Journal
MOLECULAR & CELLULAR PROTEOMICS
Volume 7, Issue 2, Pages 308-314Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/mcp.M700415-MCP200
Keywords
-
Categories
Funding
- NCRR NIH HHS [S10 RR014662-01, S10 RR14662] Funding Source: Medline
- NIDDK NIH HHS [DK52206, DK39753, R01 DK039753, P01 DK052206] Funding Source: Medline
- NINDS NIH HHS [P30 NS050276-01A1, P30 NS050276] Funding Source: Medline
Ask authors/readers for more resources
We describe a new approach for the identification and characterization by mass spectrometry of proteins that have been electroblotted onto nitrocellulose. Using this method (Blotting and Removal of Nitrocellulose (BARN)), proteins can be analyzed either as intact proteins for molecular weight determination or as peptides generated by on-membrane proteolysis. Acetone is used to dissolve the nitrocellulose and to precipitate the adsorbed proteins/peptides, thus removing the nitrocellulose which can interfere with MS analysis. This method offers improved protein coverage, especially for membrane proteins, such as uroplakins, because the extraction step after in-gel digestion is avoided. Moreover, removal of nitrocellulose from the sample solution allows sample analysis by both MALDI- and (LC) ESI-based mass spectrometers. Finally, we demonstrate the utility of BARN for the direct identification of soluble and membrane proteins after Western blotting, obtaining comparable or better results than with in-gel digestion.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available