Prolems detecting protein with antibodies

[Allison]

Trond Erik Vee Aune wrote:
> 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

In my experience with a His-tagged protein expressed in BL21 I could 
pick up the protein on a Western  with Qiagen's antibodies even if there 
was nothing obvious on a Commassie stained gel.  SO I would guess that 
you are not getting expression.  Maybe double check the sequence of your 
construct to see that everything is okay.

Also, if you can see the protein expressed from it's intrinsic promoter, 
you could try adding on a His-tag to that construct.  At least in order 
to test your detection conditions.
Allison