i used a 3mg/ml (little less) solution, i had about a (very) little
over 1ml of that, and i bound it to 1ml of column matrix. It's the
same volume as the company i outsourced the first affinity
purification to. Any help?
Thanks!
-Lou
On Thu, 17 Oct 2002 03:08:00 GMT, dk at no.email.thankstospam.net (D.K.)
wrote:
>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
>
