protein band excision/concentration

[JJ Miranda]

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.

Sincere regards,
JJ Miranda

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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