Prolems detecting protein with antibodies

[Trond Erik Vee Aune]

Dr Engelbert Buxbaum wrote:

> 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. 
> 
> 
> The protein may be insoluble, forming inclusion bodies. Incubate the
> lysate with high concentrations of urea (6--8 M) to solubilise those and
> then do a western (or even a simple spot blot).

This is a good idea and there are some reasons so suspect that my 
protein will aggregate if expressed at high levels. I will certainly do 
this after I've confirmed that the gene is expressed with a biological 
activity assay.

> If the His-tag gets removed by proteases, an anti-poly-His antibody may
> not work. Are your anti-epitope antibodies sequence- or conformation
> specific? Conformation-specific antibodies may not work after protein
> denaturation during SDS-PAGE. The natural source of the expressed
> protein should be usable as positive control for antibody function. 

My anti-His is the Tetra-His antibody from Qiagen. It's supposed to work 
under both native and denaturated conditions.

I use a his-protein ladder as a positive control of the antibody, and I 
get a clear signal from this ladder.

> If you still get no signal, you'd have to go one step backward and
> verify that the bacteria actually have been transformed and make the RNA
> with Southern and Northern blots, respectively. But usually the problem
> is inclusion body formation. 

My guess now is that something is wrong with the construct. If not, I 
will certainly test the insouble and the soluble fraction more 
thouroughly, and solubilizing the insoluble fraction with urea is a good 
advice.

> BTW: Inclusion body formation can sometimes be prevented by expressing
> the protein with tags that act as a kind of chaperone to keep the target
> protein soluble, like maltose binding protein (MBP). The His-tag may be
> used in addition to make purification easier.

We've considered using other tags to enhance the solubility, but 
postponed it for now.

Thanks for your suggestions.

Trond Erik

-- 
Trond Erik Vee Aune
Department of Biotechnology, NTNU
http://www.biotech.ntnu.no/molgen

- Must be sad being a dyslectic, agnostic insomniac, lying
   awake during the night, wondering if there really is a dog