In article <2q8u89$skp at news.iastate.edu> yqhuang at iastate.edu () writes:
>From: yqhuang at iastate.edu ()
>Subject: immunoaffinity purification
>Date: 4 May 1994 19:49:29 GMT
>Dear netters,
>I have been trying to purify a cell surface glycoprotein by immunoaffinty
>chromatography.I tried protein G-sepharose for both purification and
>covalent coupling of the monoconal antibody.That worked just fine.I
>solubilized the membrane proteins in detergent,without first purifying the
>membrane.The problems were:1)elution:I tried high pH,low pH,high salt(4 M
>MgCl2),NaSCN,urea,and even guanidine.HCl,no one satisfied me for recovery.
>2)purity.I used proteinG-sepharose precolumn,but still could not get rid of
>the contaminants.Lentil lectin column did not help either.
>Thank you for your advice in advance.
>Yueqiao Huang
>3178 Molecular Biology Bldg
>Iowa State University
>Ames,IA 50011
>fax:515-294-0345
>email:yqhaung at iastate.edu>--
> Recovery in Affinity Chromatography:
I don't understend if the low recovery is in the coupling or in the elution?
Probably You should be able to answer to this question.
For my experience if the cupling is poor you still have many things that can
be tried, Pierce or Biorad sell many different reagents and/or kits for this
porpose. There is no rule for deciding what is the best coupling method.
For the elution may be necessary 8M urea. Have you tried?
The best precolumn for getting resd of aspecific binding is the same column
coupled with an other antibody ( one no very expensive) of the same isotype.
I hope the above suggestion will be helpfull. Good Luck.
Salvatore Sechi
Laboratory of Molecular Allergy and Immunology
NIH, NIAID
email: ssechi.helix.nih.gov
