protein band excision/concentration
atlantis at netcom.com
Fri Jul 31 22:09:00 EST 1998
In-gel digestion followed by ESI can get a little ugly b/c the 5% TFA you
need to extract the peptides isn't very nice to HPLC columns. If you do
decide to do ESI, use 5% formic acid instead since it still works. IMO
though, it might be better to do an in-gel digest and then use MALDI.
This way, you can also sequence the peptides.
A caveat on the Cohen and Chait method. 1. It only extracts whole
protein. 2. You need many picomoles to do it. If you have only a small
amount of protein (even into the high femtomole range) you can still get
a sequence with in-gel digests and MALDI.
D. Pearton (pearton at saul5.u.washington.edu) wrote:
: 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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