Constitutive probes (again?)
Joel Andrew Krepps
jkreps at SCRIPPS.EDU
Thu Oct 17 08:18:18 EST 1996
On Thu, 17 Oct 1996, Jonathan Richard Howarth wrote:
> I am currently studying gene expression in Arabidopsis roots,
> stems, leaves, flowers and siliques and am looking for a constitutive
> probe for my northerns. I have previously tried an 18S ribosomal probe
> which worked fine but I found I could not strip and reuse the filters
> afterwrds. I then tried Actin (AAc1, Nairn et al., (1988) Gene, 65.
> p247-257) but this seems to be more highly expressed in flowers and
> perhaps less in leaves i.e. its not constitutive.
> Does anyone who has done similar studies in the past have any
> suggestions as to a better probe and if so where I could get it from.
> Hope some one can help. My e-mail is jrh2 at st-andrews.ac.uk
> Thanks for any advice,
> Jonathan Howarth.
Another option is use a probe for beta-tubulin (eight? family members in
Arab.) but do the hybridization/washes at low stringency. The idea is to
pick up signal from all the isoforms, the total of which should not
change for the same number of cells. I have used the ribosomal approach
to normalize but with a slight twist- I use the rDNA genes from
Pisum sativum (Pea), hybridize under normal stringency but wash under low
stringency. When I have 4 ug of total RNA per lane and transfer to
nitropure (nylon reinforced nitrocellulose from MSI, Woburn MA USA), I
can strip the blot to ~ 100 to 200 cpms. I then leave the blot to decay
to background. I usually try to get all of my anticipated probing done
prior to the rDNA probing but have often had to do additional probings
later and other than the time factor, it has not been problematic for
Dept. of Cell Biology
The Scripps Research Institute
La Jolla CA
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