RE ELUTION OF ANTIGENS FOR WESTERNS

mdjlsc at cc.newcastle.edu.au mdjlsc at cc.newcastle.edu.au
Wed Dec 20 05:22:48 EST 1995


This is always a problem using ip's and then westerns. Acid elution with
glycine will also strip the antibody off the protein-A as well as the antigen.
The only real solution is to use covalently coupled beads. If you have plenty
of the antibody then directly couple it to CnBr activated bead or equivalent.
If your source of antibody is commercial and therefore expensive, you can
prepare covalently bound Sepharose-Protein-A-Antibody beads in small quantities
using a small crosslinking molecule such as DMP (Pierce). The western then
becomes much cleaner. Antigens can be eluted by a variety of means including
acid elution as you have suggested (Other methods 3.5M MgCl2 or pH 5.5 Acetate
buffer for example).

Hope this helps,

RICK THORNE
CANCER RESEARCH UNIT
THE UNIVERSITY OF NEWCASTLE
AUSTRALIA



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