DIG-labelled RNA probes

Ed Beaty ebeaty at firecraker.com
Wed Aug 14 15:33:59 EST 1996


If you're using a chemiluminescent detection kit, it's possible that you are "burning out" your signal.  If too 
much probe is binding to your blot, it will bind a huge amount of enzyme conjugate.  When you add the 
chemiluminescent substrate, the conjugate will react with the substrate, using up all the reactive chemicals 
(dioxetane or luminol) before you get a chance to put the blot on film.  As a result, you will only see signal 
on the periphery of the blotted band, where the concentration of probe and conjugate is much lower.  

You can try reexposing your blot to a colorimetric substrate; if you're burning out a chemiluminescent 
substrate, you probably have enough signal to get a strong band with a less sensitive colorimetric substrate.  
Also, you don't see the "halo" effect you were describing. The next time, you could try cutting back on the 
probe concentration or the enzyme conjugate concentration.

Hope this helps,
Ed Beaty
Research Associate
Pierce Chemical Co.


GILLIAN NI BHARLO BOTANY PG wrote:
> 
> Hi there!
> I have recently started doing northerns using DIG-labelled RNA
> probes, and have noticed some strange binding patterns with some of
> them. The probe appears to encircle the band of interest rather than
> actually binding to it. This particularly seems to be a problem with
> heavily loaded gels - is overloading masking the RNA hybrid from the
> antibody? I hope to use these probes in in-situs and am afraid a
> similar effect there would distort an attempt at localising signals.
> I will be exceedingly grateful if anyone can cast some light on this
> one.....!
> THanks in advance
> Gillian



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