in-nitrocellulose trypsin digest

[Laine]

Hi,
I'm relatively new to this. Has anyone ever done a trypsin digest and extraction on nitrocellulose blotted proteins before?
Any tips would be helpful.
Thanks!

[molecule2005]

Hey there!

Yes, this can be done.

Tryptic digestion can be done with either NC, Nylon, or Immobilon. Nitrocellulose seems to give more reproducible yields of peptides.

see
http://pingu.salk.edu/~sefton/Hyper_...s/Tryptic.html
for a detailed protocol

welcome to the forum!
please please let us know how you did!

[kiki06]

Hey there!

Another method I found is detailed here in this abstract:
http://pubs.acs.org/cgi-bin/abstract...ac060344t.html

Use of Nitrocellulose Membranes for Protein Characterization by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

We present an improved method for MALDI-MS analysis of proteins that have been electroblotted onto a nitrocellulose (NC) membrane. With this approach, electroblotted proteins can be analyzed directly for intact molecular weight determination or after on-membrane digestion by dissolution of the nitrocellulose in MALDI matrix solution containing 70% acetonitrile and 30% methanol. This solution helps maintain solubility of proteins and peptides while dissolving the NC membrane, which is dissolved by 100% acetone in other protocols. On-membrane tryptic digestion using this method requires half the time of in-gel digestion and results in fewer missed cleavages and better protein coverage. For the membrane proteins studied, bovine uroplakins II and III, the protein coverage was almost twice that provided by conventional in-gel digestion, and the transmembrane domains of both uroplakins were detected only after on-membrane digestion. We also demonstrated the compatibility with MALDI-MS of a new dye, MemCode, which is specifically designed for staining NC membrane-immobilized proteins and is faster and more sensitive than Ponceau-S. Our improved on-membrane digestion protocol greatly improves the study of soluble and, particularly strikingly, integral membrane proteins by mass spectrometry.

[Laine]

Cheers for that guys,
I'll have a go and let you know how I get on.
Laine.

[admin]

Great stuff guys!

Yes please let us know how you do Laine