would suggest doing a step-wise elution with increasing concentration of immidazole, this allows you to was away any contaminating proteins with the lower concentration of imidazole but get your protein of interest at the high concentration of imidazole. I think the youtube video below gives an excellent starting point on how to fix yoru problem. I hope this helps.
https://www.youtube.com/watch?v=y0ffJBFQswk
Hi,
I had a similar problem and the following youtube video was a huge help and solved my problem. I think the amount of imidazole you are using is too low, esp the 20mM concentration. I would suggest doing a step-wise elution with increasing concentration of immidazole, this allows you to was away any contaminating proteins with the lower concentration of imidazole but get your protein of interest at the high concentration of imidazole. I think the youtube video below gives an excellent starting point on how to fix yoru problem. I hope this helps.
https://www.youtube.com/watch?v=y0ffJBFQswk
Hi,
what sort of column are you using? Are you loding it yourself with Ni? If so have you tried other Ions? Also are you doing it by hand or with an automated system (FPLC)? If so, how fast and with what pressure are you loading and washing?
When is your protein eluting? Does it bind at all, meaning it stays on the column while loading or does it run right through? If so all of it or are you getting some product still in the elution?
Have you addressed tge possibility of His-Tag degradation? If your His-tag is being degraded during preparation of your cell lysate then you would see your protein of interest bind in the same way as non-tagged proteins.
If you run a western for your protein from the cell lysate (or bound fraction on beads) does this band also react to an anti-Histag?
If it does not, you can consider upping the concentration of protease inhibitors in your lysis buffer; this could fix the problem.
I use 10mM Imidazole in my lysis buffer, 20mM in my wash buffer to get rid of nonspecifically bound proteins and then elute with 250mM imidazole.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology