Mr Somchai Sangamnadech
(ssan at mail.nerc-oxford.ac.uk) asked about free Con A leaking from Con A-Sepha-
rose.
As we know that mature Con A is a non-glycosylated protein consisting 4 homo-
logous subunits, and not all of them were directly linked to Sepharose.
Therefore, leakage of Con A subunits from the tetramer is a common problem,
especially when one uses binding buffers with pH value below 5.6. It is
advised to use washing buffers with near neutral pH.
For elution fractions missing the expected bands, I suspect whether your bindingbuffer contain the necessary divalent metal ions, e.g., Ca(2+) and/or Mg(2+),
which is involved in binding between glycoprotein and Con A (once the protein isbound, even 5 mM EDTA can't compete the bound metal).
Con A-Sepharose (produced for Sigma by Pharmacia) works fine in my case of init-ial purification of a recombinant glycoprotein from CHO cell secretion.
Please feel free to ask any additional questions should you encounter more pro-
blems.
*******************************************************************************
* Ke-Wei Zhao, Ph.D. Phone: (310)825-8722
* Department of Biological Chemistry Fax: (310)206-5272
* UCLA School of Medicine, CHS 33-257 e-mail: kzhao at biovx1.biology.ucla.edu
* Los Angeles, CA 90095-1737
********************************************************************************
