His-tag protein purification - contamination with other... - ...His-rich proteins (Nov/04/2004 )

Welcome everyone. I'm having a problem with purification of a his-tagged recombinant protein. After affinity chormatography step I have good protein recovery, but contamination with other his-rich proteins. I tried ultracentrigugation (100 000 g, 1 hour) of a lysate before purification and extensive washing of a column (after washing protein absorbance at 280 nm was 0.000), but it didn't help. I wonder whether the additional wash step with a wash buffer of a lower pH (about 6, now it is 7,9) could help, or maybe the gradient of imidazole concentration during elution, or the gel filtration (my protein is smaller than all the contaminating proteins in my sample). Thanks for any suggestions.

Are you including imidazole in your wash buffer? If not, do an experiment to determine how high a concentration of imadazole you can include in the wash buffer before your protein elutes from the column. You should be able to remove the majority of his-rich proteins by this method.

I would also use gel filtration as the final step in your purification method. You may need to concentrate your sample afterwards though, since the fractions are usually quite dilute.

In my wash buffer I have 22,5 mM imidazole, in elution buffer it is 500 mM. I'll try with raising the concentration of imidazole in wash buffer as you suggested. Thanks for your help

You could also put some Imidazole in your Binding Buffer so that these other His-proteins may not bind that much... I used a concentration of Imidazole of 5 mM in Binding Buffer, 60 mM in Wash Buffer and 1 M in Elute Buffer.

I also have 5 mM imidazole in binding buffer. I'll try with 60 mM imidazole in wash buffer (instead of 22,5 mM in my buffer).

Finally I decided to make an linear gradient of imidazole from 22.5 mM to 500 mM. Hope this will work.

Hello, I'm new here...and affinity chromatography is also new for me. What is exactly the function of the imidazole here? What I know is that imidazole is an organic base and a histamine inhibitor. What caused this substance able to elute the his-tagged protein from the column? Thanks

Hello, I'm new here...and affinity chromatography is also new for me. What is exactly the function of the imidazole here? What I know is that imidazole is an organic base and a histamine inhibitor. What caused this substance able to elute the his-tagged protein from the column? Thanks

Imidazole has higher affifnity for metal ions - nickel - than the His motifs in his-tagged recombinant proteins - that's why this compound is able to elute these proteins.

Hi,

Are your His tag is on C-ter or N-ter of your protein. If your His-tag is on N-ter, I think your protein is troncated or the transcription was partially completed or you have a degradation problem. Did you have an antibody for your protein? (Not a antibody for 6xHis). Try a western blot on your eluate. If you get a lot of band.

In all case it is better to have His-tag at the end if your protein.

ffaucher