western blot question

[Dr Engelbert Buxbaum]

Am 11.07.2007, 10:05 Uhr, schrieb Rebecca Pickin <RPickin from cvm.msstate.edu>:

> Hello all!
>
> I am trying to get an immunoprecipitation protocol up and running and am
> in need of some help.  I have plenty of IP protocols, but I'm running my
> results on a gel before I proceed with the next step.  Eventually, we
> would like to be able to skip the gel.

A Western blot without running a gel?

> But as of now, there is nothing
> detectable on the gel, so we dont' want to proceed until we determine  
> what is wrong.

How much protein do you expect to precipitate? Most often IP is run with  
metabolically labelled cells (35-S Cys/Met), the label is detected by  
autoradiography. Even silver-staining may not be sensitive enough to  
detect that small amount of protein.

> Here's what is up so far:  I am running duplicate samples on both native
> and SDS-PAGE gels.  When we silver stain the native gel, there are smears
> and bands visible.  However, the only bands visible on the SDS-PAGE
> western blot (detected with my antibody) are the markers.

IP is always a balance between being stringent enough to avoid background  
and not being so stringent that you destroy the antibody/antigen complex.  
Therefore the optimal detergent concentration in the washing buffer needs  
to be determined experimentally for each system.

One thing that I found useful was Pansorbin (Calbiochem, killed Staph.  
aureus cells) instead of Protein-A agarose, it comes cheaper and works  
better.