In answer to: Removing GuHCl prior to PAGE

Jonas jonas at CAJAL.MBB.KI.SE
Thu Jun 17 10:57:44 EST 1999


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Hello


The method book "Current protocols in Protein chemistry" provides the
following protocol, which worked wonderful in my hands to precipitate
proteins after size exclusion chromatography in the presence of 4 M
GuHCl (Chapter 6.3.6). The precipitate you observe is due to the
insolubility of a GuHCL-SDS complex what means that GuHCl has to be
completely removed prior to PAGE:


25 ul sample solution
+ 225 ul ice cold EtOH
vortex, incubate 10 min -80°C (or -20°C)
spin down 5 min max. setting, 4°C
withdraw supernatant, save pellet
suspend pellet in 250 ul ice cold 90 % EtOH to remove remaining traces
of GuHCl
mix thoroughly
spin again same settings
air dry pellet
proceed


You can upscale the method



Good Luck


Jonas



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