2D gel question

Hiranya Roychowdhury hroychow at NMSU.EDU
Wed May 10 15:20:57 EST 1995


On 9 May 1995, DAVID MISZTAL 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?
> 
> 


Hi,
	Two-dimensionals can be run with the regular reducing buffer, 
however it is best suplemented with 50mM DTT. The protein can also be 
pptd with the addition of 5-6 vol of chilled ethanol (-20C) and leaving 
it at -20 for 30 min. Spin the pellet down, dry in a speed vac or under 
steady N2 flow, and redissolve the pellet in 5 - 10uL of 10%SDS and 50mM 
DTT, heat to 90C for 5 min. Cool to RT and add water to the volume that 
the tubes can accomodate (generally about a total of 50uL). The reason 
not to use 2ME is that it is unstable.

good luck
			>>>>>>>>>>>>>>>>>>>>>>>>>>>
			  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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