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another GST story #2

Dietmar.Tietz at agrar.uni-giessen.de Dietmar.Tietz at agrar.uni-giessen.de
Tue Jul 19 04:17:31 EST 1994


Dear Terry:
 
It is nice to hear from somebody who worked with Hedrick!
 
I hope I got it right when I wrote 1988 in a review in 
Adv. Electrophoresis that Ferguson published such plots
in Metabolism 1964, 13, 985-1002.  Hedrick & Smith
later popularized them in Arch. Biochem. Biophys. 1968, 126,
155-164 and other papers.  My former bosses at the NIH, 
Rodbard and Chrambach, did some mathematics and presented 
a theory based on Ogston's earlier work that explains that semi-
logarithmic relation between electrophoretic mobility and gel 
concentration.  They found that Ferguson was the first one who 
ever published such plots, and they called them Ferguson plots.  
I believe this is now the only name used in the literature.  
Ferguson himself did not suggest any name.
 
As far as the different binding ratios of SDS to proteins 
are concerned, I would suggest that it depends very much on
the charged surface groups.  Proteins with an unusual
composition behave anomalously.
 
By the way, do you have a reference for glycoproteins
absorbing to polyacrylamide?
 
Talk to you some other time, Dietmar




> 
> Dear Dietmar,
> Thanks for your note to me.  I thought those plots were called
> Smith-Hedrick plots.  That may be due to my having Hedrick as my
> lab instructor at the University of California, however.  I must
> say that your discussion is much more thorough than his handout
> or old paper in Anal Biochem back in the '70s.  I think it came
> out the same time as Fergoson's paper and it appears that
> Fergoson won out.
> 
>  But your reply, like mine, does
> not deal with the real question--why does SDS not bind at the
> proper ratio to certain proteins?  Also, I think glycopoteins interaction with
> acrylamide matrix and not with SDS is responsible for the
> analomous migration of these proteins.  This GST#2 thread is
> leading into strange places.  I am getting a lot of requests for
> refs on PEST proteins.
> 
> Well met on the Net!
> 
> Terry
> 
> > 
> > > 
> > > On 15 Jul 1994 15:14:16 GMT, Stephen R. Lasky wrote:
> > > 
> > > (some stuff deleted)
> > > 
> > > >>In general though,  we use denaturation in the presence of SDS so that a
> > > >>protein will migrate at a rate relative to its mass and not be effected by
> > > >>amino acid composition.
> > > 
> > > >That's the generally accepted therory and, no doubt, many if not most 
> > > >proteins fit nicely into that scheme. But there are exceptions, just one 
> > > >example (might be interesting for Jean-Marc and Emma): There is a protein 
> > > >called dTAF40 because on a SDS gel it has the apparent molecular mass of 40 
> > > >kd. Molecular cloning of the cDNA revealed a molecular mass of only 29 kd 
> > > >(Cell 75, 519-530, 1993). Could anybody kindly give an explanation for this 
> > > >unusual migration on a SDS gel? 
> > > 
> > > >Matthias Zeiner
> > > >Inst. Biol. Chem.
> > > >Heidelberg
> > > 
> > > Dear Matthias,
> > > 
> > > It is highly likely that the proteins in your question
> > > that migrate as larger proteins on SDS-PAGE have a high proline
> > > content.  This phenomenon involving proline has been noted many 
> > > times before; for
> > > example see Pham and Sivasubarmanian in GENE 122, 345 (1993) or
> > > Ziemer, Mason and Carlson in JBC 257, 11176 (1982).  My own
> > > personal experience with such a protein is one from an insect 
> > > small RNA virus which is 17 kDa yet migrates at Mr = 24k (>140% 
> > > more apparent MW).  
> > > It is a PEST protein (a property correlated with rapid turnover
> > > and possessed by many regulatory genes and oncogenes) and has a 
> > > 49% of P, E, S, and T.  
> > > However, to the nub of your question as to why proline does this,
> > > I and the literature I've seen are still unaware.  Anybody else
> > > have a handle on this?
> > > 
> > > Terry Hanzlik, terryh at ento.csiro.au
> > > CSIRO Division of Entomology
> > > Box 1700
> > > Canberra, ACT  2601
> > > Australia
> > > 
> > > 
> > > 
> > Dear Terry Hanzlik:
> >  
> > Certain proteins, amoung them glycoproteins and possibly prolin as well,
> > absorb SDS at a different ratio than other proteins.  Consequently,
> > those proteins have a different surface net charge density and one
> > cannot compare them with standard proteins by using just one SINGLE
> > gel concentration.  A way out is the Ferguson plot technique which
> > I have just explained 30 minutes ago in a detailed message to Matthias
> > and to the methods-and-reagents group.
> >  
> > If you have more questions or cannot find my detailed message on the
> > network, please contact me.
> >  
> > Best regards, Dietmar Tietz
> > 
> >  
> > ******************************************************************
> > *           Dietmar Tietz, Ph.D.,  Research Scientist            *
> > *            Biostatistics, Justus-Liebig-University             *
> > *            Ludwigstr. 27, D-35390 Giessen, Germany             *
> > *                  Phone: +49-(641)-702-6015                     *
> > *                   Fax: +49-(641)-702-5995                      *
> > *              Email: Dietmar.Tietz at Uni-Giessen.de               *
> > ******************************************************************
> > 
> > 
> > 
> 
> 




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