What determines the qualitity of an antibody?

Frederik Börnke ricky_boernke at gmx.net
Fri Feb 25 03:11:32 EST 2000


Hi all,

yesterday we had this discussion about antibodies in the lab. We 
routinely produce antibodies against various proteins from recombinant
material produced in E. coli. However, the quality of the  obtained
ab's differs dramaticly. Abs generated against plant proteins often
don't work at all although they recognize the recombinant protein
quiete nicely. On the other hand when the protein comes from other
organisms, like bacteria, the serum quality is surprisingly good, 
even in detecting low amounts of recombinant protein in transgenic
plants. We were discussing the reasons for that, i.e. plant proteins
don't express very well in E. coli (at least in our experience) most
of the proteins are extremely unsoluble. Hence, purification under
denaturing conditions is the method of choice (pQE-system). Now when
the protein is injected into the rabbit it might form larger complexes
which creates artificial epitopes that are not present under, for
instance,
western blot conditions. Proteins from bacteria usually express quiete
good in E. coli and large amounts can be recovered from the soluble
fraction so that the native conformation/epitopes is (are) retained.
Does that mean that the initial solubility of the recombinant protein
determines the quality of the ab? Another thoerie was that bacterial
proteins are much more antigenic than plant ones, possible?
If the solubility thing is true this would mean that under western blot
conditions proteins are more or less in there native conformation, which
must be true at least to some extend otherwise one could not do 
overlays or phosphorylation assays and stuff. Does anybody know how the 
proteins look like after transfer on a membrane?
Any thoughts on that?

Cheers
Ricky

*******************************************************************

Frederik Boernke
Research Group of  Molecular Plant Physiology
Institute for Plant Genetics and Crop Plant Research (IPK)
Corrensstr. 3
06466 Gatersleben
Tel.  039482 -5 321
Fax. 039482 -5 515
http://www.ipk-gatersleben.de




More information about the Methods mailing list