2-D gels with phosphoproteins

Stephen P. Driska PhD driska at astro.ocis.temple.edu
Fri Oct 20 13:53:36 EST 1995


Patsy RENARD (prenard at biocell.fundp.ac.be) wrote:
: Is there anyone who has already been successfull wit bi-dimensional gels
: run on with 32P-labelled proteins ? If so, HOW ?

: I am using the Pharmacia Biotech system with precast immobiline dry strip
: gels for the first dimension and precast SDS polyacrylamide gradient gels
: for the second dimension. I get very good results when I stain the gels
: with silver. But if I run on phospho-labelled proteins (derived from
: classical in vivo labelling protocols), I get a important background on
: the autoradiography, making any analysis of the gel impossible.
: Could you please indicate me how to decrease this background ?
: Thanks for your attention.

: Patsy

: -- 
: Patsy Renard (PhD student)
: Cellular Biochemistry, FUNDP
: 61, rue de Bruxelles
: B-5000 Namur
: Belgium
: Tel : 32-81-724321
: Fax : 32-81-724135

	Sure, lots of people have.  The hardware we used was different
(older) but the concept is the same.  The answer may lie in your 32P
incubation conditions.  If you label tissue with inorganic phosphate
32P, all sorts of things get labeled, and it is often said that
phospholipids, nucleic acids, etc get labeled in addition to proteins
being phosphorylated.  Many people do a protein precipitation before
gels, that way eliminating some of this background  when they just run the
protein precipitate.   Also, some people don't show the whole
autoradiograph.  
	More sophisticated scanners/analysis programs allow
backgrounds to be subtracted.

	Some people also get very different results if they incubate
with 32P a very long time compared to a short time.  Since most people
aren't interested in following the technical details, but instead are
interested in specific protein phosphorylation, it may be hard to find
explanations of why tissue is incubated one way or another; you'll
probably just read about the method that works.  Good Luck with it....

--
Steve Driska, Physiology Department, Temple University Medical School
Philadelphia, PA 19140 USA (215) 707-3283 
driska at astro.ocis.temple.edu  
If I could think of something witty and clever to say, it would go right 
here ----->>    .........................



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