nicholas_theodorakis at urmc.rochester.edu
Tue Nov 16 09:39:52 EST 1999
In article <3831084F.C5096FA3 at musica.mcgill.ca>, Mano
<mdep at musica.mcgill.ca> wrote:
> YES, I am trying to localize a specific message. However, i am also
> wondering what to expect to see by ethidium bromide staining. In
> fractions would I see the most intense 28 and 18 S rRNAs?
> Mano wrote:
> > Hi again,
> > If I fractionate polyribosomal RNA and RNPs on a sucrose
> > and perform a northern blot, what profile of RNA would I expect
> to see
> > on the gel??
> > Thanks
> > Emmanuel Petroulakis
Manoli, do you collect the gradient by pumping through a UV flow cell
to record the absorbance? If so, that should give you a clue as to
where to expect the most intense rRNA staining.
In general, it will depend how translationally active your particular
cells are. Some have a whopping big absorbance peak at 80S monosomes,
but for some, most of the RNA is in the polysome fraction.
As to where those peaks are, it depends on your gradient and spin time.
If I do a 15-45% gradient for 90 min. in a SW41, and collect 16
fractions, the 80S is around fraction 3. The polysomes are spread
throughout the rest, but the distribution and peak will depend on the
cell. To spread out the "top" of the gradient, but still keeping
polysomes from pelleting, I do 10-50% gradient for 2 hrs, which should
spread out the 40S, 60S, and 80S peaks well, and bunches the polysomes
in the bottom half of the gradient. This is where EtBr staining or
Methylene Blue staining of the blot will help in determing which peak
is 40S, etc.
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