ljd3 at duke.edu (Lou D'Amico) wrote:
>I'm trying to affinity purify an AB i generated in guinea pig to an
>insect enzyme (JH-esterase). It's approximately 60kD. When we had a
>company originally do the affinity purification, the yield was low
>(maybe 500 ug out of 20mls of serum). The serum that passed through
>the column was still chock full of reactive Ab (confirmed by Western
>Blot). I did another purification in house on an IgG purified extract
>of the serum, and still got low yields in the purification (maybe
>300ug). I don't have tons of the antigen to work with, and I was
>curious if someone could help me with two questions.
>1) Does the size of the antigen impact yield this severly? I was
>coupling 2-3mg on a ml of column matrix.
>2) Are there standard alternative techniques to purifying Ab's of
>large mol weight proteins that might be more appropriate?
3 mg/ml bound protein is typically optimal for affinity
chromatography. What was the volume of column used in
affinity purifucation?
DK
