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another GST story #2

Dietmar.Tietz at agrar.uni-giessen.de Dietmar.Tietz at agrar.uni-giessen.de
Mon Jul 18 07:59:36 EST 1994

> 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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