protein/gel question

Bob Steinberg rsteinbe at etowah.uokhsc.edu
Sun Jul 6 16:47:08 EST 1997


John C. Robinson wrote:
> 
> To all:
>         This question recently came up, and as my experience with protein work
> is a bit sketchy (to be charitable), I thought I would throw it out to
> you all for comments.
>         We're in the process of purifying a large mol. wt. protein to use to
> generate an antibody.  Our traditional way to isolate the protein is to
> run it out on a gel, stain with a Coomassie B.B./MeOH/AcOH sol'n,
> destain, and cut out the protein of interest.  The suggestion was put
> forth that simply staining the edges (where the standards are) would be
> better - then you could re-assemble the gel and cut out the (unfixed and
> unstained) chunk presumably containing your desired protein.
>         Is this, in fact, preferable?  Does fixing or staining the gel have any
> adverse affect on future uses of the protein (subsequent to purification
> from the gel, of course)?  Might there be any other ways to get the
> protein even better than these two?  We await your collective wisdom
> with bated breath.
> 
>         TIA,
>                 John, for the lab
You might try staining with copper (about 5 min in 0.3M CuCl2 followed
by a few seconds destaining in 0.25M Tris, 0.25M EDTA)-- this gives
fairly sensitive "negative" staining-- after locating and excising the
band(s) of interest, the copper can be removed by more extensive washes
in the Tris-EDTA solution-- this procedure has the advantage that the
only fixation is by copper cross-linking, and this is entirely reversed
by the chelator treatment. The protein can then be recovered by
electroelution.



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