2d gel cleanup

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Fri May 19 20:34:24 EST 2000


In article <3925B034.23D636E9 at uiuc.edu>, Andrea Beckel-Mitchener
<amitch at uiuc.edu> wrote:
>I'm not sure what you mean by "fixing".  I dehydrate with 50%
>Methanol/2%glycerol for 10-15 min before drying.  If the
background is due to
>unincorporated label, this treatment doesn't get rid of it.
>A.
>

Well, that would fix the proteins, but I'm not sure that would
allow enough time for the unincorporated label to diffuse out.

For comparison, in the "olden days" we either
(1) stain the gel with Coomassie Blue in 50% MeOH, then destain
with several changes of 10% MeOH/10% HOAc, and/or
(2) fix in 50% MeOH, then do PPO/DMSO fluorography

Either of those had extensive washing steps that would allow
free label to diffuse out. Perhaps you could follow your 50%MeOH
step with a couple of 20-30 min. washes of 10% MeOH/10% HOAc
(with or without the glycerol as you please)?

As for precipitation, I agree that TCA might be a dodgy choice
before IEF, but perhaps acetone pptn, would be ok.

Also, are these proteins labeled from intact cells, or from an
in vitro translation? The latter can be a little tricky, because
of the high protein content. Also, IVTs have a ton of charged
[35S]met-tRNA that might give a problem in a 2D.

Nick


Nick Theodorakis

nicholas_theodorakis at urmc.rochester.edu
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