His-tag protein purification - contamination with other... - ...His-rich proteins (Nov/04/2004 )
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.
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?
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.
I am having a similar problem with my recombinant protein. I was wondering what brand imidazole you all use
Thank you for any suggestions
I use from Sigma (cat # I-0250).