15 replies to this topic

[Bednaar]

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.

[gmcg]

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.

Edited by gmcg, 04 November 2004 - 04:39 AM.

[Bednaar]

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

[Janina]

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.

[Bednaar]

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).

[Bednaar]

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

[merien]

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  

[merien]

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  

[Bednaar]

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.

[ffaucher]

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

[Bednaar]

In pRSET vector I have his-tag on N-term, I tried Western Blot but with anti his-tag antibody (Abs for my protein are hard to get commercially, only Calbiochem had them but now they do not sell them because of so few customers interested) - it was positive, I had good signal, the Ab did not react with other his-rich proteins, I had one band, also the MW of positive band was good, so I don't think I have these problems. If I had a degradation, the bands would be of lower MW than my protein - due to all of my SDS-Page checkings they are of higher MW. Now I'm going to purify another version of my recombinant protein - in pTrcHis2-TOPO vector it has his-tag on c-term, but this is due to biological activity of my protein - the N-term is highly active part of my molecule, and I'm going to incubate a cell line with his-tagged protein due to my future research plans.

[merien]

What would happen if the column doesn't has adequate capacity or the loaded sample is too much? will it cause the expected protein (the recombinant proteins) can't be eluted?

[heigor]

If your columm has not enough capacity for all your protein, you will losse protein, but the purification conditions would be the same. May be you need increase the ellution time and buffer vol., but nothing else

I have another question about his-tag protein. Do you know if is possible to purifie proteins witha His-tag in the middle of the protein? Any experiences?

I think that the principal problem is that the His-groups are not accesible to the column, and purificacion would be very poor.

bye

[Leannett]

Hi all,
I am having a similar problem with my recombinant protein.  I was wondering what brand imidazole you all use
Thank you for any suggestions

[Bednaar]

I use from Sigma (cat # I-0250).