multiple bands on GAPDH S. blot

Andrew Hobbs andrewh at uniwa.uwa.edu.au
Mon Sep 20 03:33:51 EST 1993


Darin Obrien (IHG) (darin at gene.med.umn.edu) wrote:

: 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?

Hi, 
	Is there any possibility that one of your reagents or even the
genomic DNA itself is contaminated.  eg a plasmid or lambda DNA.  You
need very small amounts to swamp the normal hybridization signal.  And
it doesn't even need to be a GAPDH clone if your probe DNA contains
vector sequences.  

Andrew Hobbs
Biochemistry
University of Western Australia
andrewh at uniwa.uwa.edu.au


: 	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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