Help destaining NBT/BCIP Western blot on Nitrocellulose

Harold Rees hrees at mail2.sas.upenn.edu
Mon Oct 7 14:24:58 EST 1996


Bradley Turner (bsturner at MBCRR.HARVARD.EDU) wrote:
: Does anyone know of a method to destain (or reduce the amount of
: over-staining) of a western blot that was detected with alkaline-
: phosphatase secondary antibody and stained with NBT/BCIP on
: nitrocellulose?  The blot was overdeveloped in the dye reagent
: and it is difficult to see the bands of interest.

: Or is there a way to wash off the current stain and antibodies
: leaving the blotted proteins on the NC thus allowing re-probing
: of the original blot?

Yes.  Washing in dimethylformamide (available from Sigma and others) removes 
the NBT/BCIP product from the membrane.  You have to be careful, though,
because dimethylformamide also likes to do strange things to certain 
plastic containers that may be used for the wash!  Glass is OK, though.  
I'm not sure how long to wash the membrane, but over-washing should not 
be a problem. 

--
Hal Rees                                ********************************
Monell Chemical Senses Center           *"Oh well, whatever, nevermind"*
Philadelphia, PA                        *  -Kurt Cobain                * 
hrees at mail.sas.upenn.edu                ********************************



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