Does anyone have experience with identification of protein from blotting membrane? I tried to follow several methods, but I have found very less robustness and high protein loss during the processing. Any suggestions?
Maybe you have to consider the use of a specific PVDF for MS analysis. Merck Millipore has a series of membranes named Immunobilon-PSQ transfer membranes that can be useful for your necessities.
I spent a lot of time doing this several years ago, and finally I gave up. The few proteins that I could extract from the protein were heavily modified pointing to protein damage, oxidation, etc during electrophoresis and blotting and staining procedure.
I think you must try the protocols for phosphopeptide analysis. Here is the ref: http://www.jbc.org/content/284/17/11090.full.pdf
When I extracted proteins from PVDF membrane, I used then LC-ESI-MS. For MALDI-TOF it's quite complicated sample because of membrane. But I used ammonium bicarbonate and dimethylformamide for disruption of membrane and protein extraction.
Probably it may be an approach to put a cutted piece or strip directly in the MALDI after treating the whole membrane withe the matrix solution?
Please see the references:
If you can use a high sensivity MS you can identify the proteins by theese kind of approaches.
Analysis of Electroblotted Proteins by Mass
Spectrometry: Protein Identification after
Western Blotting
Western Blotting Method: Direct On-Membrane Identification
of Target Molecules Detected by Western Blotting
Comparison of in-gel and on-membrane digestion
methods at low to sub-pmol level for subsequent
peptide and fragment-ion mass analysis using matrixassisted
laser-desorption/ionization mass spectrometry
Maybe you have to consider the use of a specific PVDF for MS analysis. Merck Millipore has a series of membranes named Immunobilon-PSQ transfer membranes that can be useful for your necessities.
There are also some procedures for running gels and transferring for other types of analysis that may help cut down on modification. There are few that are relatively easy to find for N-terminal (old) analysis of proteins/peptides as this is very sensitive to any modifications at the terminus. I'm going to check out some of these references as well. Thanks everybody.
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