2D gel question

Hiranya Roychowdhury hroychow at NMSU.EDU
Fri May 12 14:55:55 EST 1995


On 11 May 1995, Cornelius Krasel wrote:

> DAVID MISZTAL (P1034294 at CSDVAX.CSD.UNSW.EDU.AU) wrote:
> > 	Someone has asked me to run a protein sample which is in
> > normal reducing buffer used for 1D gels.  Has anyone ever tried to
> > add 2D sample buffer to such a sample to obtain successful separation
> > on a 2d gel?  Alternatively, is there a method for precipitating
> > proteins from reducing buffer?
> 
> The problem is probably not the reducing agent but the SDS which will
> make isoelectric focusing next to impossible.
> 
> --Cornelius.
> 
> --
> /* Cornelius Krasel, Institut f. Pharmakologie, Versbacher Str. 9, D-97078 */
> /* Wuerzburg, Germany                email: phak004 at rzbox.uni-wuerzburg.de */
> /* "Science is the game you play with God to find out what His rules are." */
> 
> 

We use a combination of SDS/DTT buffer to resuspend precipitated protein
samples to be used for 2-D analysis. The protein is first dissolved in a
minimal amount of the SDS/DTT buffer and immersed in 90 C water bath for 2
min. To this the regular O'Farrel's 2-D buffer is added to a final vol. of
50uL. In fact in O'Farrels paper he mentions the use of SDS.  Logically the
use of SDS to solubilize proteins for 2-D seems a bit self-defeating,
considering what the first dimension is all about.  However, this treatment
(as mentioned above) does not affect the isoelectric separation to any
significant degree. And since the usefulness of the system lies primarily in
the identification of polypeptides showing identical Mr values in a 1-D
SDS-PAGE, a slight alteration of the pI of the species(if any) should not
pose a major problem. Furthermore, as someone that is working with membrane
proteins, I have found it even more useful a method in preparing 100,000xg
pellets for 2-D analysis. I suspect that the short heating time does not
alter the charges in the polypeptides, yet the treatment solubilizes the
proteins nicely. In fact streaking is also minimal as a result.
			>>>>>>>>>>>>>>>>>>>>>>>>>>>
			  Hiranya S. Roychowdhury
   			  Plant Genetic Engineering Lab.
			  Box 3GL, NM State Univ.
			  Las Cruces, NM 88003
			  Phone: (505) 646-5785
			  hroychow at nmsu.edu
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