Prolems detecting protein with antibodies

[Trond Erik Vee Aune]

Hi,

I'm trying to detect a protein, but get no result. The gene is from 
Pseudomonas but has been cloned in pET16b (with N-term. His-tag) and 
expressed in E.coli BL21(DE3). I've used both antibodies against the 
His-tag (Penta anti-his from Qiagen) as well as antibiodies designed 
specifically against different epitopes in my protein. Unfortunately I 
have no idea if the antibody against my protein works, since I have no 
positive control. Neither antibody gives any positive result for my 
protein. I induced the cells (10 ml) with 1 mM IPTG and let it grow at 
20 degrees for 4 hours or overnight. I've looked for the protein in both 
the medium, soluble cytoplasmic and insoluble cytoplasmic fractions. The 
proteins contains no signal peptide. The culture grows well after 
induction and shows no sign of lysis.

I know the protein is expressed in E.coli from it's intrinsic promoter, 
allthough with low frequency. I suspected at first that there may be 
some form of negative feedback to keep the protein concentration down, 
but I still don't get any result from the pET promoter, which removes 
any negative feedback regulation of expression.

I'll try to concentrate the protein extract and do it again, as well as 
testing BL21CodonPlus(DE3)RIPL, but I fear that there will be no 
positive result now neither. Should I try scaling up the process to 
maybe 100 ml and concentrating? Would you recommend purification (using 
the his-tag) before western/dot blot? What about stability, is there any 
way of minimizing the chances that the protein is degraded after lysis?

Trond Erik