Prolems detecting protein with antibodies

Trond Erik Vee Aune trondaun at
Sat Jun 3 02:59:20 EST 2006

Dr Engelbert Buxbaum wrote:

> Trond Erik Vee Aune wrote:
>>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 

> 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

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

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