Help: Modified Western.

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Fri Feb 13 09:49:25 EST 1998


In article <6btj3e$tpd$1 at news3.tufts.edu>, jdimos at tufts.edu (John T.
Dimos) wrote:

> I am not having any luck with the following protocol, and would appreciate 
> any feedback or even ridicule if appropriate.
> 
> After standard SDS-PAGE and transfer, I block my blot thoroughly (I've used a 
> variety of blockers - homemade and commercial).  I then incubate with my 
> ligand of interest.  After washing I add a mAb against said ligand,
wash, then 
> incubate in a secondary conjugated to HRP.  After ECL (supersignal, 
> Pierce) development my entire blot is black, even with a 5 sec film
exposure.  
> All dilutions are in PBS, and all washes are in PBS, and should be sufficient 
> (4-6x for 10 mins each).  The ligand is ~50aa if that is of interest.  The 
> antibodies work as they should.  If I run the above protocol but do not add 
> the ligand my blot is perfect - has the expected bands.  Any suggestions
would 
> be appreciated.
> Thank you.
> John Dimos.

Hi John, 
Sounds as if the peptide ligand binds strongly to either the membrane
itself, or a component of the blocking agent.
First of all, I would replace PBS with a more stringent solution, e.g.
TNT, TBST, or even RIPA buffer. Then, try changing the type of membrane,
i.e. use nitrocellulose if your blots were on PVDF/Immobilon, or vice
versa. If the problem persist, change your blocking solution. It is
certainly worth then to try different blocking agents, ranging from dried
milk to polyvinylpyrrolidone (PVP) or tween. You can also try the
commercial Rotiblock solution from Carl Roth GmbH in Karlsruhe, Germany.
It is some chemical, non- proteinaceous compound that works great in our
hands.

Good luck,
Frank



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