Constitutive probes (again?)

[Joel Andrew Krepps]

On Thu, 17 Oct 1996, Jonathan Richard Howarth wrote:

> Hello,
> 	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.
> 
> 
> 
Dear Jonathan,

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 
me.  

Joel Kreps
Postdoc
Dept. of Cell Biology
The Scripps Research Institute
La Jolla CA