multiple bands on GAPDH S. blot

Darin Obrien IHG darin at gene.med.umn.edu
Wed Sep 15 09:24:46 EST 1993


Hi,

	I recently probed a genomic southern blot (10ug mouse DNA from
various cell lines) with a mouse GAPDH (glyceraldehyde phosphate
dehydrogenase)  probe for normalization of the amounts of DNA loaded in
each lane.  I got about 20 distinct bands ranging in size from
about 15-20kb to about 400bp with a predominant band at about 10kb.  My
final wash of the membrane was in 0.1XSSC/0.1%SDS at 65oC for 30 minutes
which is usually stringent enough in my hands to ensure good specificity. 
These bands were easily detected even after a 6hr exposure (usually takes
me a 48hr exposure). This looks to me like I'm picking up high copy number
stuff.  Any ideas? Do you think it could be cross hybridization to other
dehydrogenases?  Has anyone else observed this? I have heard that there are
GAPDH pseudogenes which look like spliced products.  Does anyone know how
many there are?
	More importantly, as I have done this with the intention of standardizing
loading, has anyone used an actin probe for standardization of southern
blots or could you recommend a good probe.

Thanks much,

darin

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