I am trying to assist a researcher who wants to confirm a protein ID she has from a western blot. She'd like to run just the western band, and rather than the full in-gel she'd like to run after a Western detection to verify. However, it's a very low abundance protein and I am concerned about sensitivity. Does anyone have any advice about the working sensitivity range you typically observe from an on-membrane digestion? Or even a recovery estimate?
Any other tips or suggestions also welcome.
I have not tried on-membrane digestion, but i have done In-gel digestion.
I think entire protein removal from membrane is not possible, even tough partial protein can be obtained using on-membrane digestion which enough for identification purpose. But following out comes can be expected
1. masking of peaks form you protein of interest by complex mixture of proteins you used for blocking the membrane during western blot development
2. Again you will be digesting not only your protein but also antibodies used for development during western blot. They will too contribute peaks during MS identification
keeping these out comes in mind you will have to perform sample preparation for MS analysis.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques