svetlov at oncology.wisc.edu
Fri Nov 13 18:49:28 EST 1998
In article <F42CB9DBDC42D211B39F00AA00AF32820233BB84 at Keiki.Hpu.Edu>,
"Tatyana Nikitina" <TN3532 at TN3532.spb.edu> wrote:
: Vladimir Svetlov wrote:
: >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
: >antibodies etc.)?
: The subunits were labelled with P32.
Aga! - skazali my s Petrom Ivanovichem... I may be wrong since you did not
give enough specifics but anyway... There were a couple of occasions when
my colleagues labelled cells with P32 inorganic (ortho) phosphate in their
quest for protein phosphorylation. Most of label in such situation ends up
in nucleic acids and if one is not careful during the purification
procedure nucleic acids are going to be predominant P32 carriers. These are
usually not getting into protein SDS-PAGE gels well and could make it
appear as if most of the label is stuck in the well. Gamma-P32 ATP is not
gonna be as bad but prolonged incubation with the cell would eventually
make P32 end up in nucleic acids also. If you think this could be a problem
treat your proteins with some nonspecific nuclease like P1 and see if it
does the trick.
: >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.
: We also think that our problem is with protein aggregation but we can not
: reveal the nature of this aggregation. It seems that only phosphorylated
: subunits aggregate because when we stained the gel after autoradiography
: with silver we could see that nonlabelled subunits had been separated.
I'd try to separate unlabelled prep (no P32) and prob it with
phosphorylation-specific antibodies just to make sure that this is indeed
the case. Treating the sample with nucleases (above) could also help to
reduce aggregation/trapping if nucleic acids are present. As I mentioned
before loading in 6 M urea should help too.
V. Svetlov, Ph. D.
McArdle Laboratory for Cancer Research
University of WI, Madison
1400 University Ave.
Madison, WI 53706
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