Mass spectrometric identification of proteins from Western blots

Eppendorf tubes with protein
May 2. 2016


We have an unknown protein recognized in Western blot by autoantibodies that we need to identify.

Is it possible to send in a piece of a PVDF western bloting membrane for mass spectrometric protein identification, do you have any special requirements or protocols that needs to be followed?


Proteins detected by antibodies in Western Blots cannot be directly analyzed by mass spectrometric peptide mapping. The main reason is that the Western Blot protocol adds other proteins to the PVDF membrane for detection. This includes proteins for blocking the membrane, typically milk protein powder or bovine serum albumin (BSA), as well as primary and secondary antibodies for staining [1, 2].

Instead, to identify the unknown protein, a parallel 1D or 2D gel should be run at the exact same conditions, stained by a sensitive silver stain optimized for compatibility with mass spec analysis, the protein spot of interest cut out and analyzed by trypsin digestion and nano-LC MS/MS or MALDI MS/MS peptide analysis for protein identification [1, 3, 4].

Also the antibodies used for detection can be very specific and able to identity minute amounts of a specific protein even though other proteins may be present at the same position in the 1D or 2D gel. The mass spectrometric analysis requires that the protein of interest is relatively pure at the gel position [2, 4].

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[1]          Shevchenko et al: Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels” Analytical Chemistry1996

[2]          Jonsson et al: Recovery of gel-separated proteins for in-solution digestion and mass spectrometry“, Analytical Chemistry, 2001

[3]          Liang et al: “Characterization of SDS–PAGE-separated proteins by matrix-assisted laser desorption/ionization mass spectrometry“, Analytical Chemistry1996

[4]         Jørgensen et al: Efficacy and compatibility with mass spectrometry of methods for elution of proteins from sodium dodecyl sulfate-polyacrylamide gels and polyvinyldifluoride membranes“, Analytical Chemistry2004

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