Ab purification problem

Lou D'Amico ljd3 at duke.edu
Thu Oct 17 13:03:24 EST 2002

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?



On Thu, 17 Oct 2002 03:08:00 GMT, dk at no.email.thankstospam.net (D.K.)

>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? 

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