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?

	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

More information about the Proteins mailing list