protein band excision/concentration

Conrad R Fjetland fjetland at bcm.tmc.edu
Tue Jul 28 11:02:25 EST 1998


Antonin Tutter (atutter at aim.salk.edu) wrote:
: Hi all.

: i'm new to this group, i hope my question is not inappropriate:

: does anyone have a tried-and-true protocol for excising a protein band from
: a gel, electroeluting it onto a membrane, and precipitating it so as to
: concentrate it?  i would like to silver stain the gel first if
: re-solubilization is possible.  the key here is minimal loss.  i am trying
: to concentrate a peptide that i am pulling down from nuclear extract using a
: his-tagged protein pulldown protocol.  the band is detectable by silver
: stain.  i need to load at least 50 or so lanes to have enough peptide to
: obtain sequence data.  

: please e-mail me your reply, i don't have access to the newsgroup form work.

: thanks in advance,

The way we extract proteins from gels is to cut the band with a real fine
razor blade.  Place the gel slice into some dialysis tubing with a
small volume of buffer and seal it
with clips.  Then we tape the tubing flat onto a agarose gel box and fill
it up with SDS-PAGE running buffer, or whatever buffer you are using.  Run
the extraction of the protein from the gel at a low voltage at least 4
hours at 4 degrees.  Then to concentrate the protein, you can use several
different methods.  We use this method for 50 kD proteins, so I don't
know if it will work for very small proteins.I don't think that the silver
stain can be removed,
so you may not be able to stain the gel before cutting.  You can stain a
strip of the gel that has a duplicate sample of the ones you wish to cut
out and line the strip up.  Hope this helps.

Conrad R. Fjetland
Baylor College of Medicine
fjetland at bcm.tmc.edu 



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