SDS PAGE and phosphorylation

Jim Woodgett jwoodget at oci.utoronto.ca
Wed Jul 31 11:03:49 EST 1996


In article <4tmeof$5s6 at d2.tufts.edu>, rfeldber at emerald.tufts.edu (Ross S 
Feldberg) writes:
 
>   I am curious if anyone knows why phosphorylated proteins can 
> be separated from their non-phosphorylated forms on SDS gels. I 
> would assume that the introduction of 1 or 2 more negative 
> charges wouldn't have a significant effect on a protein that has a 
> large amount of SDS already bound. Can anyone point me to a review 
> article or techniques article which discusses this ?  thanks 

It's been rationalised that the addition of negatively charged phosphate 
interferes and reduces SDS binding thus actually reducing the net negative 
charge/mass ratio of the protein which is relied upon to neutrilise the effect 
of charge and allow separation of proteins to be largely dependent on the 
sieving effect of the gel.

I don't know about a good review  on this but the effect is well-known and 
often exploited to monitor phosphorylation (e.g. shifting of ERK1 or ERK2 upon 
phosphorylation/activation by MEK).  The problem is that the underlying effect 
is highly dependent on the protein (presumably the amino acids surrounding the 
phosphorylated residue(s)). Some highly phosphorylated proteins don't shift at 
all whereas other shift by >20 kDa.  The magnitude of the effect is also 
dependent on the conditions and type of gel (%BIS, overall % acylamide, etc.).

In general, SDS gel mobility shifts should not be relied upon for estimation 
of phosphorylation due to the empiricism of the effect and alternative 
possibilities for the apparent mass change (such as glycosylation, 
ubiquitinylation, etc.).

Hope this helps,

Jim Woodgett


Associate Professor: University of Toronto
Senior Scientist: Ontario Cancer Institute mailto:jwoodget at oci.utoronto.ca
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