protein band excision/concentration

D. Pearton pearton at saul5.u.washington.edu
Fri Jul 31 19:04:57 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.  

Something to try might be reverse staining with Copper stain (Biorad) and
excission of the band followed by destaining.

See Cohen and Chait (1997) Analytical Biochem 247, 257-267

I've managed to recover test proteins in this manner that were analysable
via MALDI.

As an alternative, if you're trying to identify the peptide why not
silver stain followed by in-gel digestion and elctrospray MS/MS?

Yet another method might be to blot onto PVDF, coomaasie stain and
N-terminally sequence the band.

There are also charged versions of PVDF that are designed for elution of
proteins.  I must admit I've not had that much success with them.

--
Dave Pearton
pearton at u.washington.edu
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Down he sank in a chair-ran his hands through his hair-	+
     And chanted in mimsiest tones			+
Words whose utter inanity proved his insanity,		+
     While he rattled a couple of bones.		+
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"The Hunting of the Snark" Lewis Carroll		+
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