South-western blot ?

Frank O. Fackelmayer Frank at Fackelmayer.de
Fri Jun 8 09:09:04 EST 2001



Bertrand Collet wrote:
> 
> Hello,
> 
> This might be a stupid question:
> 
> Is it possible to bind a DNA-binding protein to its target DNA sequence
> immobilised on a  southern membrane ? In other word, how accessible is a
> DNA molecule bound to a southern membrane ?
> 
> Is there anybody who tried this and/or who has a good reference for this
> ?
> 


South-western blots are usually done the other way round: immobilize the
protein on a membrane and incubate with radioactive DNA. We´ve done that
routinely for years, and I can send you a detailed protocol.
South-westerns can either be done after SDS-PAGE (but then, of course,
the protein is denatured and has to "refold" to an active conformation),
or as dot-blot assays where the native protein is blotted to the
membrane by vacuum. 
I have never done it the way you suggest, but it may work as well. The
accessibility should be sufficiently good, because southern blots work
well and DNA molecules are much bigger than proteins. However, the
protein could be washed off the membrane during the detection step.

Frank




More information about the Methods mailing list