svetlov at oncology.wisc.edu
Thu Nov 12 13:54:35 EST 1998
In article <364AC571.1DFCA772 at TN3532.spb.edu>, "Òàòüÿíà Íèêèòèíà"
<TN3532 at TN3532.spb.edu> wrote:
> I would be very grateful if you would give me some advice. The
> point is that
> we study the influence of RNA polymerase III phosphorylation on the
> level in vitro and have faced one problem. When being analysed by
> gel electrophoresis (by Laemmli, 1970), the phosphorylated RNA
> polymerase III
> subunits are arrested at the start line.
What led you to believe that this is the case (were the phosphorylated
subunits labelled with P32/33, were they assayed with anti-phosphoY/S/T
> At the same time
> subunits of the enzyme run throug the gel freely. Would you let me know
> whether you
> had the problem like ours in your work and if that's the case how you
> solved it.
> Thank you in advance.
No one in our lab could recall a situation when phosphorylated protein was
selectively retained in the well. I've been working with Pol II, which is
differentially phosporylated at CTD of its largest subunit (Rpb1) to what
appears to be much greater degree than any of the Pol III subunits,
nevertheless, phosphorylated Rpb1 gets into the SDS-PAG without much
problems. The conditions we use for the full Pol II run are 4-20% gel
(either homemade or Novex precast) in Tris-Glycine buffer at 100 V for
couple of hours. Other folks routinely use SDS-PAGE to separate
phosphorylated and unphosphorylated Rpb1 and you can see for yourself that
substantial phosphorylation is not barring rather large molecules of Rpb1
from entering the gel (Archambault et al., JBC, 1998, 273:27593-27601;
Sakurai & Fukasawa, JBC, 1998, 273: 9534-9538 - these just from the top of
the pile and you can view them online from JBC site). I believe that the
problem you are experiencing is not with the SDS-PAGE of phosphorylated
subunits per se but either with using too high percentage of the gel (they
do tend to run slower than non-phosphorylated ones at least in some gel
systems) or with protein agrregation. Make sure you have fresh sample
buffer with plenty of BME. You can also try loading your proteins in 6 M
urea (unlike GuaHCl it does not precipitate and does not significantly
affect the separation) - this should pull most of your subunits apart. I'd
try to use 4-20% or 6% gel with Tris-glycine or phosphate buffer while
making sure that all the components/operations affecting denaturation of
the sample (heating, BME, and SDS) have been added/carried out.
P.S. When The Man (Dick Burgess) gets back I'll ask him whether somewhere
in that inexhaustable supply of stories of protein purification and
analysis there is a fable about phosphorylated proteins that would not get
into the gel.
> Tatyana Nikitina (TN3532 at TN3532.spb.edu),
> St. Petersburg, Russia
Vladimir Svetlov, PhD
McArdle Lab for Cancer Res.
1400 University Ave.
Madison, WI 53706
More information about the Methods