2D gel question
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.
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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