Destaining NBT-BCIP -northern-blot

Paul Klosen klosen at neurochem.u-strasbg.fr
Tue Jan 23 11:30:38 EST 2001


Hi,

I don't know about Nylon resisting chloroform, on PVDF Western blots the
best and cleanest elution I know is with chloroform. I know this
certainly won't work with Nitrocellulose, but PVDF resist nicely. Don't
be afraid of the PVDF turning translucent in the chloroform, it will
turn back to normal once go go back to water through alcohol rinses. Oh
yes, I almost forgot, to get to chloroform you either have to dry the
blots or better rinse them in ethanol or methanol to get rid of the
water. Of course, you do all this in a chemical hood because of the
health risks associated with chloroform (one of the drawbacks of the
technique)

Paul

Michael Witty wrote:
> 
> Dear Francoise, I did not know that you could remove the indole coloured
> precipitate from NBT-BCIP stained membranes.  How did you find out about
> this?  It sounds good for my NBT-BCIP stained western blots!
> Regards, Mike.
> 
> On 23 Jan 2001 francoise.martz at metla.fi wrote:
> 
> > Non-radioactive northern-blot, colorimetric NBT-BCIP detection:
> > To reprobe a northern membrane (nylon N+) using the DIG and the
> > colorimetric system, I am removing the precipitate by incubation in DMF,
> > 50oC, but there are 2 problems:
> > - strong staining is not removed,
> > - I have the feeling that RNA are also removed (they are fixed by backing
> > 80oC, 2h).
> > So is there any other possible treatment to remove the color precipitate,
> > that keeps the RNA on the membrane...?
> > Thanks for your help.
> > Francoise
> >
> > ___________________________________
> > Françoise Martz
> > The Finnish Forest Research Institute
> > Rovaniemi Research Station
> > P.O. Box 16
> > 96301 Rovaniemi, Finland
> > Tel: +358 16 336 4215 - Fax: +358 16 336 4640
> >
> >
> > ---
> >
> >

-- 
Paul Klosen, PhD
CNRS UMR 7518 Neurobiologie des Fonctions Rythmiques et Saisonnieres
Universite Louis Pasteur  12, rue de l'Universite F-67000 Strasbourg,
FRANCE
Tel. 03.88.35.85.04  Fax. 03.88.24.04.61  klosen at neurochem.u-strasbg.fr






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