Protocol for gel silver staining – mass spec & protein identification

Aug 6. 2015

Problems arising from the use of commercial staining kits:

Scientists use silver staining for sensitive detection of proteins separated by 1D and 2D SDS PAGE. The detection limits should be between 0.5 and 5 ng [1, 2].

Many silver staining protocols and commercial staining kits are not compatible with mass spectrometry. It happens because they use cross-linking reagents. Therefore, it is essential to use a silver stain protocol compatible with mass spec if one wishes to identify proteins (protein identification) from bands and spots isolated from SDS PAGE gels [1, 2].

Our recommendations for selecting a proper staining method:

We recommend that you use a silver staining method that is optimized for compatibility and sensitivity with trypsin digestion and MS analysis for low protein amounts. Many silver staining protocols are incompatible and thus do not work with protein identification using mass spec (mass spectroscopy) [1, 2].

This protocol (follow the link below) describes how to use the Alphalyse method for SDS Page gel silver staining, which we optimized for mass spectrometric analysis.

How to get confident protein identification and high sequence coverage:

By using the protocol, you get confident protein identifications. In addition, the protocol provides a high sequence coverage by MALDI MS and ESI MS due to the high recovery of peptides from the stained gel. The protocol was tested and documented in many publications [3] (see examples on the Alphalyse website).

Get the full protocol here

Protocol for silver staining before LC-MS analysis


[1]          Chevallet et al.Silver staining of proteins in polyacrylamide gels,” Nature Protocols2006

[2]          Rabilloud, T.: “Mechanisms of protein silver staining in polyacrylamide gels: a 10-year synthesis“, Electrophoresis1990

[3]          Mortz et al.Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis,” Proteomics2001

About the author:

Ejvind Mørtz, Alphalyse

Ejvind holds a PhD degree from the University of Southern Denmark in Protein Chemistry & Molecular Biology. He has more than 20 years of experience in the development of protein analyses and mass spectrometry methods in the research & development of protein biologics; vaccines, monoclonal antibodies, therapeutic proteins as well as cell and gene therapies. He is experienced in project management and outsourcing collaboration projects with pharma/biotech, CMO/CROs as well as university research groups.

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