2D gel question

Philp Robin John mcbrp at leonis.nus.sg
Sun May 21 23:44:55 EST 1995


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 with the 1D buffer is the amount of SDS present and this will 
interfere with the iso-electric focussing step of your 2D run. I have not 
tried loading directly in Laemmli loading buffer or having diluted it 
first. However, I have carried out cell lysis, in preparation for 2D 
PAGE, using a buffer containing SDS, Tris, 2ME so it is similar in its 
composition to the 1D gel loading buffer. After lysing cells in this I 
treated the gloop with Dnase and Rnase to break down nucleic acids and 
then carried out an acetone ppt. This worked very well and the pellet 
could be resuspended in the urea buffer for 2D and excellent separations 
were obtained. Only point to note is that the acetone ppt should be done 
at room temperature to prevent the SDS coming out of solution and ppt'ing 
with the protein. Hope this is useful.

Robin J. Philp
Institute of Molecular and Cell Biology
Singapore, 0511.



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