Good rabbits or bad rabbits

[Michael Mason]

To who ever,

I am a PhD student at the University of Queensland, and my project involves 
raising antibodies against a particular protein. 

I started off injecting two rabbits with Freunds adjuvant mixed 
with 100ug of un-dialysed protein which contained approximately 1M Urea. After 
4 sets of injections (conducted every 2 weeks) the blood serum was not 
detecting any proteins on a Western Blot at 1/100 dilution. After this I 
decided to dialyse the protein against PBS, and repeat the immunization 
protocol again, using 200ug of protein on the same two rabbits. Other than the 
controls, and the purified antigen, no bands were seen on the Western. I 
continued to inject every two weeks for another 6 weeks. After this I started 
to get some faint bands on a Western using 1/100 diluted serum.

I do know however that the protein I am trying to detect is on the western 
membrane. So it can not be a simple fact of it not coming out in the 
extraction process.

Could it be that I have bad rabbits?, a little to old maybe? or did the Urea 
in the protein solution destroy antibody production? or could it be the 
protein itself? 

I would greatly appreciate it if anyone can come up with some sugguestions, as 
I am now starting to inject two new rabbits with dialysed protein.


Thanks in advance
Mike Mason
M.Mason at Botany.uq.edu.au